Samples from 93 of 115 consecutive cadaveric renal transplants were selected to define polymorphisms in both IFN-γ and IL-10. A 12 CA repeat IFN-γ polymorphic allele was found in 73 patients (70 in patients analysed further). This polymorphism was associated with high IFN-γ production in vitro. According to the presence or not of the 12 CA repeat allele patients were separated in high and low producer genotype groups. The incidence of acute rejection was 54.3% in this high IFN-γ genotype group, contrasting with 44.4% in the low IFN-γ. Requirement for ATG therapy was greater in the high IFN-γ group (odds ratio [OR]=2.5). Among HLA-DR-mismatched patients, IFN-γ high producer genotype was more strongly associated with rejection (OR=1.6). In the cyclosporine monotherapy subgroup, 11 out of 14 patients with IFN-γ high genotype (78%) had acute rejection (OR=2.88, p=0.09). Graft survival was similar between the two IFN-γ groups. When the analysis was controlled for the presence of delayed graft function, 40.5% of the high IFN-γ genotype patients had serum creatinine levels above 200 micromoles/L contrasting with only 14.3% of the low IFN-γ genotype recipients at 5 years after transplantation (p=0.05). In a regression model of creatinine at 1 year the significant variables were the presence of DGF, donor age greater than 50, greater than two rejection episodes, DR mismatch, donor female to male recipient sex, IL-10 high genotype, and IFN-γ high genotype. Conclusion: The 12 CA repeat IFN-γ polymorphic allele is associated with high IFN-γ production. We have shown that this high producer genotype for IFN-γ influences acute rejection in kidney transplantation, particularly in high-risk groups; it is also associated with worse long-term graft function.

The acquisition of renogenic competence (the ability to give rise to kidney) during embryonic development is not yet fully understood. Clarifying the temporal and molecular aspects of this process is equally essential for understanding excretory system development and for devising methods for successful differentiation of embryonic stem cells (ESCs) to renal cells for disease modeling, toxicology screening and potential cell replacement therapies. In embryo development, the metanephric (permanent) kidney arises as a result of inductive interactions between two embryonic structures that arise in the intermediate mesoderm - the ureteric bud (UB, a diverticulum of the Wolffian duct) and the metanephric mesenchyme (MM). The UB develops into the collecting duct system and the MM undergoes an epithelial-to-mesenchymal transition to form the secretory units of the kidney - the nephrons. In this thesis, I used a tissue disaggregation-reaggregation method that allows the reconstruction of mouse organotypic kidney rudiments to place different embryonic cells in the environment of a developing kidney and assess their potential to integrate into kidney epithelia and differentiate to renal cells. First, the suitability of this method was evaluated and a quantitative assay for evaluating the numbers of test cells integrating in various renal compartments was developed. Second, the reaggregation method was used to characterise the renogenic potential of undifferentiated mouse ESCs, ESC-derived cells after Notch inhibition, and cells derived from the presumptive nephrogenic regions of embryos at various stages of development. ESCs are isolated from the inner cell mass of an embryo and have the potential to differentiate to any tissue of the body when injected into mouse blastocysts. Strategies have successfully been devised for ESC differentiation to many lineages, but very few studies reported any success with the differentiation of ESCs to a renal lineage. Undifferentiated ESCs showed a very good ability to form chimeric structures with developing kidney tubules (both nephrons and extending UBs). Nevertheless, the resulting structures were morphologically different from renal epithelia in most cases and integrated ESC-derived cells were not positive for several combinations of kidney markers. These results suggested that the influence of the niche was not sufficient for a successful ESC differentiation to renal cells. Treatment of ESC with an inhibitor of the Notch pathway to increase the proportion of mesodermal cells did not improve this outcome. On the basis of these results, it was speculated that the earliest lineage to which embryonic stem cells must be differentiated in order to become competent to make renal cells should first be identified. I addressed this by determining the developmental stage at which cells able to contribute to the formation of metanephric epithelia first appear in mouse embryo development. When mixed in embryonic kidney reaggregates labelled cells isolated from the nephrogenic regions of E9.5 embryos integrated into various renal compartments. These cells were seen in UBs, nephrons, glomeruli and the condensing mesenchyme. Marker expression studies showed that the exogenous E9.5 cells expressed an array of kidney markers - Pax2 in renal epithelia and the condensing mesenchyme, Wt1 in glomeruli and Six2 in the condensing mesenchyme. Furthermore, exogenous E9.5 cells also co-expressed Pax2/Wt1 in the condensing mesenchyme, Megalin/Ecadherin in the proximal tubule and Pax2/E-cadherin in renal epithelia. This provides evidence that challenges the existing model and suggests that some cells from the intermediate mesoderm at a stage where the metanephric blastema is yet formed are competent to contribute to kidney structures. Furthermore, experiments with E8.5 embryos showed that such a renocompetence could be acquired even before the specification of intermediate mesoderm. These findings contribute to our knowledge about kidney cell specification and provide valuable information to guide future attempts to develop an efficient method for deriving renal cells from ESCs. Furthermore, the reported ability of ESC-derived non-kidney cells to form chimeric structures with renal tubules provides a proof-of-principle that it might be possible to use exogenous types of cells for physiological support to injured kidney tubules, thus offering a possible novel approach for cell replacement therapies.

