Indiana University-Purdue University Indianapolis (IUPUI) / Diabetic Kidney Disease (DKD) is a major complication of diabetes. Incomplete understanding of its molecular mechanisms is highlighted by the limited treatments options. We hypothesized that inhibition of protective endogenous mechanisms plays major role in the pathogenesis of DKD. While renoprotection is mediated by cyclic nucleotides (cAMP and cGMP), phosphodiesterases (PDEs) lead to cyclic nucleotide degradation. Our investigation focused on the role of calcium/calmodulin activated PDE1 in DKD. Three isoforms of PDE1 are differentially expressed in vascular smooth muscle cells, renal tubular epithelial cells, podocytes, and mesangial cells. We used highly potent and selective PDE1 inhibitor LY1 to explore systemic hemodynamic and local renal role of PDE1. LY1 reduced systolic and diastolic blood pressure in normotensive and spontaneously hypertensive rats. Renal protection with PDE1 inhibition was tested in mouse model of DKD, featuring a combination of diabetes, nephron loss and arterial hypertension. In this model, a PDE1 inhibitor caused a significant improvement in renal function as evident by significant reduction of albuminuria, serum creatinine and several urine biomarkers of inflammation and injury. Histopathological analysis revealed substantial improvement in the pathology of DKD in the treated group that was associated with the reduction of gene expression related to inflammation and fibrosis. Thus, we revealed the role of calcium activated PDE1 in DKD. However, the source of calcium in this context remained obscure. Our bioinformatics analysis pointed out that calcium channel TRPC6 is likely to be involved. Further in vitro studies demonstrated that TRPC6 activation induced apoptosis in human mesangial cells and isolated rat glomeruli, which was attenuated by both TRPC6 and PDE1 inhibition, thereby suggesting a functional coupling between TRPC6 (as a source of calcium) and PDE1 activation. Moving upstream, we showed that several systemic risk factors of DKD (angiotensin II, endothelin 1 and glucose) activated TRPC6 in a different manner, through generation of either reactive oxygen species or diacylglycerol. The computational modeling to relate human transcriptomic and phenotype data demonstrated the pre-clinical findings of renal benefit upon PDE1 inhibition is translatable in human. Taken together, our results suggest mechanistic link among systemic risk factors, TRPC6, calcium flux and PDE1 activation in pathogenesis of DKD. As a corollary, PDE1 inhibition leads to direct and indirect renoprotective effects.

A research report to the Faculty of Health Sciences, University of Witwatersrand, Johannesburg, in partial fulfillment of the requirements for the degree of Master Medicine in branch of Haematology / Donor specific crossmatching is performed prior to renal transplantation in order to determine the presence of pre-existing antibodies against donor HLA antigens which can result in hyperacute rejection. Flow cytometric crossmatching is reported in the literature to be a more sensitive and objective method of testing than the complement dependent cytotoxicity (CDC) method that is currently used in the Gauteng Province. A prospective analysis of the flow cytomeric crossmatch (FCXM) assay using the Luminex technology as the reference method was conducted. Forty-three samples were analysed. The T cell crossmatch (using a cutoff value of 2) revealed a sensitivity of 66.7%, a specificity of 83.8%, a positive predictive value (PPV) of 40% and negative predictive value (NPV) of 93.9%. The B cell crossmatch (using a cutoff value of 5) gave a sensitivity of 100%, specificity of 92.7%, and a PPV and NPV of 40 and100%, respectively. In addition, a retrospective analysis of clinical data for all patients transplanted during the period January 2008 to May 2009 was performed. Of a total of 50 patients assessed post transplant, none of the patients showed signs of hyperacute rejection, while twelve percent (12%) of patients revealed signs and symptoms suggestive of acute rejection. The validation of the flow cytometric crossmatch analysis was complex as there is no gold standard reference method. The assay was validated based on the clinical relevance of its high negative predictive value and the absence of hyperacute rejections in the clinical follow up. The rate of acute rejection found in this study is similar to that reported in literature.

