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Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic cause of end-stage renal disease, affecting approximately 1 in 1,000 people. The disease is characterized by the development of numerous large fluid-filled renal cysts over the course of decades. These cysts compress the surrounding renal parenchyma and impair its function. Mutations in two genes are responsible for ADPKD. The protein products of both of these genes, polycystin-1 and polycystin-2, localize to the primary cilium and participate in a wide variety of signaling pathways. Polycystin-1 undergoes several proteolytic cleavages that produce fragments which manifest biological activities. Recent results suggest that the production of polycystin-1 cleavage fragments is necessary and sufficient to account for at least some, although certainly not all, of the physiological functions of the parent protein. Keywords Autosomal dominant polycystic kidney disease * Transcription * Proliferation * Apoptosis * Wnt * CHOP * Proteolytic cleavage, Molecular pathogenesis of autosomal dominant polycystic kidney disease Autosomal dominant polycystic kidney disease (ADPKD) affects approximately 1 in 1,000 people and is the most common potentially lethal genetic disease [1-3]. [...]

Endogenous vasopressin regulates Na-K-ATPase and [Na.sup.+]-[K.sup.+]-[Cl.sup.-] cotransporter rbsc-1 in rat outer medulla. Am J Physiol Renal Physiol 282: F265-F270, 2002; 10.1152/ ajprenal.00354.2000.--Previous reports have shown a stimulatory effect of vasopressin (VP) on Na-K-ATPase and rBSC-1 expression and activity. Whether these VP-dependent mechanisms are operating in vivo in physiological conditions as well as in chronic renal failure (CRF) has been less well studied. We measured ATPase expression and activity and rBSC-1 expression in the outer medulla of controls and moderate CRF rats both before and under in vivo inhibition of VP by OPC-31260, a selective [V.sub.2]-receptor antagonist. OPC-31260 decreased Na-K-ATPase activity from 11.2 [+ or -] 1.5 to 3.7 [+ or -] 0.8 in controls (P < 0.05) and from 19.0 [+ or -] 0.8 to 2.9 [+ or -] 0.5 [micro]mol [P.sub.i] * mg [protein.sup.-1] * [h.sup.-1] in moderate CRF rats (P < 0.05). CRF was associated with a significant increase in Na-K-ATPase activity (P < 0.05). Similarly, CRF was also associated with a significant increase in Na-K-ATPase expression to 164.4 [+ or -] 21.5% compared with controls (P < 0.05), and OPC-31260 decreased Na-K-ATPase expression in both controls and CRF rats to 57.6 [+ or -] 9.5 and 105.3 [+ or -] 10.9%, respectively (P < 0.05). On the other hand, OPC-31260 decreased rBSC-I expression in both controls and CRF rats to 60.8 [+ or -] 6.5 and 30.0 [+ or -] 6.9%, respectively (P < 0.05), and was not influenced by CRF (95.7 [+ or -] 5.2%). We conclude that 1) endogenous VP modulated Na-K-ATPase and rBSC-1 in both controls and CRF; and 2) CRF was associated with increased activity and expression of the Na-K-ATPase in the outer medulla, in contrast to the unaltered expression of the rBSC-1. The data suggest that endogenous VP could participate in the regulation of electrolyte transport at the level of the outer medulla. vasopressin; rbsc-1; sodium-potassium-adenosine 5'-triphosphatase; chronic renal failure

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Regulation of the number of ion channels at the plasma membrane is a critical component of the physiological response. We recently demonstrated that the Ca2+ -activated K+ channel, KCa2.3 is rapidly endocytosed and enters a Rab35- and EPI64C-dependent recycling compartment. Herein, we addressed the early endocytic steps of KCa2.3 using a combination of fluorescence and biotinylation techniques. We demonstrate that KCa2.3 is localized to caveolin-rich domains of the plasma membrane using fluorescence co- localization, transmission electron microscopy and co-immunoprecipitation (co- IP). Further, in cells lacking caveolin-1, we observed an accumulation of KCa2.3 at the plasma membrane as well as a decreased rate of endocytosis, as assessed by biotinylation. We also demonstrate that KCa2.3 and dynamin II are co- localized following endocytosis as well as demonstrating they are associated by co-IP. Further, expression of K44A dynamin II resulted in a 2-fold increase in plasma membrane KCa2.3 as well as a 3-fold inhibition of endocytosis. Finally, we evaluated the role of Rab5 in the endocytosis of KCa2.3. We demonstrate that expression of a dominant active Rab5 (Q79L) results in the accumulation of newly endocytosed KCa2.3 on to the membrane of the Rab5-induced vacuoles. We confirmed this co-localization by co- IP; demonstrating that KCa2.3 and Rab5 are associated. As expected, if Rab5 is required for the endocytosis of KCa2.3, expression of a dominant negative Rab5 (S34N) resulted in an approximate 2-fold accumulation of KCa2.3 at the plasma membrane. This was confirmed by siRNA- mediated knockdown of Rab5. Expression of the dominant negative Rab5 also resulted in a decreased rate of KCa2.3 endocytosis. These results demonstrate that KCa2.3 is localized to a caveolin-rich domain within the plasma membrane and is endocytosed in a dynamin- and Rab5-dependent manner prior to entering the Rab35/EPI64C recycling compartment and returning to the plasma membrane. [ABSTRACT FROM AUTHOR]

