Indiana University-Purdue University Indianapolis (IUPUI) / Type II nephronophthisis (NPHP2) is an autosomal recessive renal cystic disorder characterized by mutations in the inversin gene. Humans and mice with mutations in inversin have enlarged cystic kidneys. Increased kidney size in NPHP2 may involve altered cell growth, apoptosis, electrolyte transport and fluid accumulation in the cysts. To test this hypothesis, histology and transcriptome analysis were performed on one-day old wild-type and inv/inv mice to uncover molecular pathways altered in the mutant mice. Histology of inv/inv mice kidneys showed dilated cystic tubules compared to wild type. Pathway analysis of transcriptome data showed that inversin exerts its effects on kidneys, at least in part, through the transcriptional regulation of genes implicated in inflammation, immune response, cellular metabolism, cell cycle and ion transport. Genes involved in inflammation or immune response were upregulated whereas the genes involved in cell cycle progression and ion transport were downregulated. To validate the array findings from inv/inv mice kidneys, functional consequence of inversin loss on transepithelial ion transport was measured by electrophysiological techniques in shRNA mediated inversin-depleted renal cell type isolated from mouse cortical collecting duct (mCCD). Depletion of inversin decreased vasopressin-induced Na+ absorption, but did not alter Cl- secretion in mCCD cells. Addition of amiloride, a specific blocker of the epithelial sodium channel (ENaC), abolished basal ion transport in both inversin knockdown and control cells indicating ENaC involvement. Loss of inversin decreased Na+ absorption and this effect, in part, was mediated by transcriptional and post-translational regulation of ENaC mediators. To better understand inversin function in renal cells, transcriptome analysis was performed in control and inversin-depleted mCCD cells. Pathway analysis showed that inversin-depletion altered the genes represented in cell cycle, cellular assembly and organization, DNA replication, cell proliferation and ion transport in this isolated renal cell type. In concordance with the array data from inv/inv mice kidneys, a decrease in the expression of cell cycle, ion transport and apoptotic genes were observed accompanied by an upregulation of genes implicated in inflammatory or immune response indicating a direct effect of inversin on renal cells. Together, this study utilized a combination of transcriptome and functional analyses to unravel the role of inversin in renal cells. These data demonstrate that loss of inversin can cause a delay in cell cycle progression with a decrease in cell proliferation and apoptosis which in turn can perturb the development of the renal tubule. Also, a decrease in Na+ reabsorption together with differential regulation of other ion transporters can result in altered electrolyte transport contributing to cystogenesis, cyst growth, fluid accumulation and cyst expansion in NPHP2.
| Identifer | oai:union.ndltd.org:IUPUI/oai:scholarworks.iupui.edu:1805/12513 |
| Date | 05 1900 |
| Creators | Kulkarni, Nalini H. |
| Contributors | Blazer-Yost, Bonnie L. |
| Source Sets | Indiana University-Purdue University Indianapolis |
| Language | en_US |
| Detected Language | English |
| Type | Thesis |
| Rights | Attribution-NonCommercial-NoDerivs 3.0 United States, http://creativecommons.org/licenses/by-nc-nd/3.0/us/ |
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