CAMP can be either mitogenic or anti-mitogenic, depending on the cell type. We demonstrated previously that cAMP inhibited the proliferation of normal renal epithelial cells and stimulated the proliferation of cells derived from the cysts of polycystic kidney disease (PKD) patients. The protein products of the genes causing PKD, polycystin-1 and polycystin-2, are thought to regulate intracellular calcium levels, suggesting that abnormal polycystin function may affect calcium signaling and thus cause a switch to the cAMP growth-stimulated phenotype. To test this hypothesis, we disrupted intracellular calcium mobilization by treating immortalized mouse M-1 collecting duct cells and primary cultures of human kidney epithelial cells with calcium channel blockers and by lowering extracellular calcium with EGTA. Calcium restriction for 3–5 h converted both cell types from a normal cAMP growth-inhibited phenotype to an abnormal cAMP growth-stimulated phenotype, characteristic of PKD. In M-1 cells, we showed that calcium restriction was associated with an elevation in B-Raf protein levels and cAMP-stimulated, Ras-dependent activation of B-Raf and ERK. Moreover, the activity of Akt, a negative regulator of B-Raf, was decreased by calcium restriction. Inhibition of Akt or phosphatidylinositol 3-kinase also allowed cAMP-dependent activation of B-Raf and ERK in normal calcium. These results suggest that calcium restriction causes an inhibition of the phosphatidylinositol 3-kinase/Akt pathway, which relieves the inhibition of B-Raf to allow the cAMP growth-stimulated phenotypic switch. Finally, M-1 cells stably overexpressing an inducible polycystin-1 C-terminal cytosolic tail construct were shown to exhibit a cAMP growth-stimulated phenotype involving B-Raf and ERK activation, which was reversed by the calcium ionophore A23187. We conclude that disruption of calcium mobilization in cells that are normally growth-inhibited by cAMP can derepress the B-Raf/ERK pathway, thus converting these cells to a phenotype that is growth-stimulated by cAMP.

Despite the recent launch of tolvaptan, the search for safer polycystic kidney disease (PKD) drugs continues. Ciclopirox (CPX) or its olamine salt (CPX-O) is contained in a number of commercially available antifungal agents. CPX is also reported to possess anticancer activity. Several mechanisms of action have been proposed, including chelation of iron and inhibition of iron-dependent enzymes. Here, we show that CPX-O inhibited in vitro cystogenesis of primary human PKD cyst-lining epithelial cells cultured in a 3D collagen matrix. To assess the in vivo role of CPX-O, we treated PKD mice with CPX-O. CPX-O reduced the kidney-to-body weight ratios of PKD mice. The CPX-O treatment was also associated with decreased cell proliferation, decreased cystic area, and improved renal function. Ferritin levels were markedly elevated in cystic kidneys of PKD mice, and CPX-O treatment reduced renal ferritin levels. The reduction in ferritin was associated with increased ferritinophagy marker nuclear receptor coactivator 4, which reversed upon CPX-O treatment in PKD mice. Interestingly, these effects on ferritin appeared independent of iron. These data suggest that CPX-O can induce ferritin degradation via ferritinophagy, which is associated with decreased cyst growth progression in PKD mice. Most importantly these data indicate that CPX-O has the potential to treat autosomal dominant PKD.

