ii RENAL ALOGRAFT HISTOPATHOLOGY IN DOG LEUKOCYTE ANTIGEN (DLA) MISMATCHED OGS FOLOWING RENAL TRANSPLANTATION Kristyn Donnely Broaddus Permision is granted to Auburn University to make copies of this thesis at its discretion, upon request of individuals or institutions and at their expense. The author reserves al publication rights. ____________________ Signature of Author ____________________ Date of Graduation iv VITA Kristyn Donnely Broaddus, daughter of Richard M. and Susan L. Donnely, was born on August 30, 1975, in Royal Oak, Michigan. She atended Detroit Country Day School and graduated in 1993. In August of 1993, she atended Georgetown University and graduated in May 1997. She atended Michigan State University?s College of Veterinary Medicine from August 1997 until graduation May 2001. From 2001 to 2002 she completed a smal animal rotating internship in smal animal surgery and medicine at the College of Veterinary Medicine, Auburn University, Auburn, Alabama. From 2002 to 2005, she completed a residency in smal animal surgery at Auburn University. While completing her residency requirements, she was enrolled at Auburn University for a Master of Science degre in Veterinary Biomedical Science. v THESIS ABSTRACT RENAL ALOGRAFT HISTOPATHOLOGY IN DOG LEUKOCYTE ANTIGEN (DLA) MISMATCHED OGS FOLOWING RENAL TRANSPLANTATION Kristyn Donnely Broaddus Master of Science, August 8, 2005 (D.V.., Michigan State University, 2001) (B.S.L.A., Georgetown University, 1997) 90 Typed Pages Directed by D. Michael Tilson Reciprocal renal transplantation and bilateral nephrectomy were performed in 10 healthy, adult, mongrel dogs to evaluate alograft histopathology in dog leukocyte antigen (DLA)-mismatched dogs undergoing renal transplantation with transient imunosuppresion. Imune conditioning consisted of nonmyeloablative (200cGy) total body iradiation (TBI), bone marow transplantation (BMT) (7/10 dogs), cyclosporine (CSA)(15 mg/kg BID), mycophenolate mofetil (MF) (10mg/kg BID) and intermitent prednisone. Biopsies were collected at transplantation, during full imunosuppresion (44 to 90 days), once medications were reduced or discontinued (228 to 580 days) and at necropsy or open surgical biopsy. Biopsies were evaluated for interstitial, tubular, vascular, and glomerular lesions. Blood urea nitrogen (BUN), vi creatinine (Cr), and clinical score were determined at each biopsy. Seven of 10 dogs survived > 200 days (average of 600 days). Transient CSA toxicity was suspected in 6 dogs. Lymphocytic, plasmacytic interstitial inflamation and tubulitis progresed when imunosuppresive medications were reduced. Al seven dogs had histologic lesions consistent with some degre of alograft rejection at biopsy thre. Four dogs were euthanized due to persistent azotemia and histologicaly end-stage organ failure was confirmed. Two dogs are stil alive at 500+ and 1500+ days post-transplantation. vii ACKNOWLEDGEMENTS The author expreses appreciation for the funding provided by the Auburn University Department of Clinical Sciences necesary to engage in this investigation. Appreciation is given to D. Michael Tilson, Stephen Lenz, Glenn Niemeyer and Clinton Lothrop for the guidance in, support of, and commitment to this study. Special thanks are given to Bridget Dean, Johnathan Mahorny, Steve Waters, and Dr. Susan Goodman for their exceptional care of the dogs included in this study, and Beth Landreth for her help with eforts with in the histopathology laboratory. Finaly the author would like to thank Dr. Charles C. Broaddus for his support, patience and understanding during the completion of this research and thesis. vii Style Manual or Journal used Veterinary Surgery Computer software used Microsoft word 2000 for Apple ix TABLE OF CONTENTS LIST OF FIGURES????????????????????????..?xi LIST OF TABLES?????????????????????????...xii I. INTRODUCTION???????????????????.........1 I. LITERATURE REVIEW????????????????????..3 Renal Transplantation Renal Alograft Rejection Histocompatibility Rejection at the celular level Imunosuppresion and Transplantation Imune tolerance Discovery of imune tolerance Induction of imune tolerance Veterinary Studies II. STATEMENT OF RESEARCH OBJECTIVES???????????.21 IV. MATERIALS AND METHODS?????????????????.22 Dogs Bone Marow Transplantation Transplantation Surgery Surgical biopsies Imunosuppresive regimen and monitoring Serum cyclosporine concentrations Clinical status monitoring Histopathological evaluation of biopsy specimens Imunophenotyping of infiltrating lymphocytes V. RESULTS??????????????????????.........30 Clinical Results Biopsy One (normal specimens) Biopsy Two (full imunosuppresion) Biopsy Thre (modified imunosuppresion) x Fourth Biopsy Cyclosporine levels Imunohistochemistry VI. DISCUSION????????????????????..........58 VI. BIBLIOGRAPHY???????????????????????71 xi LIST OF FIGURES Figure 1A. Renal cortex, dog 6, biopsy 1. Microscopicaly normal???????????????????????.?41 Figure 1B. Renal cortex and outer medulla, dog 6, biopsy 2. Focal interstitial, peritubular, lymphocytic-plasmacytic inflamation??????????????????????42 Figure 1C. Renal cortex, dog 6, biopsy 3. Multiple foci of interstitial, peritubular, lymphocytic-plasmacytic inflamation??????????????????????.43 Figure 1D. Renal cortex, dog 6, biopsy 3. CD3-positive lymphocytes infiltrating tubule epithelium??????????..44 Figure 2. Renal cortex, dog 3, biopsy 2. Suspected cyclosporine toxicity. ??????????????????..45 Figure 3. Graph of dog 1: BUN/Creatinine versus Time?????????.46 Figure 4. Graph of dog 2: BUN/Creatinine versus Time????????...47 Figure 5. Graph of dog 3: BUN/Creatinine versus Time ????????...48 Figure 6. Graph of dog 4: BUN/Creatinine versus Time ?????????49 Figure 7. Graph of dog 6: BUN/Creatinine versus Time ?????????50 Figure 8. Graph of dog 7: BUN/Creatinine versus Time ????????...51 Figure 9. Graph of dog 9: BUN/Creatinine versus Time ?????????52 xii LIST OF TABLES Table 1. Clinical score, cyclosporine levels, and renal biopsy schedule in renal transplant dogs??????????..53 Table 2A. Biochemical and histopathological data in renal alograft dogs, Biopsy 1????????????54 Table 2B. Biochemical and histopathological data in renal alograft dogs, Biopsy 2????????????55 Table 2C. Biochemical and histopathological data in renal alograft dogs, Biopsy 3???????????.56 Table 2D. Biochemical and histopathological data in renal alograft dogs, Biopsy 4????????????57 xii 1 I. INTRODUCTION Chronic renal failure (CRF) is the most common disease of the kidney in dogs. 1 It can result from congenital abnormalities, toxic and infectious insults, metabolic alterations, and age related pathology. Replacement of damaged nephrons by fibrotic tisue leads to destruction of neighboring interdependent nephrons, ultimately afecting the entire organ. The exact cause of the initial insult may be dificult to define; however, once seventy-five percent of the total mas of nephrons are ireversibly damaged, end-stage renal disease (ESRD) ultimately develops. 1 Once renal pathology is deemed progresive and ireversible, supportive medical care becomes costly and unrewarding. The ideal solution for ESRD is renal transplantation. Feline renal transplantation has become a feasible therapeutic option; however, canine renal transplantation has had only limited clinical succes. Improvements in imunosuppresive drugs have helped canine renal transplantation become a short-term clinical option, but the side efects asociated with long-term imunosuppresion are significant. The development of life-threatening opportunistic infections, an increased risk of developing tumors, the high cost of imunosuppresive drugs, drug-related toxicities, and eventual chronic rejection of the kidney have al limited routine application of renal transplantation in the dog. An additional chalenge of renal transplantation is acurate post-transplantation monitoring of potential renal alograft pathology. Hematological analysis of blood urea nitrogen (BUN) and creatinine (Cr) may underestimate the extent of renal 2 alograft disease. 2 Protocol biopsies, which are taken acording to a preplanned time schedule, are an aceptable approach for diagnosing subclinical rejections in human patients. 2-5 In humans, identification and prompt treatment of subclinical rejection results in increased renal alograft survival compared to patients who are treated solely on clinical evidence of rejection. 4 Recently, it has been shown that a novel nonmyeloablative bone marow transplantation protocol which induces stable mixed hematopoietic chimerism in DLA-matched dogs can be used to induce donor specific tolerance to skin and renal alografts. 6;7 The current study uses the same imunosuppresive induction protocol which includes 200cGy total body iradiation (TBI), +/- bone marow transplantation (BMT), and short-term imunosuppresion with cyclosporine (CSA), mycophenolate mofetil (MF) and intermitent prednisone. The purpose of this study was to evaluate the progresion of alograft histopathology in relation to renal biochemical parameters (BUN and Cr), and clinical status of DLA-mismatched dogs undergoing renal transplantation. I. LITERATURE REVIEW 3 Renal Transplantation: The solution to end-stage organ failure is organ transplantation. Since 1952 when the first human kidney transplantation was performed and subsequently rejected, researchers have been deciphering the complexities of transplantation imunology. Several years later, after a succesful kidney transplantation was performed from one identical twin to another, investigators began to question the role of the imune system in transplantation medicine. Human, non-human primate, and dog models have been useful for creating protocols that promote succesful alograft aceptance. Recently both veterinarians and pet owners have pushed to offer renal transplantation for end-stage renal failure in cats and dogs. Cats have been succesfully managed with kidney transplants; however, dogs face many of the same chalenges sen in people, particularly, chronic renal alograft rejection. Within the past decade, veterinary medicine has offered several imune modulating therapies with the hope of long-term succes for transplantation in dogs. Pre-transplantation blood transfusions, antithymocyte serum, donor bone marow transplantation (BMT), and various combinations of imunosuppresive medications have al yielded useful information for transplantation medicine. 7-12 Renal Alograft Rejection Histocompatibility It is now wel known that pre-transplantation major histocompatibility complex (MHC) matching significantly improves the succes of organ transplantation. In humans, a sibling who is human leukocyte antigen (HLA) identical to the recipient, is 4 chosen for the most favorable outcome. Non-related HLA-identical matches stil incite a significant imunologic response, as HLA typing is imprecise and the MHC molecule is very complex and polymorphic. Unrelated identical matches stil require some level of imune regulation. Similarly, a dog leukocyte antigen (DLA) identical donor-recipient match can be created to minimize alograft rejection. However, it is often dificult to locate a sibling available for organ donation, and only 25% of these are probable DLA-identical. 13 Rejection at the celular level Given that leukocyte antigen identical siblings are rarely available for organ donation, renal alograft rejection is a very real isue in both human and veterinary medicine. Alograft rejection has been studied extensively in order to understand and control the recipient?s imune response to the donated organ. This rejection proces can be broken down into cel-mediated and humoral components. The cel-mediated imune response requires the binding of antigen to CD4-positive or CD8-positive T cels. CD4-positive cels, sometimes caled T-helper cels, help to coordinate the various activities of the imune system. CD4-positive T cels also generate cytokines IL-2, IL-4, and IL-5 which increase vascular permeability and atract lymphocytes and macrophages. CD8-positive T cels are clasified as T-suppresor cels or kiler T- cels. T-suppresor cels inhibit or suppres imune responses. Kiler T cels atack cancerous cels and cels infected with viruses. In order for these cels to initiate rejection they must first receive specific celular signaling. Na?ve T cels require two independent signals from the same antigen presenting cels (APC) (macrophages, B cels and dendritic cels) for activation. The 5 MHC binding to the na?ve T cel provides the first antigen-dependent signal. The MHC receptor presents antigen to the T cel receptor (TCR) and asociated CD3 molecule. The second antigen-independent signal is the result of B7 (CD80/86) molecules on the APC binding to CD28 molecules on the T cel. This binding is required for clonal activation. Once activated, a T cel can expres CD40L which binds to CD40, a glycoprotein expresed on APCs and thymic epithelial cels. The binding of CD40-CD40L further sustains the costimulatory response. 