Podocyte or endothelial cell VEGF-A knockout causes thrombotic microangiopathy in adult mice. To study the mechanism involved in acute and local injury caused by low podocyte VEGF-A we developed an inducible, podocyte-specific VEGF-A knockdown mouse, and we generated an immortalized podocyte cell line (VEGFKD) that downregulates VEGF-A upon doxycycline exposure. Tet-O-siVEGF:podocin-rtTA mice express VEGF shRNA in podocytes in a doxycycline-regulated manner, decreasing VEGF-A mRNA and VEGF-A protein levels in isolated glomeruli to ∼20% of non-induced controls and urine VEGFA to ∼30% of control values a week after doxycycline induction. Induced tet-O-siVEGF:podocin-rtTA mice developed acute renal failure and proteinuria, associated with mesangiolysis and microaneurisms. Glomerular ultrastructure revealed endothelial cell swelling, GBM lamination and podocyte effacement. VEGF knockdown decreased podocyte fibronectin and glomerular endothelial alphaVbeta3 integrin in vivo. VEGF receptor-2 (VEGFR2) interacts with beta3 integrin and neuropilin-1 in the kidney in vivo and in VEGFKD podocytes. Podocyte VEGF knockdown disrupts alphaVbeta3 integrin activation in glomeruli, detected by WOW1-Fab. VEGF silencing in cultured VEGFKD podocytes downregulates fibronectin and disrupts alphaVbeta3 integrin activation cell-autonomously. Collectively, these studies indicate that podocyte VEGF-A regulates alphaVbeta3 integrin signaling in the glomerulus, and that podocyte VEGF knockdown disrupts alphaVbeta3 integrin activity via decreased VEGFR2 signaling, thereby damaging the three layers of the glomerular filtration barrier, causing proteinuria and acute renal failure. [ABSTRACT FROM AUTHOR]

The present work was designed to study Na+ K+ ATPase α1-subunit phosphorylation in rats with chronic renal failure (CRF) in comparison with normal rats. Na+ K+ ATPase α1-subunit phosphorylation degree was measured by binding the McK-1 antibody to dephosphorylated Ser-23 in microdissected medullary thick ascending limb of Henle (mTAL) segments. In addition, the total Na+ K+ ATPase α1-subunit expression and activity were also measured in the outer renal medulla homogenates and membranes. CRF rats showed a higher Na+ K+ ATPase activity, as compared with control rats (18.95 ± 2.4 vs. 11.21 ± 1.5 μmol Pi/mg prot/h, p < 0.05), accompanied by a higher total Na+ K+ ATPase expression (0.54 ± 0.04 vs. 0.27 ± 0.02 normalized arbitrary units (NU), p < 0.05). When McK-1 antibody was used, a higher immunosignal in mTAL of CRF rats was observed, as compared with controls (6.3 ± 0.35 vs.4.1 ± 0.33 NU, p < 0.05). The ratio Na+ K+ ATPase α1-subunit phosphorylation / total Na+ K+ ATPase α1-subunit expression per μg protein showed a non-significant difference between CRF and control rats in microdissected mTAL segments (2.11 ± 0.12 vs.2.26 ± 0.18 NU, p = NS). The PKC inhibitor RO-318220 10-6M increased immunosignal (lower phosphorylation degree) in mTAL of CRF rats to 128.43 ± 7.08% (p < 0.05) but did not alter McK1 binding in control rats. Both phorbol 12-myristate 13-acetate (PMA) 10-6M and dopamine 10-6M decreased immunosignal in CRF rats, corresponding to a higher Na+ K+ ATPase α1-subunit phosphorylation degree at Ser-23 (55.26 ± 11.17% and 53.27 ± 7.12% compared with basal, p < 0.05). In mTAL of CRF rats, the calcineurin inhibitor FK-506 10-6M did not modify phosphorylation degree at Ser-23 of Na+ K+ ATPase α1-subunit (100.21 ± 3.00% compared with basal CRF). In control rats, FK 506 10-6M decreased the immunosignal, which corresponds to a higher Na+ K+ ATPase α1-subunit phosphorylation degree at Ser-23. The data suggest that the regulation of basal Na+ K+ ATPase α1-subunit phosphorylation degree at Ser-23 in mTAL segments of CRF rats was primarily dependent on PKC activation rather than calcineurin dependent mechanisms. [ABSTRACT FROM AUTHOR]