A research report submitted to the Faculty of Health Sciences, University of Witwatersrand, Johannesburg, in partial fulfillment of the requirements for the degree of Master of Medicine in the branch of Internal Medicine Johannesburg, 2017 / Objective: There is limited data describing acute kidney injury (AKI) in HIV-infected adult patients in resource-limited settings where increasingly, tenofovir (TDF), which is potentially nephrotoxic, is prescribed. We describe risk factors for, and prognosis of AKI in HIV-infected individuals receiving and naïve to TDF. Methods: This was a prospective case cohort study of hospitalized HIV-infected adults with AKI (as defined by the 2012 KDIGO Clinical Practice Guideline for AKI) stratified by TDF exposure. Adults (≥18 years) were recruited: clinical and biochemical data was collected at admission; their renal recovery, discharge or mortality was ascertained as an in-patient and, subsequently, to a scheduled 3-month follow-up. Results: Amongst this predominantly female (61%), almost exclusively black African cohort of 175 patients with AKI, 93 (53%) were TDF exposed; median age was 41 years (IQR 35-50). Median CD4 count and VL and creatinine at baseline was 116 cells/mm3 and 110159 copies/ml, respectively. A greater proportion of the TDF group had severe AKI on admission (61% v 43% p=0.014); however, both groups had similar rates of newly diagnosed tuberculosis (TB) (52%) and NSAID (32%) use. Intravenous fluid was the therapeutic mainstay; only 7 were dialyzed. Discharge median serum creatinine (SCr) was higher in the TDF group (p=0.032) and fewer in the TDF group recovered renal function after 3-months (p=0.043). 3-month mortality was 27% in both groups but 55% of deaths occurred in hospital. Those that died had a higher SCr and more severe AKI than survivors; TB was diagnosed in 33 (70%) of those who died. Conclusions: AKI was more severe and renal recovery slower in the TDF group; comorbidities, risk factors and prognosis were similar regardless of TDF exposure. Because TB is linked to higher mortality, TB co-infection in HIV-infected patients with AKI warrants more intensive monitoring. In all those with poor renal recovery, our data suggests that a lower threshold for dialysis is needed. / MT2017

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Chronic kidney disease (CKD) is a major public health concern, characterized by an irreversible reduction in renal function. Currently, creatinine-based GFR estimation is predominantly used clinically to characterize CKD. However, this method is known to be an insensitive test for early losses of kidney function. Since patient prognosis relies heavily on slowing further decline of kidney function, uncovering novel biomarkers for kidney function, in conjunction with eGFR, will help improve patient outcome. Epigenetic-based biomarkers have been identified in numerous cancers, as DNA methylation changes alter cellular function. Thus, the objective of this study is to determine novel DNA methylation patterns reflecting altered kidney function. Five healthy participants that have undergone a nephrectomy have donated urine samples before and after their surgery, and global DNA methylation changes were analyzed through the 450K HumanMethylation microarray. Site- and region-level analyses were conducted to determine significant differentially methylated probes post-nephrectomy. The differential associations observed post-nephrectomy are statistically significant in both the site-level and regional analyses. Nineteen significant candidate probes have been systematically selected for validation, based on involvement in kidney function and consistent direction of methylation. Pyrosequencing assays have also been successfully designed and tested with control DNA, however replication of the microarray findings in participant DNA was unsuccessful. The inability to validate these candidate probes may be attributed to many influencing factors, and with this in mind, uncovering novel methylation patterns is still a promising biomarker for evaluating kidney function. / Thesis / Master of Science (MSc)

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

Renal dysplasia is a disease characterized by developmental abnormalities of the kidney that affect 1 in 250 live births. Depending on the severity of the renal abnormalities, this disorder can lead to childhood kidney failure, adult onset chronic kidney disease, and hypertension. Currently, the best treatment options for patients with renal dysplasia are renal dialysis and kidney transplant. Our limited understanding of the pathogenesis of renal dysplasia has prevented the development of better treatment strategies for those patients. A hallmark of renal dysplasia is an expansion of loosely packed fibroblast cells, termed renal stroma. Markedly elevated levels of β-catenin have been reported in the expanded stromal population in patients with dysplastic kidneys. Yet, the contribution of stromal β-catenin to the pathogenesis of renal dysplasia is not known. Additionally, the role of stromal β-catenin in the developing kidney is not clear. The overall hypothesis of this PhD thesis is that β-catenin in stromal cells controls key signalling molecules that regulate proper kidney development. Furthermore, we hypothesize that elevated levels of β-catenin contribute to the pathogenesis of renal dysplasia. To mimic the human condition, we generated a mouse model that overexpresses β-catenin specifically in the stroma (termed β-catGOF-S). In addition, to gain a better understanding of its role in kidney development, we generated a second mouse model deficient for β-catenin exclusively in stromal cells (termed β-catS-/-). The goal of this study is to utilize these models to understand the role of stromal β-catenin in kidney formation and investigate its contribution to renal dysplasia. The first objective defines the contribution of stromal β-catenin to the genesis of renal dysplasia. We provide evidence for a mechanism whereby the overexpression of stromal β-catenin disrupts proper differentiation of stromal progenitors and leads to an expansion of stroma-like fibroblast cells and vascular morphogenesis defects. In the second objective, we establish a mechanism where stromal β-catenin modulates Wnt9b signaling in epithelial cells to control proliferation of the nephron progenitors. In the third objective, we define a role for stromal β-catenin in proper formation and survival of the medullary stroma. Finally, in a technical report, we outline a protocol to isolate stromal cells in the developing kidney and provide potential downstream applications to further our understanding of stromal β-catenin in the developing kidney. Taken together, our findings establish a crucial role for stromal β-catenin in the genesis of renal dysplasia and demonstrate, using two mouse models, that stromal β- catenin must be tightly regulated for proper formation of the stroma lineages and development of the kidney. / Thesis / Doctor of Philosophy (PhD)

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

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