14 Dendritic cels of the donor and recipient are key players in alograft rejection. Dendritic cels are the most efective APCs because of their expresion of MHC I, MHC I, and costimulatory molecules. Dendritic cels present antigen to na?ve T cels and stimulate a T-cel response to these antigens. Consequently, their removal promotes alograft survival. Recipient dendritic cels generate an imune response by presenting donor aloantigen in the cleft of ?self? MHC to T cels in the lymph nodes. Once these dendritic cels bind to recipient T cels with the corresponding receptor, the T cel is then primed, initiating clonal expansion. This provides a large population of antigen specific T cels that are able to bind and kil alograft cels. Donor dendritic cels (pasenger leukocytes in alograft) can also stimulate recipient?s aloreactive T cels by migrating to a local lymph node and presenting donor antigen. A close MHC match, i.e. similar MHC structure betwen donor and recipient, should promote a les vigorous imune response against the alograft. An antibody-mediated imune response is also a component of alograft rejection. A hyperacute rejection episode can ocur if preformed antibodies to the donor are present in the recipient at the time of transplantation. Prexisting 6 aloantibodies within the recipient against donor blood group antigens and donor MHC antigens wil cause alograft rejection within minutes of transplantation. The vascular endothelium of the graft is the main target of this humoral atack. Complement and coagulation cascades are rapidly initiated. This is visible imediately as a blood engorged organ with extensive hemorrhage. Soon the alograft is deoxygenated and rejected. With current blood typing and MHC matching hyperacute rejection in human transplant patients is a rare event whereas chronic rejection is the main factor typicaly precipitating renal alograft dysfunction and eventual loss. At a histopathologic level, the halmarks of renal alograft rejection in humans are tubulitis and vasculitis. Most rejection proceses begin subclinicaly as interstitial mononuclear cel infiltration, edema and potentialy interstitial hemorrhage. Clinical rejection becomes apparent as renal tubular and vascular changes occur. Renal tubule invasion by lymphocytes and narowing of the interlobar, interarcuate, and interlobular arteries by intimal thickening occurs. CD4-positive, CD8-positive and IL-2 staining al reveal an increased response. Later an antibody-mediated endothelial response occurs causing necrotizing vasculitis and eventual thrombosis. Eventualy a majority of renal transplant patients wil suffer from the consequences of chronic rejection which are primarily vascular injury, tubular atrophy, interstitial fibrosis and los of renal parenchyma. 15;16 Widespread aceptance of human renal transplantation created a need for a standardized system of renal alograft biopsy evaluation. This ideology evolved into the ?Banff 97? scheme which has been acepted as a universal system of renal 7 alograft analysis among human pathologists. This system of clasification has helped the renal transplant community ?to guide therapy and establish an objective end point for clinical trials.? 17 It also provides a system of communication betwen clinician, pathologists and researchers. The ?Banff 97? scheme is based upon the second, third, and fourth Banff conferences, and the Syntex/Roche mycophenolate mofetil trials 17 . It serves as the universal language for interpreting rejection episodes as wel as predicting a prognosis. It aims to provide ?international uniformity? in the evaluation of renal alograft biopsies 5 ?Banff 97? clasifies renal alograft rejection into thre categories (I, I, II) based on the specific location of inflamation. Type I rejection is tubulointerstitial inflamation without arteritis. Type I rejection is vascular rejection with intimal arteritis. Type II rejection is severe rejection with transmural arterial changes. Tubulitis and intimal arteritis in more than one focal region are considered halmarks for alograft rejection. Interstitial inflamation alone is not a signal of rejection. Clasic infiltrates are T lymphocytes, monocytes, and macrophages. The presence of other inflamatory cel types such as eosinophils, neutrophils, or plasma cels should be noted. Vasculitis is defined as lymphocyte infiltration beneath the vascular endothelium and ranges from arteritis characterized by focal inflamation in the vascular media to transmural fibrinoid necrosis of the vesel wal. The total number of arteries in the renal alograft biopsy specimen and the presence of hemorrhage or infarction should be noted. Glomerulitis is defined by mononuclear infiltration and endothelial cel enlargement, and is clasified as segmental or global based on the 8 extent of these changes. Under current Banff 97 guidelines, the significance of glomerulitis is unclear. Acurate renal alograft grading using the Banff 97 scheme requires a proper biopsy specimen, specificaly, the specimen must have adequate cortical tisue with a minimum of seven glomeruli and at least one artery. Seven slides are prepared with the sections being 3 to 4 microns in thicknes. Thre slides are stained with hematoxylin and eosin (H&E), thre with periodic acid-Schif (PAS) or silver stain, and one with trichrome stain. Routine H & E staining iluminates general alograft architecture such as alterations in tubular, interstitial, glomerular, or vascular structures. Lymphoid aggregates are visualized as wel as the presence of hemorrhage, edema, and fibrosis. The PAS and silver stains are useful for identification of glomerulitis, tubulitis, and any destruction of tubular basement membranes. Furthermore, chronic features such as arteriolar hyaline, increased mesangial matrix, double contours in glomerular capilaries, and thickened tubular basement membranes are enhanced by PAS and silver stains. The silver stain highlights basement membranes of tubules and mesangial matrix as wel as the basal lamina of smooth myocytes and elastic fibers. Trichrome stains for collagen, iluminating major vesels and the renal pelvis as wel as emphasizing the location and the extent of alograft fibrosis. 17 The ?Banff 97? clasification system encourages routine alograft biopsies to help identify episodes of subclinical rejection and enable early intervention. It is wel established that hematological analysis of urea nitrogen and creatinine often underestimates renal alograft disease, requiring biopsies to document the pathology of 9 renal alograft. 2 . Based on biopsies, subacute rejection episodes occur in about 30% of human patients within the first thre months of transplantation. 4 Subclinical rejection at 6 months has been proven to be an independent predictor of long-term graft dysfunction. 18 Additionaly, treatment of subclinical rejections with prednisone at 1 and 3 months resulted in a decreased rate of early and late graft rejection, les tubulointerstitial disease, and lower creatinine values. 4 There is not an established system of renal alograft analysis for veterinary patients. A few veterinary studies have tried to adapt the ?Banff 97? scheme with moderate succes. 1;12;19;20 A recent feline study looked at renal biopsies and necropsies of feline alograft recipients on a CSA and prednisone protocol. The average mean survival time of renal alografts was 270 days (1 day to 9 years). Out of 77 cats, 69% of recipients showed signs of chronic alograft nephropathy and 51% had evidence of CSA toxicity. Eighty-percent of biopsies revealed histopathologic evidence of rejection, whereas clinical rejection was evident in only 50% of these cats 21 . Vasculitis, tubulitis, and lymphocytic glomerulitis, al major criteria for rejection in humans, were not sen in the cats. Overal, the alograft rejection in cats appears diferent from humans and potentialy involves a humoral component 21 . Most veterinary acounts are descriptions of necropsy specimens from patients with clinical evidence of rejection or at the timed end of a study. The lack of intermediate stage biopsies specimens; e.g. scheduled needle biopsies, makes the predictive use of Banff 97 dificult to ases. Serial biopsies in dogs and cats would be useful to document the progresion of renal pathology before end-stage renal disease occurs. 10 Imunosupresion and Transplantation Historicaly, chronic medical imunosuppresion has been the primary modality used to suppres the recipient?s imune response in order to avoid alograft rejection. Imunosuppresive drugs such as prednisone, azathioprine, calcineurin inhibitors, mycophenolate mofetil (MF), and others, have been used alone or in combination to prevent renal alograft rejection in people and dogs. Prednisone was the first imunosuppresive agent to be used in solid organ transplantation. Prednisone stabilizes the cel membranes of endothelial cels and inhibits the chemotaxis of neutrophils, monocytes, and lymphocytes. Monocytes and lymphocytes are sequestered to the lymphatics and bone marow. T cel activation and macrophage function are impaired. Prednisone inhibits collagenase, elastase, and tisue plasminogen activator. The release of arachidonic acid by cel membrane phospholipids is also inhibited resulting in decreased production of prostaglandins, thromboxanes, and leukotrienes. Although cheap and efective, prednisone alone does not prevent rejection. Furthermore, the long-term complications of prednisone such as gastrointestinal bleding, iatrogenic hyperadrenocorticism, diabetes melitus, and increased susceptibility to infection are al significant side efects. 14;2 Azathioprine replaced prednisone and remained a mainstay of imunosuppresive therapy for approximately 30 years. Azathioprine is a purine antagonist that interferes with DNA synthesis, kiling actively dividing lymphocytes. It is metabolized to 6-thioinosinic acid which blocks de novo synthesis of adenosine monophosphate and guanosine monophosphate. However, azathioprine afects al 11 rapidly dividing cels in the recipient, resulting in hepatotoxic efects, bone marow suppresion, anemia, and damage to intestinal epithelium. 23 2 The development of newer, more tolerable imunosuppresive agents such as cyclosporine (CSA) and tacrolimus signaled a new age for transplantation medicine. CSA, a cyclic decapaptide derived from the soil fungus, Tolypocladium inflatum, was introduced in 1983. It inhibits T-lymphocyte proliferation by inhibiting the phosphatase activity of the calcium activating enzyme calcineurin and prevents select cytokine synthesis (interleukins-2,-3,-4, GM-CSF, TNF-?) 2;23 . The suppresion of interleukin-2 further diminishes T cel proliferation. 23 Cyclosporine is a mainstay in transplantation medicine; however, nephrotoxicity due to CSA has ben documented in people. A histologic description of isometric tubular vacuolation, arteriolar myocyte vacuolation, arteriolar endothelial cel necrosis, arteriolar subendothelial hyaline deposits, arterial fibrointimal proliferation, and glomerular microthrombi are consistent with CSA toxicity in people 16;17 . Chronic nephrotoxicity is characterized by interstitial fibrosis, tubular atrophy, and non-obliterative arteriolopathy. 16 The widespread use of calcineurin-inhibitors has decreased early acute rejection episodes but paradoxicaly has induced nephrotoxicity and eventual graft loss. Several authors claim calcineurin-inhibitor-induced nephrotoxicity to be almost universal at ten years post-renal transplantation in people. 24;25 Tacrolimus, a T-lymphocyte inhibitor, was initialy approved for liver transplants and later for kidney transplant patients in 1997. It blocks T-lymphocyte activation genes using a mechanism similar to cyclosporine but is 10 to 100 times 12 more potent; however tacrolimus has more side efects such as severe vasculitis and intussusception. 2 In 1995, mycophenolate mofetil (MF) was FDA approved as an imunosuppresive agent. Similar to azathioprine, MF inhibits de novo purine biosynthesis, afecting rapidly dividing cels. 14 Side efects of MF include vomiting, diarhea, gastrointestinal hemorrhage and petechiation, and les frequently pancreatitis (CelCept ? [Mycophenolate Mofetil] Labeling, Roche) . 8;26 The irony of modern imunosuppresive agents is that short-term graft survival has experienced huge strides; however, long-term survival remains largely unimproved. Imunosuppresive agents generaly afect the entire imune system impairing the host versus graft response (HVG) as wel as lowering the recipient?s response to opportunistic infections. The development of life-threatening infections, an increased risk of developing tumors, drug-related toxicities, and eventual chronic rejection of the kidney have al limited the capabilities of long-term imunosuppresive medications. Imunologic Tolerance Idealy, an imunotherapeutic agent would be used temporarily to alow the imune system to recognize an alograft as ?self? until the recipient?s body could maintain this status without exogenous treatment. The imune system would not atack a graft while maintaining al other normal imune functions. This selective alteration of the imune system is caled imune tolerance. Donor specific alograft tolerance is defined as the indefinite survival of a normaly functioning alograft in the 13 absence of maintenance imunosuppresion. 