MS, University of Cincinnati, 2023, Medicine: Biomedical Research Technology

It has been established that the early presence of Glial cell line-derived neurotrophic factor (GDNF) in the metanephric mesenchyme is essential for the outgrowth of the ureteric bud from the nephric duct. Also, GDNF in the cap mesenchyme promotes branching of the collecting duct throughout kidney development. We have shown that GNDF is expressed by self-renewing nephron progenitor cells (NPCs) at mouse embryonic day 10.5 (E10.5); however, single-cell RNAseq by Magella et al., demonstrated that Gdnf is strongly expressed by stromal progenitor cells at E14.5. To reconcile these seemingly opposed results, we used a transgenic GDNF-CreERT2 and R26R-tdTomato mouse model to conduct lineage tracing experiments activated through tamoxifen injections at different time points during development. We hypothesized that the expression of Gdnf was dynamic, with early expression in NPCs transitioning into stromal progenitors at later stages. Our lineage studies demonstrate that Gdnf is expressed primarily by NPCs at early stages (from E9.5 to E12.5) and primarily by stromal cells during the later stages of development (E13.5 and later). Unexpectedly, we revealed that Gdnf is also expressed by nephric duct (ND) progenitors at even earlier stages. Expression of Gdnf was transient in the ND, and quantification of recombination shows that expression in the ND progenitors starts as early as E7.5 and peaks around E8.5. Altogether, our results indicate that Gdnf is expressed by all kidney progenitor cells (ND progenitors, NPCs, and stromal progenitors) but at different time points during kidney development. The physiological relevance of these transitions remains to be elucidated.

Committee: Cristina Cebrian-Ligero Ph.D. (Committee Chair); Kyle McCracken M.D. Ph.D. (Committee Member); Samantha Brugmann Ph.D. (Committee Member) Subjects: Developmental Biology

PhD, University of Cincinnati, 2020, Medicine: Molecular and Developmental Biology

Nephron segmentation is a poorly understood process that forms four distinct regions of the nephron: the renal corpuscle, the proximal tubule, the loop of Henle, and the distal tubule. Each segment has a specialized function necessary for proper renal filtration. The proximal tubule is the main site of active reabsorption in the nephron, responsible for approximately 65% of total reabsorption. Proximal tubule dysfunction has been implicated in many nephrology disorders, such as renotubular acidosis and Fanconi renotubular syndrome (FRTS), which can lead to chronic kidney disease in adulthood. In order to provide better treatments, it is necessary to understand the molecular mechanisms underlying proximal tubule development. The goal of this dissertation is to determine the molecular mechanisms regulating proximal tubule development. Hepatocyte nuclear factor 4 alpha (Hnf4a) is a transcription factor that is only expressed in the proximal tubules of the kidney. A heterozygous mutation in the HNF4A gene has been identified in patients with FRTS. FRTS is defined as generalized proximal tubular dysfunction characterized by polyuria, polydipsia, glucosuria, proteinuria, and phosphaturia. This suggests that Hnf4a is a key regulator of proximal tubule development and function. However, the role of Hnf4a in proximal tubule development is unknown and there was no mouse model for kidney-specific Hnf4a deletion. Therefore, to investigate the role of Hnf4a in the kidney, we generated two mouse models of nephron-specific Hnf4a deletion. The first mouse model studied featured mosaic deletion of Hnf4a in Six2-expressing nephron progenitors. In this model, Hnf4a mutant mice showed a paucity of proximal tubules in the developing kidney. This paucity led to mutant mice developing FRTS-like symptoms. Hnf4a mutant cells were unable to mature into LTL-high proximal tubules cells, indicating that Hnf4a is required for proximal tubule maturation. In the second mouse model, we investigated (open full item for complete abstract)

Committee: Joo-Seop Park Ph.D. (Committee Chair); Elif Erkan M.D. (Committee Member); S. Steven Potter Ph.D. (Committee Member); Katherine Yutzey Ph.D. (Committee Member); Aaron Zorn Ph.D. (Committee Member) Subjects: Developmental Biology

PhD, University of Cincinnati, 2018, Medicine: Molecular and Developmental Biology

