The collecting duct of the distal nephron marks the final location where adjustments to Na+ excretion can be made, therefore determining the final concentration of Na+ conserved in the extracellular fluid which plays a role in governing overall blood volume and pressure. This transport of Na+ is subject to hormonal regulation but the signalling pathways underpinning this regulation however, are not fully understood. In this thesis the signalling pathways allowing both basal and insulin-stimulated Na+ absorption were explored in the murine collecting duct cell line, mpkCCDcl4. The effects of two insulin-sensitizing drugs, TZDs, on ENaC-mediated Na+ transport were investigated and the signalling pathways underlying two other hormonal regulators of ENaC, dexamethasone and vasopressin, were also examined. Unstimulated monolayers of mpkCCDcl4 cells generated spontaneous Na+ absorption which was quantified by measuring equivalent short circuit current (Ieq). Selective inhibition of PI3-kinase, mTORC2 and SGK1 left ~80 % of the current intact, indicating these signalling molecules are not required for basal Na+ transport. Acute addition of insulin stimulated Ieq and this occurred with a concomitant increase in mTORC2, SGK1 and Akt activity. Inhibition of PI3-kinase abolished the insulin-stimulated response as well as phosphorylation of downstream substrates, indicating a crucial role of PI3-kinase. Inhibition of mTORC1 with rapamycin did not alter basal or insulin-stimulated Na+ transport. The mTOR inhibitors TORIN1 and PP242 could therefore be used to evaluate the role of mTORC2. These inhibitors greatly reduced insulin-stimulated ENaC-mediated Na+ transport and also abolished SGK1 and mTORC2 activity, indicating a novel role of mTORC2. An inhibitor of SGK1, GSK650394A abolished insulin-stimulated Na+ transport and specifically inhibited SGK1 acitivty demonstrating the importance of SGK1 in insulin signalling. The inhibitor Akti-1/2 also abolished insulin-mediated Na+ transport but this compound inhibited both Akt and SGK1 activity. The TZDs pioglitazone and rosiglitazone did not alter basal or insulin-stimulated Na+ transport and had no effect on SGK1 activity indicating these drugs do not alter Na+ absorption in this cell line. Dexamethasone stimulated ENaC-mediated Na+ transport in a similar manner to insulin and this could be blocked with rapamycin. This drug did not alter phosphorylation of NDRG1 indicating that dexamethasone stimulates Na+ transport in an mTORC1-dependent manner but without altering SGK1 activity. Arginine vasopressin also stimulated Ieq but did so by reducing Rt with an associated depolarisation of Vt. Ieq could be blocked with amiloride and vasopressin-stimulated Ieq was insensitive to TORIN1 and PP242. Vasopressin suppressed SGK1 phosphorylation of NDRG1 but did stimulate protein kinase A (PKA) activity. Therefore vasopressin stimulates Ieq via a PKA-dependent but mTOR- and SGK1-independent pathway.

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.