<p>Given the emerging epidemic of renal disease in HIV+ patients and the fact that HIV DNA and RNA persist in the kidneys of HIV+ patients despite therapy, it is necessary to understand the role of direct HIV-1 infection of the kidney. HIV-associated kidney disease pathogenesis is attributed in large part to viral proteins. Expression of Vpr in renal tubule epithelial cells (RTECs) induces G2 arrest, apoptosis and polyploidy. The ability of a subset of cells to overcome the G2/M block and progress to polyploidy is not well understood. Polyploidy frequently associates with a bypass of cell death and disease pathogenesis. Given the ability of the kidney to serve as a unique compartment for HIV-1 infection, and the observed occurrence of polyploid cells in HIV+ renal cells, it is critical to understand the mechanisms and consequences of Vpr-induced polyploidy. </p><p>Here I determined effects of HIV-1 Vpr expression in renal cells using highly efficient transduction with VSV.G pseudotyped lentiviral vectors expressing Vpr in the HK2 human tubule epithelial cell line. Using FACS, fluorescence microscopy, and live cell imaging I show that G2 escape immediately precedes a critical junction between two distinct outcomes in Vpr+ RTECs: mitotic cell death and polyploidy. Vpr+ cells that evade aberrant mitosis and become polyploid have a substantially higher survival rate than those that undergo complete mitosis, and this survival correlates with enrichment for polyploidy in cell culture over time. Further, I identify a novel role for ATM kinase in promoting G2 arrest escape and polyploidy in this context. In summary, my work identifies ATM-dependent override of Vpr-mediated G2/M arrest as a critical determinant of cell fate Vpr+ RTECs. Further, our work highlights how a poorly understood HIV mechanism, ploidy increase, may offer insight into key processes of reservoir establishment and disease pathogenesis in HIV+ kidneys.</p> / Dissertation

The Banner University Medical Center's (BUMC) renal transplant program relies on the LifePort Kidney Transporter to optimize marginal kidney organs via hypothermic machine perfusion (HMP) prior to transplantation. Hemodynamic parameters produced by the device followed over the duration of support, combined with clinical experience, guide decisions in determining the acceptability of a donor kidney for implantation. Thus far, statistical evidence supporting ideal parameters remain undefined. The purpose of this study is to create a logistic model that will ascertain the post-implant sustainability of LifePort® supported kidneys and predict clinical outcomes. My hypothesis is that the statistical models constructed based on retrospective LifePort® parameters and clinical outcome data will successfully predict donor organ vascular health for transplantation and the optimal support duration. A successful model will contribute to increased efficiencies in the kidney transplant process as well as improved patient outcomes. An overview of the institution’s success was weighed using a survival analysis, with delayed graft function (DGF) as the endpoint. A logistic regression model and forecast model were built to predict the outcome for rejecting or accepting the organ for transplant, as well as to predict the hemodynamic parameters hours after the start of infusion. Results concluded a flow greater than 80 mL/min had a 90% probability of transplantation. The forecast model was capable of predicting flow for up to five hours. The calculated flow was in a 10 mL/min range of the actual flow, when up to one hour parameters were entered into the model. The study concluded practicality in the clinical setting, in kidney transplantation.

The purpose of this study was to determine if a relationship existed between the change in serum osmolality in the chronic renal failure patient on hemodialysis and the number of clinical symptoms of the dialysis disequilibrium syndrome experienced during hemodialysis. The study was conducted at the Renal Dialysis Unit of an urban medical center located in a southeastern city. Data were collected from May 25, 1978 to June 30, 1978. Data were obtained during 28 hemodialysis procedures performed on 11 subjects. Serum osmolality measurements were collected prior to initiation of the dialysis procedure, at half-hour intervals during dialysis, and prior to termination of dialysis. Clinical symptoms which occurred during dialysis were recorded by the patients and nursing personnel. The hypothesis was that the number of clinical symptoms of the dialysis disequilibrium syndrome experienced by the chronic renal failure patient during hemodialysis will increase as the serum osmolality decreases.