The kidney is a complex organ that is made of many different cell types. In an effort to better understand the cell diversity within the developing kidney we have performed single cell RNA-seq on embryonic day 14.5 mouse kidneys using Drop-seq, Chromium 10X Genomics, and Fluidigm C1 platforms. AltAnalyze was used to identify sixteen cell clusters; medullary collecting duct, cortical collecting duct, ureteric bud tip, loop of Henle, distal comma shaped body, podocyte, mid S-shaped body, early proximal tubule, pre-tubular aggregate, three cap mesenchyme groups, endothelium, nephrogenic zone stroma, cortical stroma, and medullary stroma. In addition to the known identifier genes, novel specific gene associations were also discovered during analysis. One such example is the discovery of Gdnf expression from within the stromal population, which was previously thought of as being exclusively expressed from the cap mesenchyme. The wild type single cell RNA-seq data set was also used to identify Hox gene expression within the developing kidney. Interestingly there is an apparent lack of a Hox code within the developing kidney, with thirty-six of the thirty-nine Hox genes being ubiquitously expressed throughout the kidney. Previous studies have determined that Hox 10 and 11 paralogous groups have functional redundancies within kidney development. Based on the expression data it is feasible that flanking genes are also functionally redundant within kidney development. These redundancies can mask the specific functions of individual Hox genes. For this reason Hoxa9,10,11; Hoxd9,10,11 mutants were used to further elucidate the role of Hox genes during kidney development. Morphological analysis of the Hox mutants shows alterations in mature nephron segment identity, medullary zone specification, and the lack of a pelvic opening. Obtaining a comprehensive single cell RNA-seq data set allows for the visualization of the expression profile of many genes within the wild type develop (open full item for complete abstract)

Committee: Steven Potter Ph.D. (Committee Chair); Sudhansu Dey Ph.D. (Committee Member); Brian Gebelein Ph.D. (Committee Member); Ashish Kumar M.D. Ph.D. (Committee Member); Joo-Seop Park Ph.D. (Committee Member) Subjects: Developmental Biology

MS, University of Cincinnati, 2001, Medicine : Molecular and Developmental Biology

HoxA11 is one of the homeobox genes with similarity to the Drosophila homeotic gene, Abdominal-B. The Abd-B type Hox genes have been shown to be expressed in the developing limbs, axial regions and the most caudal regions of the body. From the study of HoxA11 knockout and HoxA11/D11 double knockout mice, HoxA11 has been shown to be involved in appendicular and axial skeleton development, kidney development and reproductive system development (Small et al. 1993, Davis et al. 1995, Li et al. 1995). In the HoxA11 knockout mice, there are axial skeleton phenotypes, limb phenotypes and urogenital malformation. The homozygous male and female KOs are sterile. There is no kidney phenotype in HoxA11 KOs. Hox11 paralogues are functional redundant. The HoxA11 and HoxD11 double knockout mouse have severe limb, reproductive system and kidney phenotypes. There are different kidney phenotypes in the double KO mouse: some mice have no kidney, some have one or two smaller kidneys. Though the nephrons look normal, there are fewer nephrons in the double KO kidneys. The rare adult kidneys have a thick wall of cortical tissue and poorly developed renal papillae. One wild-type allele of either HoxA11 or HoxD11 was sufficient for normal kidney development. It is not known what genes are regulated by HoxA11 in the kidney development. The goal of this project is to identify the HoxA11 downstream targets in the kidney. Gain of function and loss of function methods have been used to identify the HoxA11 downstream targets in kidney. Drosophila Engrailed repressor and HoxA11 Homeodomain fusion protein was stable expressed in mouse kidney K3 cells to inhibit expression of the HoxA11 targets. The gene expression profiles of En-A11HD positive and negative cells have been analyzed on genechips. Expression of some genes was changed in the En-A11HD positive cells. The expression of some cell proliferation factors and oncogene are decreased, while the expression of some cell differentiation factors is (open full item for complete abstract)

Committee: Dr. Steven Potter (Advisor) Subjects: Biology, Molecular

PhD, University of Cincinnati, 2006, Medicine : Molecular and Developmental Biology