27 More precisely, 1) the absence of donor-specific aloantibodies in the recipient, 2) lack of destructive lymphocytic infiltration in the alograft biopsies, and 3) verification of donor-specific unresponsivenes and third party responsivenes using functional asays define true tolerance. 28 Discovery of Imune Tolerance T cels play a pivotal role in the succes or failure of an alograft. In a ?nude mouse,? which is born without a thymus, and thus without T-cel mediated imunity, skin grafts from various species are succesful without any requirement for medical imunosuppresion. In one particular experiment, a chicken skin xenograft was succesfully performed on a mouse, and the mouse even grew feathers. 29 This emphasizes the important role of T-cel imunity in transplantation rejection; however, a person or dog without T cels wil not survive. The key to alograft and patient survival is the lack of a detrimental imune response against the alograft, not the total absence of imune reactivity. 30 This balance is the esence of tolerance. The goal of modern imune tolerance is to maintain a functional graft without ongoing therapeutic imune suppresion. 30 The elimination or inactivation of prexisting mature donor reactive T cels and the lifelong elimination or inactivation of newly developing donor-reactive T-cels are both required for tolerance. 31 The window of opportunity for tolerance induction appears to be during a period of imunodeficiency either naturaly or artificialy induced. This occurs in young patients whose imune systems are stil developing or patients who have undergone full body imune compromise i.e. total body iradiation. Peter Medawar found that 14 imature mice injected with alogeneic splen cels could later acept skin grafts from the same donor. 29 This example of tolerance showed the potential manipulation of the developing imune system for aceptance of foreign alografts. Because patients in need of transplantation generaly have mature imune systems, temporary imunologic ablation or incompetence is required to induce tolerance in an adult. Induction of Imune Tolerance It is important to note that a majority of studies that document sucesful imune tolerance induction use rodent models. In comparison to laboratory rodents, large animals and people have a much wider exposure to environmental antigens and thus maintain a more highly developed T-cel response. T-cels primed by an antigenic stimulus are more dificult to make tolerant. 31 Additionaly, large animals have MHC I molecules expresed on vascular endothelial cels, whereas mice do not. 32 This increases their susceptibility to host surveilance mechanisms. Deletion, anergy, regulation/imunosuppresion, ignorance, and hematopoietic chimerism are al potential mechanisms of imune tolerance induction. The induction of tolerance using clonal deletion can be acomplished at the central and peripheral levels. Central tolerance to ?self? is established at the thymic level. After production in the bone marow, T lymphocytes travel to the thymus for evaluation. T cels whose TCRs bind too tightly or do not bind at al to ?self? MHC molecules are eliminated via apoptosis to prevent future populations of autoreactive and dysfunctional T cels. Researchers have atempted to harnes this proces of clonal deletion to eliminate aloreactive T cels. Currently, the most robust form of tolerance is thought to be the 15 result of hematopoietic chimerism, as donor cels are continualy migrating to the thymus, promoting clonal deletion. Chimerism exists when cels from another organism i.e. the donor, exist in the recipient. A hematopoietic chimera has hematopoietic cels from a donor coexisting within the recipient?s hematopoietic population. One of the most plausible theories of tolerance requires there to be mixed chimerism, which is the existence of donor cels within a host, at levels that are greater than 1% but les than 100% of the recipient?s total cel population. Mixed chimerism is a subset of macrochimerism which has been wel established in the murine model, but is more dificult to atain in a large animal or human model. Several points argue in favor of mixed chimerism. Models for tolerance that use mixed chimerism routinely met strict experimental standards for tolerance such as the aceptance of a donor skin graft and the maintenance of an imune response to third party antigenic stimulation. Additionaly, mixed chimerism can be a quantitative measure for the presence of donor specific tolerance. 31 Hematopoietic stem cel transplantation (from harvested donor bone marow) provides the recipient?s thymus with a continual influx of donor lymphocytes to promote life- long negative selection of donor reactive thymocytes. 28 Idealy, the T cels that expres receptors (TCR) that bind alogeneic cels would be negatively selected, thus permiting long-term survival of the graft. 3 In large animal and human models, macrochimerism requires debilitation of the T cel repertoire in order to eliminate prexisting mature donor-reactive T-cels. 31 This can be acomplished within the thymus, using local or total body iradiation, or through peripheral mechanisms. Initialy full body lethal iradiation was promoted as 16 the best route to imunologic ablation. It was thought that the entirety of the recipient?s bone marow had to be ablated to create a physical space that the new progenitor cels would fil. In reality, this total ablation is only required for hematologic malignancies and does not appear necesary for alograft recipients. Les aggresive sublethal TBI resets the imune system to a lower level of surveilance as sen in imature animals or T-cel depleted adults without necesitating the total elimination of the imune response. This myelotoxic cytoreduction alows for the reception of a donor-derived bone marow transplant from the donor. 29 Local thymic and nodal iradiation have been shown to serve a similar purpose of weakening the recipient?s imune response. Induction of tolerance in peripheral tisues can be established using repetitive T cel depletion or a costimulatory blockade. Peripheral tolerance is likely most important to ?jump start? the induction phase of imunologic tolerance and becomes les important during the maintenance of tolerance. 28 Once T cel populations leave the thymus and undergo clonal expansion (proliferation of a specific and identical T cel line), they can be inactivated or eliminated in the peripheral vasculature. Using monoclonal antibodies, CD4-positive and CD8-positive T cels can be systemicaly depleted. In rodent models, the combination of monoclonal antibodies against CD8- positive T cels, 3Gy TBI, MHC disparate BMT, and one dose of cyclophosphamide led to multilineage engraftment in a majority of patients. 34 In dogs, when used without additional medical therapy, these antibodies set up an imunosuppresed environment suitable for tolerance induction; however, prolonged use (>13 days) of the antibodies led to recipient antiglobulin production and an eventual anaphylactic 17 response. When used in conjunction with azathioprine and CSA, the antibody therapy could be prolonged up to 28 days. 35 Anti-CD3 imunotoxin has been documented to induce tolerance in rhesus monkeys by depleting sesile and circulating T cels. 36;37 As mentioned earlier, costimulation via the binding of T cel surface molecules CD154 to CD40 and CD28 to B7 molecules is required for T cel activation and thus T cel directed alograft cel destruction. If costimulation is absent or blocked with CD154 antibody or CTLA4-Ig, the result is T cel anergy or inactivation, leaving the donor cels viable. The use of CD154 specific monoclonal antibody alowed for the induction and maintenance of tolerance in an outbred group of MHC-mismatched rhesus monkeys for greater then 10 months. 38 Rabbit anti-dog antilymphocyte serum in conjunction with BMT and limited CSA therapy led to long-term canine renal alograft survival (>180 days) with donor specific unresponsivenes. 39 Interestingly, if very high doses of donor bone marow (12-fold increase) are administered along with antiCD154 (+/- CTLA4Ig), then sublethal iradiation, cytotoxic drugs, and monoclonal antibodies were not required in some models. 40 Bone marow transplantation has been asociated with marginal, nonspecific imune depresion documented by the down- regulation of mixed lymphocyte reactions and cytotoxicity asays. This imune depresion was directly linked to CD34+ stem cels as wel as early progeny lymphoid cels (CD38+, CD2+, CD5+ and CD1+) and early myeloid cels (CD33+). 41 Another study was able to reduce whole body radiation to low levels (3 Gy) or eliminate iradiation entirely if very large quantities of donor bone marow were utilized. 27 Because such large quantities of bone marow are not practical in a clinical 18 seting, a course of conventional imunosuppresion, co-stimulatory blockade, low dose iradiation and standard dose BMT are often advocated with similar succes. 31 Veterinary Studies Many diferent protocols have been atempted using various pharmacologic combinations in canine models. One 100-day study found that 50% of dogs on microemulsified CSA alone (achieving 500 ng/mL) experienced alograft rejection following mismatched kidney transplantation. 1 Another study administered microemulsified CSA, azathioprine and prednisone combination to four dogs after mismatched renal transplantation with 50% fulfiling the 100 day study without clinical signs of rejection. Two dogs were euthanized, one due to an intussusception (day 8) and one due to an upper respiratory tract infection (day 64). Thre dogs had evidence of hepatotoxicity, which resolved when the azathioprine dose was decreased. Of the two survivors, one had no evidence of rejection and the other displayed acute rejection (moderate intimal arteritis). 12 In another veterinary study, a similar protocol of RADTS, prednisone, CSA, and azathioprine was followed and additionaly, thre of seven dogs also received donor bone marow (DBM). Imunosuppresion was gradualy tapered as prednisone, CSA, and azathioprine were withdrawn succesively at 200, 450 and 680 days. Four dogs survived to the end of the 2 year study but the other thre (no DBM) rejected with total drug withdrawal. One of the surviving dogs that received bone marow, survived off al drugs. 42 In another study, fiften dogs with ESRD received a kidney transplant from a dog erythrocyte antigen (DEA) match with succesful cross-matching. Dogs were 19 treated preoperatively with rabbit anti-dog antithymocyte serum (RADTS) injections, prednisone, and azathioprine and postoperatively with CSA. Rabbit anti-dog antithymocyte serum was given daily for 6 days to achieve les than 10% of normal lymphocyte population but greater than 70,000 platelets. Blood transfusions were started preoperatively and completed intraoperatively, as al dogs were anemic. Prednisone began at 1.0 mg/kg/day and was tapered until 0.25 mg/kg/2 days was achieved. Azathioprine was commenced at 1.0 mg/kg/day and gradualy by year two was tapered to an every other day dosing schedule. CSA levels were maintained at 500-600ng/ml initialy, reduced to 400ng/ml at 6 months and to 300ng/ml during the second year. The side efects of each of the drugs (CSA, azathioprine and prednisone) were monitored and the drug causing the most serious side efects was eliminated first. Using this protocol, a mean survival time of 8 months was achieved. Thre dogs died of surgical technical failures and four dogs died from rejection episodes within the first year. Thre dogs were euthanized after 12 months due to non-responsive infection, one was euthanized due to neoplasia, one due to recurrent Lyme?s disease, and one due to a fibrocartilagenous embolism. At the end of this study, two dogs had survived longer than 36 months on reduced levels of CSA, azathioprine (EOD) and prednisone (EOD). 10 The purpose of this study was to evaluate the progresion of alograft histopathology in relation to renal biochemical parameters (BUN and Cr), and clinical status of DLA-mismatched dogs undergoing renal transplantation. 20 II. STATEMENT OF RESEARCH OBJECTIVES a. Perform and evaluate renal alograft transplantation in mismatched donor-recipient mongrel pairs using a protocol that includes total body iradiation, bone marow transplantation, and short-term imunosuppresion. b. Analyze serial biopsies for evidence of renal alograft pathology using hematoxylin and eosin and imunohistochemical stains. 