ABSTRACT MOLECULAR MECHANISMS AND GENE SIGNATURES INVOVLED IN CALCINEURIN INHIBITOR NEPHROTOXICITY IN KIDNEY ALLOGRAFT By Huong Le Diem Nguyen, M.S. A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Physiology at Virginia Commonwealth University. Virginia Commonwealth University, 2011. Major Director: Valeria Mas, Ph.D. Associate Professor, Department of Surgery and Pathology Director of Molecular Transplant Research Laboratory, Division of Transplant Calcineurin inhibitors (CNI), cyclosporin A and tacrolimus, are potent immunosuppressive agents but induce toxicities causing damages and graft dysfunction, and have been suggested to contribute to late-term loss of graft in kidney transplant recipients. Even though insights on mechanism of CNI nephrotoxicity have been uncovered, prevention and treatment of these toxicities remain a major challenge in the clinical administration of CNI due to low dose-toxicity correlation, difficulty in establishing a differential patho-histological diagnosis, and varying individual susceptibility. We hypothesize that CNI nephrotoxicity follows distinct disease pathways and is characterized by significant gene signatures that differentiate it from other conditions such as acute rejection and chronic allograft dysfunction. Moreover, we postulate that CNI-induced toxicity profiles contribute to the IF/TA signatures. Microarray analysis and gene annotation were done on the study database included of tissues diagnosed with CNI nephrotoxicity (n = 9), interstitial fibrosis/tubular atrophy (IF/TA, n=10), and normal allografts (NA, n = 8). All samples were histologically classified based on the revised Banff ‘07 criteria for renal allograft pathology. Top-scored biological networks in CNI tissues were related to metabolic disease, cellular development, renal necrosis, apoptosis cell-death, immunological disease, inflammatory disease, and many others. Canonical pathway analysis emphasized oxidative stress response mediated by NRF2 and various cell-death signaling pathways including 14-3-3 signaling pathway, p53 signaling pathway, and TGF-β signaling pathway. Profiling of differentially expressed genes was done based on their statistical significance and biological relevance to the unique pathology of CNI nephrotoxicity. Among these, three genes RGS1, CXCR4, and TGIF1 were further quantitatively evaluated using real time-PCR. Between CNI group and normal allograft, t-test results showed only RGS1 gene expression level was statistically significant. Between IF/TA group in normal allograft, both RGS1 and CXCR4 showed statistical significance. The calculated relative fold changes revealed an up-regulated pattern of RGS1 and CXCR4 expression in association with pathological groups (CNI and IF/TA). We did not, however, find any association between the expression of TGIF1 in either CNI group or IF/TA group.

End stage renal disease (ESRD) is a growing epidemic impacting the United States. While the optimal treatment for ESRD is renal replacement, barriers exist making this treatment difficult and sometimes impossible for patients to pursue. One potential solution to existing barriers is to encourage patients to actively seek living donors. This is an inherently communicative and social process. The Theory of Motivated Information Management (TMIM) offers a framework for understanding factors that contribute to patients’ conversations about transplantation with their social networks. It is also possible that Patient Empowerment can add to this model, and inform future patient education. Specific variables related to the TMIM and Patient Empowerment are analyzed in bivariate and logistic regression analyses. Variables that were significant in bivariate analysis did not rise to the level of significance when included in a full logistic regression analysis. Study results and outcomes suggest that further research is warranted.

Master of Science / Biomedical Sciences / Gregory F. Grauer / Chronic kidney disease (CKD) is a potentially life-threatening disease that reportedly affects 10% of dogs and 30% of cats over the age of 15. There is no cure available for CKD, but medical management is available for patients with this disease. Research has focused on earlier detection of CKD with the goal of instituting medical management and monitoring as early in the disease course as possible. Symmetric dimethylarginine (SDMA) has recently emerged as a novel renal excretory biomarker that may aid in early detection of CKD in cats and dogs. SDMA is non-protein bound and is freely filtered by the glomerulus, is not secreted or reabsorbed, and has greater than 90% excretion by the kidneys, making it a potential target for measurement of glomerular filtration rate (GFR). Previous studies have demonstrated a close parallel between SDMA and serum creatinine (sCr), which is the currently favored serum biomarker for assessment of GFR. Research has also demonstrated a correlation between SDMA and GFR. Serum concentrations of SDMA increase above normal when GFR is decreased by 25-40%; much earlier than the 75% decrease in GFR typically required for sCr to increase above its reference interval. The studies reported here demonstrate a potential use for the SDMA:sCr ratio as a predictor of volume responsive azotemia. Furthermore, longitudinal assessment of older dogs and cats for early detection of CKD showed that SDMA was a more sensitive indicator of CKD than sCr. The evaluation of SDMA reported in this thesis presents a novel perspective on SDMA and its use clinically.

