Browsing School, Graduate by Subject "Kidney"
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Novel PC2 regulation of ezrin in renal epithelia reveals insights into ADPKD cystogenesisAutosomal dominant polycystic kidney disease is caused by the loss of function of either two transmembrane proteins, polycystin-1 or polycystin-2. In renal epithelia, the consequence of polycystin loss is the formation of progressive, focal, fluid-filled cysts. However, the function and associated downstream signaling pathways specific to the polycystins have not been defined. Therefore, a new in vitro tubuloid model was designed to investigate the proximate cellular changes in renal epithelial cells following inactivation of Pkd2, the gene that encodes for polycystin-2. This model system reinforced the relevance of proteins associated with cell junctions, adhesions, and matrix in the cyst mechanism. The impact of this model was further supported through morphometrical analysis of epithelial compartmentalization in human ADPKD tissue, demonstrating an altered apical compartment in emerging cysts compared to noncystic tubules. Seeking connection between the junctions and disrupted apical compartment led to investigation of ezrin, a master scaffold in the apical compartment in renal epithelial cells. Ezrin plays a critical role in regulation of polarity, cytoskeleton organization, and protein trafficking, and the downstream consequences of its disruption have not been elucidated. Investigation into the initiating events of cystogenesis in ADPKD revealed a dramatic change in ezrin, following loss of PC2 in our tubuloid model, cystic mouse model, and pathological human ADPKD tissue. Based on this novel regulatory relationship between PC2 and ezrin, as well as the antecedent loss of ezrin to cyst formation in mice, ezrin was overexpressed in the pkd2 morpholino zebrafish model. Increased expression of ezrin diminished the formation of pronephric cysts. This lead to the design of a cyst rescue mouse model, which has exhibited promising preliminary data for cyst area reduction with additional ezrin. The disruption of ezrin in Pkd2 inducible in vitro and in vivo model systems, changes in ADPKD patient tissue, and rescue of pronephric cysts in the pkd2 MO suggest there is a role of ezrin in renal cystogenesis. Understanding the relationship of ezrin, with PC2 in renal epithelial cells will help elucidate the mechanism of ADPKD cystogenesis and define important downstream pathways necessary for epithelial functions.
Transcriptional and Post-Transcriptional Regulation of Genes Critical for Sodium Reabsorption and Blood Pressure Control in the KidneySerine/threonine kinases (WNK1, WNK4, SPAK, OSR1) and cation co-transporters (NKCC2, NCC) are members of a multi-kinase network that determines renal Na+ reabsorption and blood pressure (BP) regulation. The importance of these proteins is highlighted by their roles in monogenic forms of hyper- and hypotension, animal models, and by the efficacy of BP-lowering medications that target this pathway. While post-translational regulation of these proteins has been well established, regulation of these genes at the transcript level is not completely understood. In this study, we examined both human and mouse kidney transcriptomes to uncover novel transcriptional and post-transcriptional regulation of two genes in this pathway. First, STK39 encodes for Ste20-related proline alanine rich kinase (SPAK), which phosphorylates and activates cation co-transporters. Variants within STK39 are associated with susceptibility to essential hypertension, and SPAK null mice are hypotensive and mimic Gitelman syndrome, a rare monogenic salt-wasting human disorder. Mice exhibit nephron segment-specific expression of full length SPAK and N-terminally truncated SPAK isoforms with impaired kinase function. We established that while humans also express transcript isoforms similar to those found in mice, they differ in abundance and are transcribed from human-specific promoters. Second, SLC12A3 encodes for the thiazide-sensitive Na+-Cl- co-transporter (NCC), and rare mutations in this gene cause Gitelman syndrome. In humans and mice, alternative polyadenylation of NCC pre-mRNA results in a longer 3'UTR isoform, while alternative splicing within the final exon leads to an exon-exon junction downstream of the termination codon. Both of these events generate potential substrates for nonsense-mediated mRNA decay (NMD). By suppressing NMD, we demonstrated that NCC transcript abundance is partially determined by post-transcriptional processing of its final exon. Finally, dietary K+ manipulation differentially alters SPAK and NCC transcript and protein isoform abundance, demonstrating dynamic physiological regulation of gene expression in response to salt reabsorptive needs. In summary, genes in this pathway undergo complex transcriptional and post-transcriptional regulation, resulting in the differential expression of novel alternative transcripts that contribute to the fine-tuning of BP control.