Sodium transport correlates with varying Na+-K+-ATPase activity rates along the nephron. Whether differences in Na+-K+-ATPase regulation by protein kinase C-dependent phosphorylation are also present has not been tested. We measured the degree of Na+-K+-ATPase α1 subunit phosphorylation by the binding of McK-1 antibody to dephosphorylated Ser-23 and Na+-K+-ATPase activity in medullary thick ascending limb of Henle (mTAL) and proximal tubules (PCT). The degree of Na+-K+-ATPase phosphorylation at Ser-23 was lower in mTAL than in PCT (DU 13.43±1.99 versus 2.3±0.20, respectively, P<0.01) while Na+-K+-ATPase activity was higher in mTAL (3,402±83 vs 711±158 pmol/mm tubule per hour in PCT, P<0.01). PKC inhibitor RO-318220 10-6 M decreased phosphorylation in PCT to 125±10% (P<0.05). In mTAL, RO-318220 did not modify the phosphorylation degree or the activity of Na+-K+-ATPase. Both calcineurin inhibitor FK-506 10-6 M and phorbol 12-myristate 13-acetate (PMA) 10-6 M increased the degree of Na+-K+-ATPase phosphorylation (P<0.05) and inhibited Na+-K+-ATPase activity to 657±152 and 1,448±347 pmol/mm tubule per hour, respectively, in mTAL (P<0.01). Increase in [Na+]i to 30, 50 and 70 mM resulted in no changes in Na+-K+-ATPase phosphorylation degree or activity in mTAL. Conversely, in PCT increments in [Na+]i were paralleled by decreased phosphorylation (from 120±7 to 160±15% of controls, P<0.05) and increased Na+-K+-ATPase activity (from 850±139 to 1,874±203 pmol/mm tubule per hour, P<0.01). Dopamine (DA) 10-6 M decreased both Na+-K+-ATPase dephosphorylation to 41.85±9.58% (P<0.05) and Na+-K+-ATPase activity to 2,405±176 pmol/mm tubule per hour in mTAL (P<0.01). RO-318220 reversed DA effects. Data suggest that regulation of the degree of Na+-K+-ATPase α1 subunit phosphorylation at Ser-23 and enzyme activity have different mechanisms in mTAL than in PCT, and may help us to understand the physiological heterogeneity of both segments. [ABSTRACT FROM AUTHOR]

Measures ATPase expression and activity of rBSC-1 expression in the outer medulla of controls and moderate chronic renal failure (CRF) rats both before and after in vivo inhibition of vasopressin. Ability of endogenous vasopressin to modulate Na-K-ATPase and rBSC-1; Connection between CRF and increased activity and expression of the Na-K-ATPase in the outer medulla.

The small conductance calcium-activated potassium channel (KCa2.3) has long been recognized for its role in mediating vasorelaxation through the endothelium-derived hyperpolarization (EDH) response. Histone deacetylases (HDACs) have been implicated as potential modulators of blood pressure and histone deacetylase inhibitors (HDACi) are being explored as therapeutics for hypertension. Herein, we show that HDACi increase KCa2.3 expression when heterologously expressed in HEK cells and endogenously expressed in primary cultures of human umbilical vein endothelial cells (HUVECs) and human intestinal microvascular endothelial cells (HIMECs). When primary endothelial cells were exposed to HDACi, KCa2.3 transcripts, subunits, and functional current are increased. Quantitative RT-PCR (qPCR) demonstrated increased KCa2.3 mRNA following HDACi, confirming transcriptional regulation of KCa2.3 by HDACs. By using pharmacological agents selective for different classes of HDACs, we discriminated between cytoplasmic and epigenetic modulation of KCa2.3. Biochemical analysis revealed an association between the cytoplasmic HDAC6 and KCa2.3 in immunoprecipitation studies. Specifically inhibiting HDAC6 increases expression of KCa2.3. In addition to increasing the expression of KCa2.3, we show that nonspecific inhibition of HDACs causes an increase in the expression of the molecular chaperone Hsp70 in endothelial cells. When Hsp70 is inhibited in the presence of HDACi, the magnitude of the increase in KCa2.3 expression is diminished. Finally, we show a slower rate of endocytosis of KCa2.3 as a result of exposure of primary endothelial cells to HDACi. These data provide the first demonstrated approach to increase KCa2.3 channel number in endothelial cells and may partially account for the mechanism by which HDACi induce vasorelaxation.

Autosomal dominant polycystic kidney disease (ADPKD), a most common genetic cause of chronic renal failure, is characterized by the progressive development and enlargement of cysts in kidneys and other organs. The cystogenic process is highly complex and involves a high proliferative rate, increased apoptosis, altered protein sorting, changed secretory characteristics, and disorganization of the extracellular matrix. ADPKD is caused by mutations in the genes encoding polycystin-1 (PC-1) or polycystin-2 (PC-2). PC-1 undergoes multiple cleavages that intervene in several signaling pathways involved in cellular proliferation and differentiation mechanisms. One of these cleavages releases the cytoplasmic C-terminal tail of PC-1. In addition, the C-terminal cytoplasmic tails of PC-1 and PC-2 interact in vitro and in vivo. The purpose of this review is to summarize recent literature that suggests that PC-1 and PC-2 may function through a common signaling pathway necessary for normal tubulogenesis. We hope that a better understanding of PC-1 and PC-2 protein function will lead to progress in diagnosis and treatment for ADPKD.