Renal ischemia/reperfusion (I/R) is a main cause of acute kidney injury (AKI) and delayed graft function after renal transplantation. Previous studies in human and experimental models have identified that inflammation and oxidative stress are two key players in renal I/R injury. However, the underlying mechanisms remain speculative. The overall objective of the study was to investigate the biochemical and molecular mechanisms of I/R-induced renal injury and the effect of tyrosol supplementation on I/R-induced kidney oxidative stress damage. In the present study, renal I/R was induced in Sprague-Dawley rats and in a human kidney proximal tubular cell line. A significantly elevated expression of pro-inflammatory cytokine expression (MCP-1, IL-6) was observed. There was a significant decrease in mRNA and protein levels of two hydrogen sulphide (H2S)-producing enzymes, CBS and CSE, with a concomitant reduction of glutathione and H2S production. In the cell culture model, hypoxia–reoxygenation of proximal tubular cells led to a decrease in CBS and CSE expression and an increase in pro-inflammatory cytokine expression. Supplementation of glutathione or H2S donor (NaHS) effectively abolished cytokine expression in tubular cells. Experiments were conducted to detect oxidative stress markers. It was demonstrated that there was a significant increase in peroxynitrite formation and lipid peroxidation in the kidney after I/R insult, which might be caused by the elevation in nitric oxide (NO) metabolites and inducible nitric oxide synthase (iNOS). Administration of tyrosol, a natural phenolic compound, reduced peroxynitrite formation, lipid peroxidation and the level of NO metabolites via inhibiting NF-B activation and iNOS expression. Tyrosol treatment improved kidney function and had a protective effect against I/R-induced AKI. The present study has clearly demonstrated that (1) there is a reduction of H2S production via inhibition of CBS and CSE expression, which contributes to increased pro-inflammatory cytokine expression in the kidney and in tubular cells upon I/R insult; (2) restoration of endogenous H2S production would be of therapeutic value in regulating inflammatory response in I/R-induced kidney injury; (3) tyrosol treatment has a beneficial effect against renal I/R-induced oxidative stress, in part, through its inhibition on NF-B activation and iNOS-mediated NO production. / May 2016

Shiga toxin-producing enterohemorrhagic Escherichia coli (EHEC) is an emerging food- and water-borne pathogen, causing approximately 73,000 annual infections in the United States and an estimated 1.5 million infections globally. E. coli O157:H7, the most frequently associated EHEC strain, is primarily transmitted through consumption of contaminated ground beef and produce and leads to hemorrhagic colitis in humans. In 5% to 15% of infected patients, circulating Shiga toxins (Stx1, Stx2) cause hemolytic uremic syndrome (HUS), characterized by the presence of thrombocytopenia, hemolytic anemia, and thrombotic microangiopathy, contributing to acute kidney injury (AKI). Current treatment is supportive and antibiotic therapy is contraindicative as it increases toxin production. Therapeutics for EHEC-induced HUS need to be identified to minimize kidney injury and uncontrolled coagulopathy. Well-characterized animal models of HUS and EHEC infection are available and provide avenues for potential therapeutic discovery. Baboons (Papio) challenged with endotoxin-free Shiga toxins develop full spectrum HUS, and mice infected with Stx2-producing Citrobacter rodentium (Cr Stx2+), a genetically modified enteric mouse pathogen, develop severe Stx2-mediated kidney injury. Initial studies have shown that soluble thrombomodulin (sTM), an anti-coagulant, is a promising therapeutic in preventing severe kidney injury in pediatric patients. In these studies, we determined whether complement was activated in baboons challenged with Shiga toxins, and evaluated whether intraperitoneal injection of sTM would reduce disease severity from mice infected with Cr Stx2+. D-dimer and cell injury markers (HMGB1, histones) confirmed the presence of coagulopathy and cell injury in Stx challenged baboons. Studies revealed that complement activation is not required for the development of thrombotic microangiopathy and HUS induced by EHEC Shiga toxins in these pre-clinical models. Soluble thrombomodulin treatment in Cr Stx2+ infected mice significantly decreased colonization but did not alter mortality. However, gene expression of kidney injury markers (NGAL, KIM-1) decreased significantly compared to no treatment indicating sTM-associated cytoprotectivity. The C. rodentium mouse model does not develop the coagulopathy seen in HUS patients and sTM treatment may be more effective in the baboon toxemia model. Soluble thrombomodulin is a promising therapeutic for EHEC-induced HUS and should be further evaluated in Stx challenged baboons.