21 IV. MATERIAL AND METHODS Dogs Ten mixed bred dogs weighing 8.0 to 27.6 kg were included in the study. The dogs (7 intact females and 3 intact males) were considered normal based on physical examination, prolonged observation (> 6 months), complete blood counts (CBC), serum biochemistry profiles, and urinalyses. Two dogs received kidneys from DLA- mismatched siblings. DNA typing was used to ensure related dogs were mismatched at the clas I and I major histocompatibility complex (MHC) using microsatelite analysis through polymerase chain reaction (PCR) techniques acording to published protocols. 10 The remaining 8 dogs received a renal alograft from a mismatched, unrelated donor. This project was approved by the Auburn University Institutional Animal Care and Use Commite. Dogs were housed in USDA/ALAC acredited facilities. Bone Marrow Transplantation Seven of 10 dogs received bone marow transplantations. Bone marow was harvested from humeri and femora of the donor dogs while under general anesthesia and imediately prior to renal transplantation surgery. Bone marow was collected 22 into 12 ml heparinized syringes. Bone marow mononuclear cels were purified by Ficoll-Hypaque (1.077) density gradient centrifugation (150 x g) for 30 minutes. The low-density cels were washed with Hanks balanced salt solution and infused into the recipient?s cephalic vein after the transplantation surgery but within 8 hours of total body iradiation. Transplantation Surgery Ten dogs underwent surgery for renal transplantation. The anesthesia protocol consisted of premedication with butorphanol (0.4mg/kg) and midazolam (0.2mg/kg), followed by anesthesia induction with thiopental, propofol, or isoflurane mask induction. General anesthesia was maintained with isoflurane (1-4%), oxygen, and intermitent hydromorphone (Hydromorphone HCL, Baxter, Healthcare Corp., Derfield, IL) boluses. Epidural anesthesia, using morphine (Astramorph PF ? , Astra, Westborough, MA) (0.1 mg/kg) and bupivicaine (Bupivicaine, Abbott Labs, N. Chicago, IL) (1.0 mg/kg) was performed prior to surgery. The dogs were maintained on balanced crystaloid fluid therapy (11ml/kg/hr) and dopamine (3mcg/kg/hr) while under general anesthesia. Dogs were prepared for aseptic surgery and given intraoperative cefazolin (22 mg/kg IV q2h). A ventral midline incision was made and the smal intestines were enteroplicated in gentle loops using simple interupted sutures of 4/0 polydioxanone (PDS I, Ethicon, Inc., Sommervile, NJ). The native left kidney was removed from the recipient dog. Approximately 4 cm of aorta and caudal vena cava were cleared just cranial to the aorto-ilial bifurcation in the recipient dog. Next, the donor kidney 23 was harvested and flushed with room temperature heparinized saline. The renal artery was dilated and exces adventitia was removed. The recipient dog received heparin (75 U/kg IV) prior to placement of the aortic clamp, in preparation of the donor kidney anastomosis. The prepared recipient aorta was clamped and the donor kidney positioned on the left side. A 4 m aortic punch (Alegiance Healthcare Corp., McGaw Park, IL) was used to create the arterial stoma. The aortic anastomosis was completed with 6/0 polypropylene (Prolene, Ethicon, Inc., Sommervile, NJ) using two simple continuous suture lines. The caudal vena cava was occluded with vascular clamps. The recipient caudal vena cava was incised to match the diameter of the donor kidney renal vein. Care was taken to avoid twisting the vein of the alograft and the venous anastomosis was completed using simple interupted suture of 6/0 polypropylene. Once the artery and vein were anastamosed, the vascular clamps were removed, and alograft reperfusion was established. A gelatin sponge (Gel-foam, Pharmacia-Upjohn, Kalamazoo, MI) was placed around the anastamotic site to aid with hemostasis. Once hemostasis was satisfactory, the urinary bladder was opened and the alograft ureter was inserted through the wal of the bladder using blunt disection. The ureter was spatulated and sutured with simple interupted suture of 6/0 polypropylene. One to two additional sutures were placed on the serosal surface betwen the ureter and bladder. The renal alograft was stabilized with the mesovarium, mesotesticular or mesocolon. The native right kidney was then removed from the recipient dog, leaving the heterotopic alograft as the only kidney. Post-operative analgesia was provided by the epidural anesthesia, intravenous administration of hydromorphone (0.10 mg/kg IV as needed but no les than q4-6hrs) 24 and/or butorphanol (0.1mg/kg/hr CRI or 0.2-0.4 mg/kg IV PRN but not les than q4hrs). Acepromazine (0.02-0.05mg/kg) was used for supplemental sedation as needed. Post-operative analgesic administration and fluid therapy (2-4 ml/kg/hour of a crystaloid dextrose solution) were typicaly continued for 4-7 days. Additional supportive care was provided as needed. Surgical biopsies The unused, nephrectomized kidney from each recipient and a needle biopsy of the remaining donor kidney were examined microscopicaly to determine if sub- clinical pathology was present at the time of the transplant. These specimens were designated as biopsy one. Alograft biopsies were collected at thre additional time periods: a) while recipients were on full imunosuppresive therapy (biopsy 2: betwen 44 and 90 days), b) after maximal drug reduction (biopsy 3: 228-580 days) and c) a final biopsy (biopsy 4: obtained at necropsy [dogs 4, 5, 6, 7, and 9] and as an open surgical biopsy [dog 1]). Alograft biopsies were obtained with a 14 to 18-gauge Tru-cut biopsy needle (Achieve, Alegiance Healthcare Corp., McGaw Park, IL) via a mini-laparatomy. A larger wedge biopsy of the renal alograft was obtained from the initial biopsy (biopsy 1) and from necropsy specimens. Imunosupresive Regimen and Monitoring Imediately before surgery, recipient dogs received TBI using a linear acelerator to deliver a nonmyeloablative total dose of 200 cGy at 28.5 Gy/min. Cyclosporine [CSA] (Sandimune, Novartis, E.Hannover, NJ) was initiated the day 25 before surgery (15 mg/kg PO) and continued BID. Mycophenolate mofetil [MF](Celcept, Roche Laboratories, Nutley, NJ) therapy was initiated the day of transplantation. Six dogs were initiated at a full dose of MF (10 mg/kg BID) and 4 dogs were started at a half dose (5 mg/kg PO BID) with al dogs receiving the full dose (10mg/kg BID) by wek 2. CSA and MF were given at these doses until the first alograft biopsy was obtained. Al dogs were on antibiotics to minimize infections during the imunosuppresive period. Imunosuppresive therapy was slowly tapered beginning 24 to 48 hours after biopsy 2. Cyclosporine was tapered first with reductions of 25-30% of the full dose (15 mg/kg). Once the cyclosporine dose was reduced by greater than 50% of the starting dose, oral prednisone was added to the imunosuppresive protocol (1 mg/kg/day). If the BUN and serum creatinine concentrations (Cr) were within normal limits 30 days after starting prednisone, then MF was reduced by 25-30%. Every 30 days the dose of CSA or MF was again reduced by 25-30% until prednisone was the only medication being given. After 30 days of prednisone alone, al imunosuppresive drugs were discontinued. If Cr rose to greater than 3.0 mg/dl, in the absence of other causative factors, imunosuppresive therapy was reinitiated. Depending on the severity of the azotemia, concurrent biopsy findings and clinical signs, CSA and MF dosages were restarted at 50 to 100% of the full dosage. Al drug dose calculations were based on current weights. Complete blood count and serum chemistry profiles were monitored every 24 to 48 hours for the first 7 to 10 days. CBC, BUN, and Cr were then evaluated twice wekly until neutrophil and platelet counts normalized, and wekly thereafter. 26 Indirect blood presure (Doppler) was monitored wekly beginning at the time of drug reduction. Serum Cyclosporine Concentrations Trough CSA levels were measured from stored serum samples taken prior to each surgical biopsy and submited to the Auburn University Clinical Pharmacology Laboratory. The CSA asay was performed using a monoclonal fluorescence polarization imunoasay. Clinical Status Monitoring Each dog was monitored twice daily for atitude, appetite, and GI signs. Weight was monitored wekly. Eating the designated amount of food, bright atitude, lack of vomiting/ diarhea and lack of significant weight loss (>3kg) earned a score of 4. Each abnormality would subtract one point from a total score of 4. For example, bright atitude (+1), vomiting (-1), eating meals (+1) and constant weight (+1) would equal a score of 3. If there was a discrepancy betwen the morning and evening clinical status, the least favorable score was recorded. The clinical score recorded (table 1) was based on the average of the daily scores the wek preceding surgical biopsy. Histopathologic evaluation of biopsy specimens 27 Al renal alograft sections were examined by the author (KDB) and a board certified veterinary pathologist (SDL). Nedle biopsy specimens were imersion fixed in 10% neutral bufered formalin for a minimum of 2 hours. Fixed specimens were routinely procesed to parafin and sectioned at 3-4?m. Duplicate sections were stained with hematoxylin and eosin (H&E) and periodic acid-Schif?s/hematoxylin stain (PASH). Additional duplicate sections were reacted with antibodies to CD3 (catalog #: N1580, Dako Corp., Carpinteria, CA) and CD79a (catalog #: M7051, Dako Corp., Carpinteria, CA), using a standardized ABC imunohistochemical procedure. Histopathology slides were evaluated for changes of the interstitium, tubules, glomeruli, and vasculature. Renal cortical inflamation was characterized based on the predominant cel type (lymphocytic, lymphoplasmacytic, or neutrophilic), location (interstitial, perivascular or peritubular), and overal severity [1+ (minimal): <10% of parenchyma; 2+ (slight): 10-25% of parenchyma; 3+ (moderate): 25-50% of parenchyma; 4+ (marked): >50% of parenchyma). Tubulitis, interstitial fibrosis, and/or vascular lesions were identified and subjectively characterized as minimal, slight, moderate, marked, or severe. Imunophenotyping of Infiltrating Lymphocytes Histopathologic sections reacted with either CD3 antibodies or CD79a antibodies were subjected to morphometric analysis. For each needle biopsy, the number of CD3-positive and CD79a-positive cels were counted and recorded. The area of each section was measured using image analysis software (Image J, National Institute of Health, Bethesda, MD). For wedge biopsies, a systematic sampling 28 technique using a random start point was employed. Beginning with a randomly selected field (cortex), CD3-positive and CD79a-positive lymphocytes were counted in every other 20X field, for a total of five fields. Counts were summed and the results expresed as the number of each lymphocyte phenotype per unit area (m 2 ). 29 V. RESULTS Clinical Results Al ten dogs had a pre-transplantation clinical score of four. Seven of ten dogs survived more than 200 days and were available for long-term follow-up. Two dogs had a series of thre biopsies and five dogs had four biopsies obtained. Thre dogs were euthanized or died betwen 5 and 20 days after renal transplantation (dogs 5, 8, and 10). Dog 5 was on a full imunosuppresive dose of CSA (15 mg/kg PO BID) and MF (10mg/kg PO BID). After the transplantation surgery, dog 5 had azotemia, persistent mucoid stool, periodic melena and vomiting. This dog did not undergo donor BMT. After 4 days of imunosuppresive therapy, MF was discontinued and a blood transfusion was given for anemia. On day 5, dog 5 had hemoptysis and dyspnea. Laboratory findings showed azotemia (BUN: 110, Cr: 7.4), severe thrombocytopenia (5,000 cels), and anemia (Hct: 21%). Dog 5 was euthanized and a gross necropsy revealed 500-600 ml of serosanguinous fluid abdominal fluid, a hemorrhagic and edematous pancreas, and difusely, petechiated, compromised bowel. The renal alograft appeared grossly normal. Histologicaly, the alograft showed T- lymphocytic multifocal perivascular, interstitial nephritis which was most severe at the corticomedullary junction. Approximately half of the lymphoid-like cels did not react 30 with CD3 or CD79a antibodies. In the corticomedullary region, tubule lumens and glomeruli are filed with a protein-like fluid. Several arteries and veins appear occluded with fibrin and there is local interstitial hemorrhage. Difuse gastrointestinal inflamation and hemorrhagic pancreatitis were noted. Histopathologic findings could not diferentiate betwen alograft rejection and progresive renal inflamation secondary to sepsis. Dog 8 was on full imunosuppresive therapy of CSA (15 mg/kg PO BID) and MF (10mg/kg PO BID) since transplantation. Dog 8 displayed vomiting, diarhea, melena and lethargy. Four days after transplantation, dyspnea was noted and thoracic films documented a generalized, severe, pulmonary interstitial patern with moderate pleural efusion and cardiomegaly. Furosemide (1mg/kg) was administered and MF was discontinued. On day 5, a whole blood transfusion was administered for anemia (16%) and 500ml of fluid evacuated from the chest via thoracocentesis. On day 7, dog 8 was euthanized due thrombocytopenia (15,000 platelets with gingival petechiation), persistent azotemia (BUN: 49-126; Cr: 3.2-4.5), protein:creatinine ratio of 6.5, anemia (18%) and lethargy, intermitent neurologic deficits and inability to stand. Gross necropsy revealed a hemorrhagic pancreas, grey to black petechiated intestinal tract, and fluid in the abdomen and chest. The renal alograft appeared normal. Histopathology was consistent with an acute hypoxic event. Renal histopathologic analysis revealed multiple, prominent intravascular fibrin formation and local coagulative necrosis. Scant neutrophilic and T-lymphocytic interstitial nephritis, tubular atrophy and regions of tubule dilation with proteinaceous material were noted. Arteries and veins were congested. There was minimal inflamation. 