Autosomal-dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease and is characterized by progressive growth of fluid-filled cysts. Growth factors binding to receptor tyrosine kinases (RTKs) stimulate cell proliferation and cyst growth in PKD. Nintedanib, a triple RTK inhibitor, targets the vascular endothelial growth-factor receptor (VEGFR), platelet-derived growth-factor receptor (PDGFR), and fibroblast growth-factor receptor (FGFR), and is an approved drug for the treatment of non-small-cell lung carcinoma and idiopathic lung fibrosis. To determine if RTK inhibition using nintedanib can slow ADPKD progression, we tested its effect on human ADPKD renal cyst epithelial cells and myofibroblasts in vitro, and on Pkd1^f/fPkhd1^Cre and Pkd1^RC/RC, orthologous mouse models of ADPKD. Nintedanib significantly inhibited cell proliferation and in vitro cyst growth of human ADPKD renal cyst epithelial cells, and cell viability and migration of human ADPKD renal myofibroblasts. Consistently, nintedanib treatment significantly reduced kidney-to-body-weight ratio, renal cystic index, cystic epithelial cell proliferation, and blood-urea nitrogen levels in both the Pkd1^f/fPkhd1^Cre and Pkd1^RC/RC mice. There was a corresponding reduction in ERK, AKT, STAT3, and mTOR activity and expression of proproliferative factors, including Yes-associated protein (YAP), c-Myc, and Cyclin D1. Nintedanib treatment significantly reduced fibrosis in Pkd1^RC/RC mice, but did not affect renal fibrosis in Pkd1^f/fPkhd1^Cre mice. Overall, these results suggest that nintedanib may be repurposed to effectively slow cyst growth in ADPKD.

Polycystic kidney disease (PKD) results from loss-of-function mutations in the PKD1 gene. There are also reports showing abnormally high levels of PKD1 expression in cystic epithelial cells. At present, nothing is known about the molecular mechanisms regulating the normal expression of thePKD1 gene or whether transcriptional disregulation of the PKD1 gene has a role in cyst formation. We have analyzed a 3.3-kb 5′-proximal portion of the human PKD1gene. Sequence analysis revealed the presence of consensus sequences for numerous transactivating factors, including four T-cell factor (TCF) binding elements (TBEs). Transcriptional activity of the 3.3-kb fragment and a series of deletion constructs was assayed in HEK293T cells. A 2.0-kb proximal promoter region containing one of the four TBEs (TBE1) was inducible up to 6-fold by cotransfection with β-catenin. β-catenin-mediated induction was inhibited by dominant-negative TCF and by deletion of the TBE1 sequence. 15- or 109-bp sequences containing the TBE1 site, when cloned upstream of a minimal promoter, were shown to respond to β-catenin induction. Gel shift assays confirmed that the TBE1 site is capable of forming complexes with TCF and β-catenin. To determine whether expression of the endogenous PKD1 gene responds to β-catenin, HT1080 cells were treated with LiCl, and HeLa cells were stably transfected with β-catenin. In both cases, endogenous PKD1 mRNA levels were elevated in response to these treatments. Taken together, these studies define an active PKD1 promoter region and suggest that the PKD1 gene is a target of the β-catenin/TCF pathway.

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the growth of renal cysts that ultimately destroy kidney function. Mutations in the PKD1 and PKD2 genes cause ADPKD. Their protein products, polycystin-1 (PC1) and polycystin-2 (PC2) have been proposed to form a calcium-permeable receptor-channel complex; however the mechanisms by which they function are almost completely unknown. Most mutations in PKD1 are truncating loss-of-function mutations or affect protein biogenesis, trafficking or stability and reveal very little about the intrinsic biochemical properties or cellular functions of PC1. An ADPKD patient mutation (L4132Δ or ΔL), resulting in a single amino acid deletion in a putative G-protein binding region of the PC1 C-terminal cytosolic tail, was found to significantly decrease PC1-stimulated, G-protein-dependent signaling in transient transfection assays. Pkd1^ΔL/ΔL mice were embryo-lethal suggesting that ΔL is a functionally null mutation. Kidney-specific Pkd1^ΔL/cond mice were born but developed severe, postnatal cystic disease. PC1^ΔL protein expression levels and maturation were comparable to those of wild type PC1, and PC1^ΔL protein showed cell surface localization. Expression of PC1^ΔL and PC2 complexes in transfected CHO cells failed to support PC2 channel activity, suggesting that the role of PC1 is to activate G-protein signaling to regulate the PC1/PC2 calcium channel.