Renal morphogenesis involves the reciprocal inductive interactions between the ureteric bud and metanephric mesenchyme forming the collecting ducts and nephrons within adult kidney. We applied microarray technology to the study of renal morphogenesis in order to better understand the molecular mechanisms underlying development. Additionally, the techniques employed in the expression analysis of the embryonic kidney were extended to the study of renal disease. Embryonic kidneys representing different stages of renal development were analyzed using expression microarrays. Renal developmental analysis revealed many novel genes and genetic pathways involved in renal development. In addition, the normal renal development data provides a baseline for the analysis of gene targeted mice possessing disruptions in renal morphogenesis. Microarray analysis was also performed on the Hoxa11/Hoxd11 compound null renal defect throughout renal development. In conclusion, these microarray studies greatly advance our knowledge of gene expression within the normal renal morphogenesis and identify possible downstream candidate genes regulated by the Hox11 genes. Wnt signaling is crucial for normal renal morphogenesis. In Drosophila, the pygopus gene encodes a transcriptional co-activator required for canonical Wnt signaling. The targeted deletion of the mammalian orthologs of pygopus, Pygo1 and Pygo2, in mice was investigated in renal development. A disruption in ureteric number tip and morphology was identified in Pygo1/Pygo2 compound null kidneys. Additionally, canonical Wnt signaling as measure by the Bat-gal transgene is reduced within the ureteric compartment in Pygo1/Pygo2 null kidneys. Overall, these experiments suggest that Pygo function is required for activation of canonical Wnt signaling in the ureteric compartment of the developing kidney. Focal segmental glomerulosclerosis (FSGS) is characterized by the segmental scarring of the glomerulus, ultimately resulting loss of neph (open full item for complete abstract)

Committee: Dr. S. Potter (Advisor) Subjects:

Doctor of Philosophy in Clinical-Bioanalytical Chemistry, Cleveland State University, 2012, College of Sciences and Health Professions

Chronic kidney disease (CKD) is highly prevalent in the US population and has high incidence of cardiovascular and all-cause mortality. A known complication of CKD is secondary hyperparathyroidism that is caused by bone and mineral imbalances, including vitamin D deficiency. Supplementation of CKD patients with vitamin D is based on guidelines issued by the Kidney Disease Quality Outcomes Initiative (K/DOQI), which recommend administration of vitamin D2 in variable doses depending on the severity of vitamin D deficiency. Retrospective and pilot studies have shown that vitamin D2 was not as effective as vitamin D3 in treating vitamin D deficiency. In Chapter I, we investigated the effectiveness of vitamin D2 versus vitamin D3 treatment in resolving vitamin D deficiency in the pre-dialysis CKD population. This study was a double blinded, randomized, single center study that involved 22 CKD subjects. Data showed that vitamin D3 elicited a more rapid increase in 25-hydroxyvitamin D (25OHD) levels than vitamin D2, but both forms became equivalent in terms of the number of people who reached target 25OHD levels by the end of study. Glomerular filtration rate (GFR) is the best overall index of kidney function. GFR is determined by measuring the urinary clearance of a radioactive exogenous biomarker, such as iothalamate, or estimated (eGFR) by measuring creatinine and adjusting for race, gender and age using equations. There are several known limitations to using creatinine-based equations and radioactive substances exposure for eGFR and GFR determinations. In the remaining Chapters, solutions are proposed for measurement of GFR and eGFR, which involve liquid chromatography-tandem mass spectrometry (LC-MS/MS). Chapter II discusses this technique and the process of development and validation of bioanlaytical methods by LC-MS/MS. Chapter III introduces a LC-MS/MS method for the measurement of L-arginine, symmetric dimethylarginine (SDMA), and asymmetric dimethylarginine (ADMA (open full item for complete abstract)

Committee: Sihe Wang PhD (Committee Co-Chair); Aimin Zhou PhD (Committee Co-Chair); David Anderson PhD (Committee Member); Xue-Long Sun PhD (Committee Member); Nolan Holland PhD (Committee Member) Subjects: Biochemistry; Chemistry; Medicine