31 Histopathologic lesions were not considered to be consistent with alograft rejection. Based on histopathologic findings of alograft thrombosis and gastrointestinal disease, gastrointestinal toxicity was suspected as the cause of death. Dog 10 began therapy on subtherapeutic levels of MF (5mg/kg PO BID) and therapeutic levels of CSA. After one wek, MF was increased to a full dose (10mg/kg PO BID). Dog 10 had persistent vomiting and diarhea and developed an infected mamary gland. Initial prophylactic trimethoprim sulfa (Tribrisen?, Schering-Plough, Union, NJ) was changed to enrofloxacin (Baytril?; Bayer Healthcare Corp, Shawnee Mision, KS). The dog was severely thrombocytopenic (5,000 platelets) and anemic (12.7%). A whole blood transfusion was administered. Eighten days after transplantation, the dog became hyperthermic (105?F) and anuric and later died. Same day blood work showed only a mildly elevated BUN (39 mg/dl) and Cr (1.4mg/dl). Initial gross necropsy showed mild hemorrhagic changes of the serosal surface of the kidney with blood clots in the bladder. Histopathologic lesions were multifocal lymphoid aggregates at the corticomedullary junction, mild hemorrhage, acutely degenerative tubules, and distention of Bowman?s capsule and venules with protein. The lymphoid aggregates had a smal CD3-positive and CD79a- positive population but the majority did not stain. The death of dog 10 was atributed to bacterial sepsis resulting from the infected mamary gland. 32 Biopsy One (normal specimens) At the time of transplantation a kidney biopsy, either wedge or needle biopsy, was taken from al donor and recipient dogs. These biopsies were analyzed for any signs of pathology. A wedge biopsy was analyzed in 10 dogs and a needle biopsy in 4 dogs. Al slides were stained with H&E, and reacted with CD3, and CD79a antibodies. No abnormalities were noted on any slide with the exception of nine CD3- positive cels sen within the interstitium of one biopsy (dog 3)(Table 2A). Biopsy Two (ful imunosupresion) At the time of the second biopsy, the BUN for the seven dogs ranged from 5.8- 20.9 mg/dl and Cr ranged from 1.0-2.7 mg/dl. Six of seven biopsy samples had some degre of lymphocytic, plasmacytic interstitial inflamation (Figure 1b)[minimal (2 dogs) and slight (4 dogs)]. While on full imunosuppresion, no renal alograft biopsy sections had more than slight (25%) interstitial inflamation. Vacuolar degeneration of the tubular epithelium was observed in 6 dogs (Figure 2). Slight to moderate tubular atrophy (4 dogs) was also noted. One dog had focal intimal arteritis (Table 2B). Minimal tubulitis was sen in 2 alograft biopsies (dogs 2 and 3). Dog 3, which had slight tubulitis at the time of transplantation, had the most tubule invasion (35 cels). Monocytic arteritis was sen in one alograft biopsy (dog 6). Inflamatory infiltrates were composed primarily of mononuclear cels with CD3-positive lymphocytes being the major cel type. The median CD3-positive cels/m 2 was 157 (range: 63-428 cels/m 2 ) and the median CD79a ?positive cels/m 2 was 40.1 (range: 3-132 cels/m 2 ). 33 Biopsy Thre (modified imunosupresion) Timing of biopsy thre ranged from 228 days to 580 days after surgery. Renal alograft biopsies were obtained within 48 hours of imunosuppresive therapy reduction or elimination, or at necropsy (dog 3). The BUN for the seven dogs ranged from 6.1 mg/dl to 43.4 mg/dl and creatinine ranged from 1.1 mg/dl to 5.5 mg/dl in 6 dogs (Table 2C). Dog 3 was the most severely azotemic. Five of seven dogs were off of MF and CSA; 3 were stil receiving low dose prednisone. Diagnostic biopsies were obtained from two dogs (dogs 3, 7) when the serum creatinine concentration increased to greater than 4.5mg/dl despite aggresive medical management. At the time of biopsy, dog 3 had been returned to a full dose of MF and a reduced CSA dose (50%). Dog 7 had been reduced to 10% of the initial CSA dose and 60% of the initial MF dose but the drug doses were returned to full imunosuppresion when the BUN and Cr dramaticaly (140 mg/dl; 6.5 mg/dl, respectively) increased one wek after the final drug reduction. Biopsies from six of the seven renal alografts showed a histological progresion of interstitial inflamation of at least one level of severity when compared to biopsy 2. One renal alograft biopsy specimen (dog 7), with no inflamation on biopsy 2, had advanced to minimal inflamation at biopsy 3. Another alograft biopsy specimen (dog 4) with minimal inflamation at biopsy 2 progresed to slight inflamation at biopsy 3. The alografts previously graded as slight inflamation progresed to moderate (dogs 1 and 2), marked (dog 9), and severe (dog 3) inflamation (Figure 1c). One renal alograft biopsy specimen (dog 6) had been rated as minimal inflamation at biopsy 2, but then progresed to marked at 34 biopsy 3. Al biopsy specimens showed a peritubular and perivascular distribution of lymphocytic, plasmacytic interstitial inflamation. Membranoproliferative glomerulonephritis (dogs 4 and 9) and glomerulosclerosis (4 dog) were also observed. Five dogs had developed minimal (1 dog), slight (2 dogs), moderate (1 dog), or marked (1 dog) interstitial fibrosis since the second biopsy. In thre biopsy specimens neutrophilic infiltration had increased from slight to moderate from biopsy two to thre. Tubulitis developed in two dogs and progresed in 3 dogs (Figure 1d). Al alograft biopsies showed progresive in degre/severity of tubular atrophy betwen biopsy 2 and 3. Al dogs with evidence of vacuolar degeneration of tubules on biopsy 2 had les severe pathology on biopsy 3, going from multifocal to absent in one dog, difuse to multifocal in two dogs, and difuse to absent in one dog. The remaining dog stil had multifocal tubular degeneration but the severity had decreased from slight to minimal. Slight arteriosclerosis was sen in one dog. Based on morphometric analysis, the number of CD3-positive cels (range: 38- TNTC cels/m 2 ) typicaly exceded the number of CD79a-positive cels (range: 8- TNTC cels/m 2 ) in the interstitium of the biopsy. Morphometric analysis was not possible in 4 specimens (dogs 2, 3, 6, and 9) because the lymphocytes were too numerous to quantify. Dog 3 was on a reduced dose of CSA (5mg/kg/day) and MF (10mg/kg q24h), with the addition of prednisone (2mg/kg/day) and azathioprine (2mg/kg/day) as rescue agents at the time of euthanasia. This dog was mildly but persistently azotemic from the time of transplantation (average Cr: 3.16 mg/dl; range of 2.0-6.6mg/dl). Once Cr reached 5.5 mg/dl on day 228, the dog was euthanized. Samples from the 35 renal alograft obtained at necropsy showed several temporaly distinct morphologies of insult: acute, subacute and chronic. Hemorrhage and edema, lymphocytic, plasmacytic nephritis with occasional neutrophilic infiltrates, moderate tubulitis, fibrosis and sclerosis were al sen in separate regions of the alograft histopathologic samples. Fourth Biopsy Dog 1 was the only dog to have a fourth surgical biopsy. The biopsy was taken at day 1466. The dog had been off al imunosuppresive therapy for over 1300 days and had a creatinine of 1.5 mg/dl. The biopsy of this dog had minimal lymphocytic, plasmacytic interstitial inflamation with mild interstitial fibrosis. The remaining biopsy 4 specimens were obtained at necropsy. Dogs 4, 6, 7, and 9 were euthanized with suspected alograft rejections at day 550, 348, 471, and 658 days after transplantation. (Table 2D) Dog 2 was 500 days post-transplantation at this time a fourth biopsy has not been obtained. Dog 4 had been off of CSA for about 5 months and off of MF for one month while maintaining low dose prednisone (0.5mg/kg/day), when creatinine rose to 5.8 mg/dl and BUN reached 96.5 mg/dl. MF was resumed at full imunosuppresive dose and prednisone was increased (1mg/kg/day). Based on the persistent azotemia refractory to treatment and the presence of moderate glomerulosclerosis at biopsy thre, as wel as hypertension, dog 4 was euthanatized 545 days after transplantation. 36 The necropsy alograft specimen had moderate primarily T-lymphocytic interstitial inflamation, severe tubulitis with tubular atrophy, severe striped fibrosis and severe glomerulosclerosis. The capsule had a very thickened granular coating. Arteries had severe fibrointimal proliferation and more than 50% narowing of the lumens. Several artery lumens were completely obliterated. The histopathologic diagnosis was chronic alograft rejection. Dog 6 was euthanized at day 348 after transplantation with a BUN of 101.8 mg/dl and a Cr of 7 mg/dl. This dog had been off of CSA and MF for 130 days and was only on prednisone (1 mg/kg/day). Once off of CSA and MF, BUN and Cr rose steadily from 17 mg/dl and 2.1 mg/dl to an average of 43 mg/dl and 4.1 mg/dl over several months. The wedge biopsy of alograft tisue had severe lymphocytic nephritis and moderate interstitial fibrosis. The regions of fibrosis were often acompanied with lymphocytic inflamation that were most pronounced at the corticomedullary junction (perivascular). Moderate tubulitis (5-10 cels/tubular cross section), moderate to severe tubular atrophy and tubular degeneration were also noted. Mild glomerulosclerosis was present. Several large arteries had severe fibrointimal thickening which resulted in a greatly reduced lumen size. A few arteries were no longer patent. The intima of one large artery was infiltrated with CD3-positive cels, signaling arteritis. A diagnosis of alograft chronic rejection was made. Dog 7 was euthanatized 471 days after transplantation with a BUN of 93.7 mg/dl and a Cr of 8.0 mg/dl. This dog had come off of imunosuppresive therapy intermitently but was persistently azotemic and had a chronic urinary tract infection that was refractory to routine antibiotic therapy. The wedge biopsy of alograft tisue 37 had severe lymphocytic nephritis and moderate interstitial fibrosis. The regions of fibrosis were often acompanied with lymphocytic inflamation, most pronounced at the corticomedullary junction (perivascular). Moderate tubulitis (5-10 cels/tubular cross section), moderate to severe tubular atrophy and tubular degeneration were noted. Mild glomerulosclerosis was present. A diagnosis of chronic alograft rejection was made. Dog 9 was euthanized 658 days after transplantation with a BUN of 79.5 mg/dl and a Cr of 4.2 mg/dl. Imunosuppresive therapy had consisted of only low-dose prednisone for 30 days. One month prior to euthanasia a bacterial urinary tract infection was detected and treated. The wedge biopsy of the renal alograft revealed chronic degenerative changes consistent chronic rejection and end-stage kidney failure. The capsule had a thick covering of granulation tisue. The interstitium was infiltrated with severe fibrosis and moderate lymphocytic, plasmacytic inflamation. The fibrosis was difuse and somewhat striped, emanating from the medulla to the cortex. The inflamation was perivascular and periglomerular with additional lymphoid aggregates asociated with the regions of fibrosis. Tubulitis was present with severe tubular atrophy and degeneration. A large region of edema was present. Severe glomerulosclerosis was present throughout the wedge biopsy specimen. No vascular changes were evident. Chronic alograft rejection was the final diagnosis. 