| dc.description.abstract | Autosomal dominant polycystic kidney disease (ADPKD) is a genetic disorder, caused by mutations in either of the PKD1 or PKD2 genes, which leads to the development and growth of multiple fluid-filled cysts in the kidney. The cysts are present at birth and grow throughout the lifetime of the afflicted individual, eliciting pain and eventually leading to end-stage renal disease (ESRD). The PKD1 and PKD2 genes and their corresponding encoded proteins, polycystin-1 (PC1) and polycystin-2 (PC2), are necessary for proper tubulogenesis, and for the repair processes that maintain the architecture of the tubules after injury in the adult kidneys. Mutations in PC1 and PC2 results in phenotypic changes in renal epithelial cells, which consist, among others, in a more dedifferentiated state of the cells, increased proliferation, increased fluid secretion, increased apoptosis, epithelial-to-mesenchymal (EMT)-like features, abnormal activity of various cell signaling pathways, and alterations in intracellular calcium. An enzyme essential for the proper function of the kidney is the Na,K-ATPase (NKA). NKA uses the free energy from ATP to catalyze the movement of Na+ in exchange for K+ across the cell plasma membrane. Its function in the kidney is key for solute and water reabsorption and formation of urine. In addition, NKA is the receptor and signal transducer for the hormone ouabain, which regulates the sodium transport of normal renal epithelial cells. In ADPKD kidneys, the binding of ouabain to NKA has an important role in exasperating cystic progression. Specifically, ouabain stimulates both the proliferation and the fluid secretion of ADPKD cells, as well as cystogenesis in embryonic kidneys from a mouse model of ADPKD. This thesis was undertaken to determine how ouabain contributes to the overall phenotype of ADPKD cells. In particular, emphasis was placed on the effect of ouabain on the cystic phenotypes of apoptosis, EMT and intracellular calcium. Ouabain significantly increased the apoptotic rate in renal cystic epithelial cells obtained from the kidneys of patients with ADPKD (ADPKD cells). Ouabain-mediated apoptosis occurred via the intrinsic pathway, and was reliant on cytochrome c release from the mitochondria and on an altered ratio of Bcl-2 associated X protein (BAX) to B-cell lymphoma 2 (BCL-2) protein. These results agree with findings from other authors that show that apoptosis of epithelial cells is a characteristic of ADPKD cells. While ouabain can increase apoptosis in ADPKD cells, it also increases proliferation, and overall proliferation remained greater than overall apoptosis induced by ouabain. Therefore, ouabain creates a misbalance towards cell growth, which helps the development of ADPKD cysts. Additionally, epithelial to mesenchymal (EMT)-like signaling was altered by ouabain. A hallmark of EMT is the decrease in expression of E-cadherin and the increase in N-cadherin. Ouabain was able to induce this affect in ADPKD cells, without altering cadherin levels in NHK cells. Additionally, ouabain increased tumor growth factor-beta (TGF-beta) expression and SMAD3 phosphorylation, as well as increased expression of downstream targets, such as collagen-I and Snail. However, ouabain was not able to induce a complete switch to a mesenchymal phenotype, as evidenced by unchanged levels in the mesenchymal protein vimentin, the presence of beta-catenin in the plasma membrane, and the unchanged invasive properties. Interestingly, despite the decreased cell-cell adhesion, ouabain increased transepithelial electrical resistance, but did not affect the permeability of monolayers to neutral dextran, suggesting that the tightness of junctions and the permeability of the paracellular transport pathway are preserved in the cells. In contrast, epithelial cells from normal human kidneys (NHK cells) did not display any of the mentioned effects of ouabain. Together, these results reveal that while ouabain contributes to the development of the dedifferentiated phenotype of ADPKD cells, but does not lead to a complete EMT, which stimulates cyst formation and growth. Finally, intracellular calcium concentration ([Ca2+]i) is constitutively lower in ADPKD cells relative to NHK cells. NHK cells were found to respond to ouabain with an increase in [Ca2+]i, while ADPKD cells did not. Further, ADPKD cells had lower concentrations of ER calcium than NHK, but neither ER nor mitochondrial calcium levels appear to be altered by ouabain. The ouabain-induced increase in [Ca2+]i in NHK cells occurred through L-type calcium channels (LTCC). ADPKD cells did not respond to ouabain in the same manner, and this was found to be due to lower expression of full-length LTCC at the plasma membrane. Indeed, increased calpain activity was observed in ADPKD cells relative to NHK, and this correlated inversely with expression of the endogenous calpain inhibitor, calpastatin. The expression of c-Myc is elevated in ADPKD cells, and this negatively regulates calpastatin expression. Indeed, inhibition of the transcriptional activity of c-Myc resulted in increased calpastatin protein, decreased calpain activity, and decreased cleavage of LTCC yielding increased levels of basal intracellular calcium in ADPKD cells. Although we did not find that ouabain was able to further exasperate the calcium phenotype, we expect longer treatment with ouabain may have a more pronounced effect. In conclusion, both apoptosis and EMT are enhanced by ouabain in ADPKD cells and unaffected in NHK cells. Conversely, ADPKD cells do not appear to alter calcium in response to ouabain in the time points assayed. However, the knowledge that calpains are more active in ADPKD causing mid-channel cleavage of LTCC is entirely novel and potentially a benefit to the field. | |