38 Cyclosporine levels At the time of the second biopsy, al seven dogs were on full dose CSA (15 mg/kg BID). Since histopathology was consistent with reports of alograft CSA toxicity, a retrospective analysis was performed in six of the seven dogs. Two dogs had trough levels just below the serum therapeutic range of 200-300 ng/ml, two had moderately elevated concentrations, and two had extremely elevated CSA concentrations. At the third biopsy, four of six dogs had undetectable serum CSA concentrations. Of the two dogs with detectable CSA levels (dog 3 and 7) both had resumed imunosuppresive therapy when alograft rejection was suspected based on BUN and Cr levels. (Table 1) Imunohistochemistry At biopsy 1, only dog 3 had evidence of positive reactivity (positive uptake of imunohistochemical stain), defined as rare CD3-positive cels (< 10) in the interstitium. Infiltrates composed of CD3-positive lymphocytes predominated over CD79a-positive lymphocytes at the time of the second biopsy in al seven dogs? biopsy specimens. The average number of CD3-positive cels/m 2 was 157 (range: 63-428 cels/m 2 ) and the average number of CD79a-positive cels/m 2 was 40.1 (range: 3-132 cels/m 2 ). In the third biopsy specimens, CD3-positive cels (range: 38 cels/m 2 -TNTC) appeared to grossly outnumber CD79a-positive cels (range: 8- 155 cels/m 2 -TNTC). Morphometric analysis was not possible in 3 biopsy samples (dogs 2, 6, and 9) because CD3-positive and CD79a-positive cels were too numerous to quantify. At biopsy 4, dogs 4, 6, 7 and 9 alograft specimens had B-lymphocyte and 39 T-lymphocyte populations that were TNTC. Dog 1 had 103 CD3-positive cels/m 2 and 8 CD79a-positive cels/m 2 at the fourth biopsy. 40 Figure 1A. Dog 6, renal cortex, biopsy 1. Microscopicaly normal. (Stain: HE. 330x, Bar = 60?m). 41 Figure 1B. Dog 6, renal cortex and outer medulla, biopsy 2. Note 2 smal foci of interstitial, peritubular, lymphocytic, Plasmacytic inflamation. (Stain: HE. 330x, Bar = 60?m). 42 Figure 1C. Dog 6, renal cortex, biopsy 3. Note multiple foci of Interstitial, peritubular, lymphocytic, plasmacytic inflamation. (Stain: HE. 330x, Bar = 60?m). 43 Figure 1D. Dog 6, renal cortex biopsy 3. Note the CD3-positive Lymphocytes (brown) infiltrating tubule epithelium. (Stain: ABC-DAB as chromagen. 1320x, Bar = 15?m). 44 Figure 2. Dog 3, renal cortex, biopsy 2. Suspected cyclosporine Toxicity. Note markedly vacuolated tubule epithelial cels, pars recta (arowheads) (Stain: HE. 660x; Bar = 30?m). 45 Figure 3. Graph of dog 1: BUN/Creatinine versus Time. 46 Figure 4. Graph of dog 2: BUN/Creatinine versus Time. 47 Figure 5. Graph of dog 3: BUN/Creatinine versus Time. 48 Figure 6. Graph of dog 4: BUN/Creatinine versus Time. 49 Figure 7. Graph of dog 6 : BUN/Creatinine versus Time 50 Figure 8. Graph of dog 7: BUN/Creatinine versus Time. 51 Figure 9. Graph of dog 9: BUN/Creatinine versus Time. 52 Time (days) Dog Drug levels CSA levels* Clinical Score Biopsy 1 0 1 Pre NA 4 0 2 Pre NA 4 0 3 Pre NA 4 0 4 Pre NA 4 0 5 Pre NA 4 0 6 Pre NA 4 0 7 Pre NA 4 0 8 Pre NA 4 0 9 Pre NA 4 0 10 Pre NA 4 Biopsy 2 4 1 Ful ND 90 2 Ful 31 ng/ml 4 78 3 Ful 146 ng/ml 4 7 4 Ful 19.52 ng/ml 4 85 6 Ful 176 ng/ml 4 75 7 Ful 365 ng/l 4 75 9 Ful >200 ng/ml 2 Biopsy 3 580 1 Of ND 4 281 2 Of <25 ng/ml 4 28 3 Reduced 60 ng/ml 1 484 4 Of <25ng/ml 4 268 6 Of <25ng/ml 4 260 7 Ful 276 ng/ml 2 560 9 Of <25ng/ml 4 Biopsy 4 580 1 Of ND 4 50 4 pred ND 2 348 6 pred ND 3 471 7 Of ND 2 658 9 Of ND 3 CSA: cyclosporine Pred: prednisone ND: not determined NA: not applicable Table 1. Clinical score, cyclosporine levels, and renal biopsy schedule in renal transplant dogs. 53 Day Dog BUN/CR Histopathology Biopsy 1 0 1 1/1.2 normal cortical sample 0 2 15.7/0.8 normal cortical saple 0 3 14.8/1.2 rare (<10) CD3+cels, rare hemosiderin in tubules 0 4 18.6/1.4 normal cortical sample 0 6 17.9/1.6 normal cortical saple 0 7 12.3/1.0 normal cortical sample 0 9 12.8/1.2 normal cortical saple BUN: Blood urea nitrogen CR: creatinine Table 2A. Biochemical and histopathological data in renal alograft dogs, Biopsy 1. 54 Biopsy 2 Dog BUN/Cr Histopathology 4 1 12/1.7 slight lymphocytic plasmacytic interstitial inflammation 90 2 5.8/1.0 slight, multifocal, lymphocytic plasmacytic, perivascular inflammation, minimal tubulitis, slight tubular degeneration, Atrophy 78 3 14/2.7 slight multifocal, peritubular lymphocytic plasmacytic inflammation, moderate tubular degeneration, minial tubulitis 7 4 15/1.5 minimal, multifocal, perivascular, lymphocytic plasacytic inflammation, moderate tubular Degeneration 85 6 12/2.5 minimal, multifocal, perivascular, lymphocytic,plasmacytic inflammation, moderate tubular degeneration, tubular atrophy and focal moderate arteritis 75 7 15.7/1.6 no significant inflammation, moderate tubular degeneration 75 9 20.9/1.3 slight multifocal, peritubular, lymphocytic plasmacytic, inflamation, slight tubular degeneration, minimal tubular atrophy, minimal tubulitis BUN: Blood urea nitrogen CR: creatinine Table 2B. Biochemical and histopathological data in renal alograft dogs, Biopsy 2. 55 Biopsy 3 Dog BUN/Cr Histopathology 580 1 14.8/1.4 moderate, difuse, perivascular, lymphocytic, plasmacytic inflammation,minimal fibrosis, tubular atrophy 281 2 6.1/1.1 moderate, multifocal, perivascular, peritubular lymphocytic, plasmacytic inflammation, slight tubulitis, slight tubular atrophy 28 3 43.4/5.5 severe, difuse, lymphocytic, plasmacytic, neutrophilic inflammation with marked tubulitis, moderate fibrosis, tubular atrophy, marked glomerulosclerosis 484 4 61.6/4.1 slight, multifocal, perivascular/tubular plasmacytic, lymphocytic, neutrophilic inflammation with slight fibrosis, glomerulosclerosis/itis, slight tubulitis, tubular degeneration, atrophy 268 6 16/2.8 marked multifocal,perivascular, peritubular plasmacytic lymphocytic, neutrophilic, inflammation, moderate tubulitis, slight tubular atrophy 260 7 204/4.6 Minimal perivascular, plasmacytic, lymphocytic inflamation, marked fibrosis, focal arteriosclerosis 560 9 26.2/4.2 marked multifocal, peritubular, perivascular, periglomerular, lymphocytic, plasmacytic, neutrophilic, inflammation, moderate tubulitis, slight fibrosis, moderate tubular atrophy, tubular los BUN: Blood urea nitrogen CR: creatinine Table 2C. Biochemical and histopathological data in renal alograft dogs, Biopsy 3. 56 Biopsy 4 Dog BUN/Cr Histopathology 146 1 slight, difuse, perivascular, lymphocytic, plasmacytic inflammation,minial fibrosis, tubular atrophy 50 4 169/7.6 severe, multifocal, perivascular/tubular plasmacytic, lymphocytic, neutrophilic inflammation with severe fibrosis, glomerulosclerosis/itis, moderate tubulitis, severe tubular degeneration, atrophy 348 6 101.8/7.0 marked multifocal,perivascular, peritubular plasmacytic lymphocytic, inflammation, moderate tubulitis, moderate tubular atrophy, tubular degeneration 471 7 93.7/8.3 severe perivascular, lymphocytic, plasmacytic inflammation marked fibrosis, focal arteriosclerosis 658 9 marked multifocal, peritubular, perivascular, periglomerular, lymphocytic, plasmacytic, neutrophilic, inflammation, moderate tubulitis, slight fibrosis, moderate tubular atrophy, tubular los BUN: Blood urea nitrogen CR: creatinine Table 2D. Biochemical and histopathological data in renal alograft dogs, Biopsy 4. 57 VI. DISCUSION Seven of 10 renal transplant dogs conditioned with 200cGy TBI and limited imunosuppresion with CSA, MF, and intermitent prednisone achieved long-term survival (average of 600 days). Tubule epithelial vacuolar degeneration consistent with CSA toxicity was observed in six of seven biopsy specimens when the dogs were on full dose imunosuppresion. These lesions disappeared or diminished after the discontinuation of CSA. Later biopsy specimens (biopsy 3) from these seven dogs had an inflamatory component consistent with early rejection; however, the clasic vascular lesions of alograft rejection were not observed. The severity of azotemia did not correlate wel with the severity of the histopathologic lesions at biopsy thre. Five dogs had a fourth histopathologic evaluation (4 necropsy, one surgical). Biopsy 4 alograft specimens obtained at necropsy were consistent with chronic alograft rejection in al 4 dogs. Two dogs (dogs 1 and 2) are stil alive with functional renal alografts at 500 and 1500 days, respectively. In contrast to recent reports, this study provides an extended observation time in which canine renal alografts were monitored both histologicaly and biochemicaly. Al ten dogs began the study with normal BUN and Cr values and histologicaly normal kidneys. Histologic lesions of greater severity than slight 58 interstitial inflamation were not observed in alograft biopsies while dogs were on full imunosuppresive therapy; however, difuse vacuolar tubular degeneration was documented in six of seven dogs at this time. In renal alograft biopsies from humans, this is considered a sign of renal alograft cyclosporine toxicity. 16;17 Vacuolar tubular degeneration was most prominent in the proximal tubules and pars recta. These tubular lesions were most severe when dogs were on full dose CSA for 75 days or greater and lesions decreased in severity or were absent on later biopsies specimens, when CSA dosages had been reduced or eliminated (Table 1). Because of the relatively high dose and lengthy duration of CSA administration as wel as the concurrent characteristic histopathologic lesions that were reversible once CSA was reduced, CSA toxicity sems to be a likely cause for these changes. Cyclosporine alograft nephropathy in human beings is characterized by the degre of isometric vacuolization of proximal tubules. 16;17 A concurrent arteriolopathy and striped, focal, interstitial fibrosis is sen in chronic cases. 16 Cyclosporine-induced nephropathy has also been documented in cats. 20 The literature suggests CSA toxicity does not occur in dogs at comparable doses. 43;4 There was no reported increase in Cr, BUN, alkaline phosphatase, or total bilirubin in two dogs that received 5, 20 or 40 mg/kg/day of CSA for one month in one study; however, these were healthy dogs that had not undergone renal transplantation. 4 The authors of another study modeling acute alograft rejection did not report histological evidence of CSA toxicity, but these dogs were only on CSA for 20 days. 43 In contrast to these reports in which dogs were on CSA for a maximum of 30 days, the dogs in the present study had lesions consistent with CSA toxicity, as described in the human literature, and were on CSA 59 (30 mg/kg/day) for a minimum of 75 days. Thus, CSA toxicity in the dog may be time-related as wel as dose-related. In support of this conclusion, dog 1, which was on full dose CSA for the shortest period of al dogs (44 days) did not have vacuolar degeneration at biopsy 2. Other sources cite that CSA is not nephrotoxic in dogs unles whole blood levels are greater than >3000 ng/ml (>1500 ng/ml for serum). 2 Four of the six dogs with vacuolar tubular degeneration in the current study had supratherapeutic levels (>300 ng/ml) of CSA, but al six dogs were on full dose CSA for at least 75 days (average of 80 days). It is important that al difuse tubular lesions diminished in severity and distribution once the CSA dose was lowered or eliminated. It is also interesting that these dogs had more fibrosis in biopsy 3 specimens. This suggests that CSA toxicity in dogs may result in chronic alograft changes asociated with fibrosis. CSA toxicity has ben documented in 50 to 100 percent of human alograft patients 7 to 10 years after transplantation on the basis of fibrosis and hyalinosis. 2 Calcineurin-inhibitor induced nephrotoxicity has been suggested as the reason for late histologic injury and the ongoing decline in renal alograft function in human beings 25 and CSA toxicity may have contributed to the renal alograft failure in some of the dogs in the curent study. This problem deserves further investigation. CSA levels in this study were retrospectively determined from serum samples obtained at the time of biopsy two and thre. It may have been advantageous to monitor cyclosporine levels prospectively especialy when the dogs were on high dosages (15 mg/kg) of CSA. This would alowed modification of CSA dosing to reach and maintain therapeutic levels before tapering the dosage. However, the ultimate goal of this study was to initiate a standard dose of imunosuppresion and then taper 60 and eliminate the medication rather than maintain therapeutic levels of CSA. Furthermore, modifying the initial dosage could have further confounded the results as the significance of the CSA levels during the dosage reduction would have been dificult to determine. Although vacuolar tubular degeneration was diminished on the third biopsy specimens, 6 of 7 dogs had difuse perivascular and peritubular interstitial inflamation and tubulitis, al consistent with a progresive imunologic response. This inflamation was characterized as lymphocytic, plasmacytic interstitial infiltration (with occasional neutrophilic infiltration) of varying severity. In biopsies 2 and 3, T-lymphocytes outnumbered B-lymphocytes when inflamation was present. The lymphocytic, plasmacytic interstitial inflamation and tubulitis sen in the current study is consistent with alograft rejection pathology documented in previous veterinary studies. 9;43 Five dogs had a fourth histopathologic examination. Four specimens were obtained at necropsy (dogs 4, 6, 7, 9) ranging from 348 to 550 days after transplantation and one was surgicaly obtained (dog 1) at day 1466. In the necropsy alograft wedge specimens, lymphocytic, plasmacytic interstitial inflamation had progresed in al dogs. Dogs developed lymphoid aggregates populated with both B and T lymphocytes, with inflamation being the most severe at the corticomedullary junction as wel as in periglomerular and perivascular regions. Inflamation was typicaly acompanied with interstitial fibrosis, often ?striped? in nature i.e. rays emanating from the medullary region to the cortex. Amongst these four dogs, there was a definite temporal distinction in alograft pathology. The two dogs that were 61 euthanized under 500 days had more difuse interstitial inflamation and moderate fibrosis. These alografts reflected more active imunologic rejection. The two dogs that were out the longest, at days 528 and 550, had the most interstitial fibrosis, and edema, tubular atrophy and les difuse interstitial inflamation. These alografts appeared to be end-stage, displaying the efects of a diminished blood supply and dysfunctional nephrons. Only one dog (dog 1) had a regresion of interstitial nephritis, down-graded from moderate to slight, focal interstitial nephritis. One other surviving dog (dog 2) has not had a fourth biopsy. In human transplantation medicine, the importance of tubulointerstitial inflamation is deemphasized compared to vasculitis. 16;17 On human renal alograft biopsies, vasculitis is indicative of a poorer response to therapy. Vasculitis has been reported in canine renal alograft specimens during episodes of rejection. 9;12;43 Fibrinoid necrosis of large vesels has also been reported in dogs that had acutely rejected a renal alograft. 9;43 Another study documented intimal arteritis in biopsies from renal alografts in mongrel dogs receiving microemulsified CSA and azathioprine. 12 The dogs in the current study had only rare vascular lesions on biopsy 2 and 3. At biopsy 2, one dog had focal moderate intimal arteritis with medial inflamation. At biopsy 3, another dog had slight arteriosclerosis. Vascular lesions were more difuse at biopsy 4. The most prominent vascular changes at biopsy 4 reflected a difuse arteriopathy. Two dogs (dogs 4 and 6) had severe fibrointimal thickening of arterial wals ranging from fifty-percent diminished caliber to advanced obliterative arteriopathy. Both CSA toxicity and chronic rejection cause fibrointimal thickening of arteries and so discerning betwen the two etiologies is dificult. 62 Fibrointimal thickening of arteries is typicaly a focal change when asociated with CSA toxicity whereas chronic rejection results in difuse vascular changes. Additionaly, a truly obliterative arteriopathy is more consistent with chronic rejection. 16 The vascular changes of these two dogs would be more consistent with chronic alograft rejection following human clasification; however, both of these biopsies specimens have end-stage changes making specific correlations dificult. The vascular changes noted in these two renal alograft specimens could reflect a combination of insults from both CSA and chronic rejection. It should also be noted that CSA toxicity could lead to a fibrotic and compromised alograft, predisposing it to a chronic alograft nephropathy. The two dogs with arterial changes did have tubular pathology consistent with CSA toxicity at earlier biopsies as wel as the characteristic focal, striped fibrosis and tubular atrophy. These renal alograft specimens are end- stage which may make specific correlations dificult. Throughout the study, there were discrepancies betwen the histopathologic lesions and the biochemical evaluation of renal status. Although the severity of azotemia and histopathologic lesions progresed overal, individual correlations betwen biopsies and BUN and Cr levels were unreliable. Two dogs (dogs 2 and 6) had significant progresion of tubulointerstitial inflamation without concurrent change in BUN or Cr. Two dogs (dogs 4 and 7) had marked azotemia with minimal renal alograft histopathology. Only two dogs (dog 3 and 9 at biopsy 3) had consistent agrement betwen biochemical and histopathologic evaluations. The underestimation of pathology in dogs 2 and 6 is not unexpected given that greater than 75% of function must be lost to have biochemical evidence of compromise. 1 Additionaly the 63 persistent azotemia without evidence of rejection at biopsy 3 in dogs 4 and 7 may be explained by clinical factors combined with subclinical rejection. Dog 4 had documented hypertension at the time of biopsy 3 which may have increased the severity of azotemia while dog 7 had a resistant urinary tract infection that was eventualy documented on urine culture. Ultimately, at the time of the fourth histopathologic examination of renal alograft specimens, biochemical parameters and histopathologic findings were in agrement. At necropsy, al four dogs had significant azotemia and had histopathologic changes consistent with chronic renal alograft rejection. The surgical renal alograft biopsy from dog 1 had minimal interstitial inflamation and its BUN and Cr are within normal limits. This inconsistency of biochemical parameters and kidney biopsy results has been reported in both feline and human alograft recipients. In a study examining 77 feline renal transplant biopsies, 36% of biopsies showed some level of rejection despite stable renal alograft function. 20 Protocol biopsies, based on a predetermined time schedule regardles of apparent alograft function, collected from asymptomatic human transplant patients demonstrated histopathologic changes consistent with subclinical rejection in up to 30% of biopsy specimens. 3 Because tubulitis and subclinical rejection are localized proceses, the unafected portions of the alograft are able to maintain normal kidney function. 25 It is also possible that a smal needle biopsy may not sample areas demonstrating alograft pathology. Early detection and treatment of subclinical rejection has ben shown to prolong renal alograft survival. 3-5 Surgical biopsy, in conjunction with histopathologic examination, is the ?gold standard? for documenting acute and 64 subacute rejection; however, due to the invasivenes of surgical biopsies and need for a stable surgical candidate, alternative non-invasive and acurate ways to evaluate the kidney function are greatly needed. In human beings, urinary perforin and granzyme mRNA levels have been correlated with acute renal alograft rejection with 83% sensitivity and 77% specificity. 45 Evaluation of the renal segmental arterial resistance index at 3 months after transplantation was shown to be a powerful prognostic indicator of long-term graft survival in human renal transplant patients. 46 Additionaly, the usefulnes of glomerular filtration rate prediction equations for renal alograft function is being evaluated in human renal alograft recipients. 47 A recent human study linked a 25 % reduction in GFR at thre months as the strongest risk factor for subsequent graft failure. 48 The introduction of these diagnostic tests into veterinary medicine may permit more acurate and more frequent monitoring of renal alografts without the invasivenes of surgery. Such monitoring tests could fil in the gaps betwen biopsies, permiting a more complete evaluation of renal alograft function. Laparoscopic biopsy procedures may be also be helpful as a minimaly invasive technique. The initial mortality rate for this study was 30%. Two of the thre dogs that died were transplanted early in the study when MF was initiated at the ful dosage (10 mg/kg BID). These two dogs died within 7 days of surgery with gross evidence of gastrointestinal toxicity (severely petechiated bowel) and hemorhagic pancreatitis. Side efects of MF include vomiting, diarhea, gastrointestinal hemorrhage and petechiation, and les frequently pancreatitis. 8 (MF [Celcept] label) It is speculated that intestinal and pancreatic toxicity from MF is the result of reduced pancreatic 65 perfusion. 49 The use of MF at high dosages combined with TBI may potentiate gastrointestinal toxicity. It is also posible that MF potentiates bacterial translocation and subsequent sepsis. Sepsis-asociated enteritis was reported in almost 10% of patients receiving imunosuppresion with MF, CSA, TBI and fludarabine. 50 Additionaly MF is known to decrease intestinal cel turnover. 51 Gastrointestinal toxicity was les severe once dogs in the current study were initiated at a half dose of the full imunosuppresive dose of MF (5 mg/kg BID), starting one to two days after transplantation and gradualy increased to full dose (10 mg/kg BID). One dog died from unrelated sepsis, a known complication of medical imunosuppresion. The average survival in the 7 remaining was over 600 days. Considering that ESRD is 100% fatal, this protocol ofers hope that renal transplantation may become a realistic treatment option for dogs with ESRD. There were two variables, BMT (7/10) and mismatched related donors (2/10) that prevent direct comparisons among al 10 dogs. Two of the dogs that did not receive BMT died in the first 5 days of therapy of gastrointestinal toxicity. The other dog (dog 9) that did not receive BMT survived more than 658 days. The two surviving dogs which are out 1500 and 528 days did receive donor BMT. Given this broad range of survival in the thre dogs that did not undergo BMT, it is not clear if the simultaneous BMT improves survival. It might be significant that two of the thre dogs that died in the first twenty days did not receive a BMT. It is possible that donor BMT helps to minimize the number of days of severe neutropenia after surgery. 66 Two dogs in this study (dogs 1 and 6) received renal transplants from DLA-mismatched siblings. The degre of renal alograft pathology in the DLA- mismatched siblings was similar to that sen with the five unrelated DLA- mismatched recipient dogs until biopsy 4. While there was minimal interstitial inflamation at biopsy two in the two DLA-mismatched sibling dogs, the histologic lesions progresed to marked lymphocytic, plasmacytic inflamation on biopsy thre. At biopsy 2, two dogs with non-related renal alografts (dogs 4 & 7) had les inflamatory response than the two mismatched, related dogs. At 348 days, dog 6 was euthanized due to persistent, non-responsive azotemia consistent with rejection. Dog 1, however, decreased from moderate to slight inflamation on the fourth biopsy which was 1466 days after surgery. Clearly, these two similar recipients had two very diferent imune responses to their renal alografts. With only two dogs that received kidneys from mismatched siblings, it is dificult to make acurate generalizations. Unlike human transplant medicine, there is not a wel-established system of histopathologic analysis of renal alografts for veterinary transplant recipients. A few veterinary studies have adapted the ?Banff 97? scheme. 1;12;19;20 A recent feline study found the collection of biopsies for Banff 97 to be useful for the detection of subclinical rejections; however, the severity of lesions was not acurately reflected using this system. Vasculitis, tubulitis, and lymphocytic glomerulitis, al major criteria for rejection in humans, were not sen in the cats. Overal, authors suggest that the rejection of renal alografts in cats is histologicaly diferent from humans and potentialy involves a humoral component. 20 In the current study, alograft rejection 67 was indicated as moderate to severe T and B lymphocyte infiltration of the interstitium with mild to moderate tubulitis. A few cases had fibrointimal proliferation of arteries was present. Extensive interstitial fibrosis was found in 4 dogs at biopsy 4. Glomerulitis and true arteritis, clasical signs of rejection acording to Banff 97, 17 were rarely noted in these canine renal alografts. The outcome of this study points out the complexity and unpredictability of transplantation imunology despite the various atempts at manipulating the recipient imune system. Of the seven dogs that survived greater than 200 days, 5 eventualy died with profound azotemia and histopathologic evidence of alograft rejection +/- concurrent CSA toxicity. With each of these dogs the longer the survival period, the more chronic the renal alograft injury appeared. Two dogs remain survivors, but dog 1 is out significantly longer than al others at greater than 1500 days. This dog received a kidney from DLA-mismatched sibling, similar to dog 6 which was euthanized at day 348 due chronic azotemia and suspect rejection. At biopsy 2 and 3, dog 1 had minimal and moderate inflamation, respectively. At biopsy 4, when dog 1 was completely off of imunosuppresive therapy for greater than 1300 days. The minimal pathology consisted of a minimal inflamatory response present as several smal interstitial lymphoid pockets (10-20 cels). Due to this presence of T and B- lymphocytes, true imunological tolerance appears unlikely but not impossible. This focal inflamation may not a signal of rejection. Perhaps acommodation is a beter label for this recipient-alograft relationship. Acommodation can be defined as a condition where a vascularized alograft functions despite the development of a humoral imune response by the recipient. 52 Unlike tolerance, where an alograft is 68 not recognized as foreign, acommodation appears to dampen a persistent imune response for a limited period of time. Diminished antibody binding, generation of endogenous protective substances e.g. heparan sulfate, and desensitized/down- regulated response to complement are al suggested mechanisms of graft acommodation. 52 The host can acommodate by shifting from a Th1 to a Th2 response, lack of T helper response and/or via a form of peripheral tolerance. A recent review of acommodation cited several smal animal models where acommodated grafts expresed ?survival genes? (Bcl-2 and Bcl-xl) within the endothelium of the alograft vesels. These genes provided anti-inflamatory signs preventing apoptosis by alograft cels. 53 Dog 1 could be in a state of acommodation which provides indefinite recipient survival via a functioning graft; however, this period of graft aceptance is les wel defined than life-long tolerance. The negative side of acommodation is an overal decreased surveilance by the imune system resulting in increased susceptibility to environmental pathogens and neoplasia. Any infection could trigger an imune reaction, overcoming the state of acommodation leading to renal alograft rejection. In comparison to other studies in dogs which terminated at a predetermined time frame of 100 days, there was a prolonged time frame for observation of biochemical and histopathological analysis of dogs with functional renal alografts in the current study. 1;12 Crowel et al reported a 40% mortality at 6 weks and 100% mortality by one year. 9 A median survival time of eight months was achieved in a study with naturaly ocurring ESRD. 10 The current study has an average survival time greater than 600 days. 69 Overal the rejection patern established in the current study was lymphocytic, plasmacytic interstitial inflamation with severe fibrosis, moderate tubulitis, tubular atrophy, and glomerulosclerosis. Biopsies provided the most acurate asesment of renal pathology in comparison to BUN, Cr and clinical status. Factors other than primary imune rejection, such as CSA toxicity, hypertension, and urinary tract infections may also contribute to eventual alograft failure. Although dogs in the current study appeared to recover from the acute efects of CSA toxicity, long-term efects such as fibrosis and vascular compromise may have contributed to eventual end-stage failure. Starting dogs out at a lower dose or abbreviating the course of the high dose CSA may prove to have significant long-term efects on alograft survival. The dog that has survived the longest in this study was on high dose CSA the shortest period of time in comparison to the other dogs. Complete elimination of imunosuppresive medication may not be possible in unrelated DLA-mismatched mongrel dogs undergoing renal transplantation with the current conditioning protocol. However, if a significant reduction in the imunosuppresive drugs could be achieved, many of the long-term complications of imunosuppresion could be minimized and canine renal transplantation may become a widely acepted, clinical treatment for dogs with end-stage renal disease. 70 VI. REFERENCES 1. Polzin D, Osburn C, Jacob F, Ross S: Chronic renal failure, in Etinger S, Feldman E (eds): Textbook of veterinary internal medicine, chap 169. Philadelphia, 2000, pp 1634-1662 2. Colvin RB: Chronic alograft nephropathy. NEJM 349:2288-2290, 2003 3. Grim P, McKenna R, Nickerson P, Russel M, Gough J: Clinical rejection is distinguished from subclinical rejection by increased infiltration by a population of activated macrophages. J Am Soc Nephrol 10:1582-1589, 1999 4. Rush D, Nickerson P, Gough J, McKenna R, Grim P, Cheang M: Beneficial efects of treatment of early subclinical rejection: a randomized study. J Am Soc Nephrol 9:2129-2134, 1998 5. Seron D, Moreso F, Bover J: Early protocol renal alograft biopsies and graft outcome. Kidney International 51:310-316, 1997 6. Niemeyer G, Hudson J, Bridgman R, Spano J, Nash R, Lothrop C: Isolation and characterization of canine hematopoietic progenitor cels. Hematology 29:693, 2001 71 7. Niemeyer G, Welch J, Tilson D, Lothrop C: Renal alograft tolerance in DLA- identical and haploidentical dogs after nonmyeloablative conditioning and transient imunosuppresion with cyclosporine and mycophenolate mofetil. Transplantation Procedings In Pres: 2005 8. Dooley M, Cosio F, Nachman P: Mycophenolate mofetil therapy in lupus nephritis: clinical observations. J Am Soc Nephrol 10:833-839, 1999 9. Crowel W, Finco DR, Rawlings CA, Barsanti J, Rao R: Lesions in dogs following renal transplantation and imunosuppresion. Vet Pathol 24:124- 128, 1987 10. Mathews K, Holmberg D, Miler C: Kidney transplantation in dogs with naturaly occurring end-stage renal disease. JAHA 36:294-301, 2000 11. Kyles AE, Gregory C, Grifey S, Jackson J, Bernsten L, Morris RE: An evaluation of combined imunosuppresion with MNA 715 and micoemulsified cyclosporine on renal alograft rejection in mismatched mongrel dogs. Vet Surg 31:358-366, 2002 12. Bernsten L, Gregory C, Kyles AE, Grifey S, Patz J: Microemulsified cyclosporine-based imunosuppresion for the prevention of acute renal alograft rejection in unrelated dogs: preliminary experimental study. Vet Surg 32:213-219, 2003 72 13. Gregory CR, Gourley I: Organ transplantation in clinical veterinary practice, in Slater D (ed): Textbook of Smal Animal Surgery, chap 7. Philadelphia, 1993, pp 95-101 14. Janeway C, Travers P, Walport M, Shlomchik M: Imunobiology (ed Sixth). New York, Garland Science, 2005, pp 328 15. Cotran R, Kumar V, Collins T: Robbins Pathologic Basis of Disease (ed Sixth). Philadelphia, W. B. Saunders Co., 1999, pp 206-211 16. Pathology of Renal Transplantation, in Sternberg S (ed): Diagnostic Surgical Pathology, Philadelphia, 1999, pp 1767-1776 17. Racusen L, Solez K, Colvin R: The Banff 97 working clasification of renal alograft pathology. Kidney International 55:713-723, 1999 18. Nickerson P, Jefery J, Gough J, McKenna R, Grim P, Cheang M, Rush D: Identification of clinical and histopathologic risk factors for diminished renal function 2 years postransplant. Journal of the American Society of Nephrology 9:482-487, 1998 19. Kyles A, Gregory C, Grifey S, Galvez J: Evaluation of the clinical and histologic features of renal alagraft rejection in cats. Vet Surg 31:49-56, 2002 20. De Cock HEV, Kyles AE, Grifey SM, Bernsten L, Gregory CR: Histopathologic Findings and Clasification of Feline Renal Transplants. Vet Pathol 41:244-256, 2004 73 21. DeCock H, Kyles A, Grifey S: Histopathologic findings and clasification of feline renal transplants. Vet Pathol 41:244-256, 2004 22. Gregory C: Imunosuppresive Agents, in Bonagura JD (ed): Kirk's Current Veterinary Therapy XII, Philadelphia, 2000, pp 509-513 23. Janeway C, Travers P, Walport M, Shlomchik M: Imunobiology (ed Sixth). New York, Garland Science, 2005, pp 615 24. Frese P, Svalander CT, Molne J, Norden G, Nyberg G: Chronic alograft nephropathy-biopsy findings and outcome. Nephrol Dial Transplant 16:2401- 2406, 2001 25. Nankivel B, Borrows R, Chir M: The natural history of chronic alograft nephropathy. NEJM 349:2326-2333, 2003 26. Yu C, Seidel K, Nash R, Deg J, Sandmaier B, Barsoukov A, Santos E, Storb R: Syngerism betwen mycophenolate mofetil and cyclosporine in preventing graft vs host disease among lethaly iradiated dogs given DLA-nonidentical unrelated marow grafts. Blood 91:2581-2587, 1998 27. Sachs D: Mixed chimerism as an approach to transplantation tolerance. Clinical Imunology 95:S63-S68, 2000 28. Fehr T, Sykes M: Tolerance induction in clinical transplantation. Transplantation 13:117-130, 2004 74 29. Starzl T, Zinkernagel R: Transplantation tolerance from a historical prospective. Nature reviews / imunology 1:233-239, 2001 30. Kirk A: Transplantation tolerance: A look at the non-human primate literature in the light of modern tolerance theories. Critical Reviews in Imunology 19:349-388, 1999 31. Wekerle T, Sykes M: Induction of tolerance. Surgery 135:359-364, 2004 32. Kawai T, Sachs D, Cosimi A: Tolerance to vascularized organ alografts in large animal models. Current Opinion in Imunology 11:516-520, 1999 33. Rossini A, Greiner D, Mordes J: Induction of imunologic tolerance for transplantation. Physiologic Reviews 79:99-141, 1999 34. Exner B, Colson Y, Li H, Ildstad S: In vivo depletion of host CD4+ and CD8+ cels permits engraftment of bone marow stem cels tolerance induction with minimal conditioning. Surgery 122:221-227, 1997 35. Watson C, Davies H, Cobbold S, Rasmussen A, Rebelo P, Thiru S, Waldmann H, Caine R, Metcalfe S: CD4 and CD8 monoclonal antibody therapy in the dog: Strategies to induce tolerance to renal alografts. British Journal of Surgery123-124, 1995 36. Contreras J, Eckhoff D, Cartner S, Frenete L, et al.: Tolerability and side efects of anti-CD3-imunotoxin in preclinical testing in kidney and pancreatic islet transplant recipients. Transplantation 68:215-219, 1999 75 37. Asiedu C, Dong S, Lobashevsky A, Jenkins S, Thomas J: Tolerance induced by anti-CD3 imunotoxin plus 15-deoxyspergualin asociates with donor- specific indirect pathway unresponsivenes. Celular Imunology 223:103- 112, 2003 38. Kirk A, Burkly L, Baty D, Baumgartner R, Berning J, et al.: Treatment with humanized monoclonal antibody against CD154 prevents acute renal alograft rejection in nonhuman primates. Nature Medicine 5:686-693, 2005 39. Hartner W, Markees T, DeFazio S, Shafer D, et al.: Efect of early administration of donor bone marow cels on renal alograft survival in dogs treated with antilymphocyte serum and cyclosporine. Transplantation 59:131- 155, 1995 40. Starzl T, Demetris A, Murase N, et al.: Cel migration, chimerism, and graft aceptance. Lancet 339:1579-1584, 1992 41. Mathew J, Garcia-Morales R, Careno M, Jin Y, et al.: Imune responses and their regulation by donor bone marow cels in clinical organ transplantation. Transplant Imunology 11:307-321, 2003 42. Mathews K, Holmberg D, Johnson K, Miler C, Binnington A, Maxie G, Atiloa M, Smith G: Renal alograft survival in outbred mongrel dogs using rabbit anti-dog thymocyte serum in combination with imunosuppresive drug therapy with or without donor bone marow. Vet Surg 23:347-357, 1994 76 43. Haishima A, Kawakami y, Mizuno S: Acute and vascular rejection and interstitial rejection following renal alograft transplantation in dogs. J Vet Med Sci 64:1137-1140, 2002 44. Aeder M, Sutherland D, Bowers L: Cyclosporine toxicity in a canine model: clinical, histologic, and imunologic studies. Transplantation Procedings 16:1632-1633, 1984 45. Bagoui L, Hartono C, Ruchuang D, et al.: Noninvasive diagnosis of renal alograft rejection by measurement of mesenger RNA for perforin and grazyme B. NEJM 344:947-954, 2001 46. Radermacher J, Mengel M, Elis S, et al.: The Renal arterial resistance index and renal alograft survival. NEJM 349:115-124, 2003 47. Perico N, Flavio G, Remuzzi G: Asesing renal function by GFR prediction equations in kidney transplantation. Am J Transplantation 5:1175-1176, 2005 48. Kasiske B, Gaston R, Gourishankar S, Haloran P, Matas A, Jefrey J, Rush D: Long-term deterioration of kidney alograft function. American Journal of Transplantation 5:1405-1414, 2005 49. Konigsrainer A, Steurer W, et al.: Nonocclusive segmental mesenteric ischemia after combined pancreas kidney transplantation: Mycophenolate mofetil as an etiologic factor? Transplantation 70:696, 2000 7 50. Niederweiser D, Maris M, Shiruzu J, et al.: Low-dose total body iradiation and fludarabine followed by hematopoietic cel transplantation from HLA- matched or mismatched unrelated donors and postgrafting imunosuppresion with cyclosporine and mycophenolate mofetil can induce durable complete chimerism and sustained remision in patients with hematologic diseases. Blood 101:1620-1629, 2003 51. Steger A, Timoney A, Grifen S, Salem RWG: The Influence of imunosuppresion on peptic ulceration following renal transplantation and the role of endoscopy. Nephrol Dial Transplant 5:289-292, 1990 52. Koch C, Khalpey Z, Plat J: Acomodation: Preventing injury in transplantation. The Journal of Imunology 172:5143-5148, 2004 53. Salama A, Delikouras A, Pusey C, Cook H, et al.: Transplant acomodation in highly sensitized patients: A Potential role for Bcl-xL and aloantibody. American Journal of Transplantation 1:260-269, 2001