Histone deacetylase inhibitors (HDACi) increase expression of KCa2.3 (SK3) in primary microvascular endothelial cells.

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.

Plasma membrane insertion of KCa2.3 (SK3) is dependent upon the SNARE proteins, syntaxin-4 and SNAP23.

We previously demonstrated endocytosis of KCa2.3 is caveolin-1-, dynamin II- and Rab5-dependent. KCa2.3 then enters Rab35/EPI64C- and RME-1-containing recycling endosomes and is returned to the plasma membrane (PM). Herein, we report on the mechanism by which KCa2.3 is inserted into the PM during recycling and following exit from the Golgi. We demonstrate KCa2.3 colocalizes with SNAP-23 and Syntaxin-4 in the PM of HEK and endothelial cells by confocal immunofluorescence microscopy. We further show KCa2.3 can be co-immunoprecipitated with SNAP-23 and Syntaxin-4. Overexpression of either Syntaxin-4 or SNAP-23 increased PM expression of KCa2.3, whereas shRNA-mediated knockdown of these SNARE proteins significantly decreased PM KCa2.3 expression, as assessed by cell surface biotinylation. Whole-cell patch clamp studies confirmed knockdown of SNAP-23 significantly decreased the apamin sensitive, KCa2.3 current. Using standard biotinylation/stripping methods, we demonstrate shRNA mediated knockdown of SNAP-23 inhibits recycling of KCa2.3 following endocytosis, whereas scrambled shRNA had no effect. Finally, using biotin ligase acceptor peptide (BLAP)-tagged KCa2.3, coupled with ER-resident biotin ligase (BirA), channels could be biotinylated in the ER after which we evaluated their rate of insertion into the PM following Golgi exit. We demonstrate knockdown of SNAP-23 significantly slows the rate of Golgi to PM delivery of KCa2.3. The inhibition of both recycling and PM delivery of newly synthesized KCa2.3 channels likely accounts for the decreased PM expression observed following knockdown of these SNARE proteins. In total, our results suggest insertion of KCa2.3 into the PM depends upon the SNARE proteins, Syntaxin-4 and SNAP-23.

Drug Authorization for Sofosbuvir/Ledipasvir (Harvoni) for Chronic HCV Infection in a Real-World Cohort: A New Barrier in the HCV Care Cascade.

BACKGROUND: New treatments for hepatitis C (HCV) infection hold great promise for cure, but numerous challenges to diagnosing, establishing care, and receiving therapy exist. There are limited data on insurance authorization for these medications. MATERIALS AND METHODS: We performed a retrospective chart review of patients receiving sofosbuvir/ledipasvir (SOF/LED) from October 11-December 31, 2014 to determine rates and timing of drug authorization. We also determined predictors of approval, and those factors associated with faster decision and approval times. RESULTS: Of 174 patients prescribed HCV therapy during this period, 129 requests were made for SOF/LED, of whom 100 (77.5%) received initial approval, and an additional 17 patients (13.9%) ultimately received approval through the appeals process. Faster approval times were seen in patients with Child-Pugh Class B disease (14.4 vs. 24.7 days, p = 0.048). A higher proportion of patients were initially approved in those with Medicare/Medicaid coverage (92.2% vs. 71.4%, p = 0.002) and those with baseline viral load ≥ 6 million IU/mL (84.1% vs. 62.5%, p = 0.040). Linear regression modeling identified advanced fibrosis, high Model of End Stage Liver Disease (MELD) score, and female gender as significant predictors of shorter decision and approval times. On logistic regression, Medicare/Medicaid coverage (OR 5.96, 95% CI 1.66-21.48) and high viral load (OR 4.52, 95% CI 1.08-19.08) were significant predictors for initial approval. CONCLUSIONS: Early analysis of real-world drug authorization outcomes between October-December 2014 reveals that nearly one in four patients are initially denied access to SOF/LED upon initial prescription, although most patients are eventually approved through appeal, which delays treatment initiation. Having Medicare/Medicaid and advanced liver disease resulted in a higher likelihood of approval as well as earlier decision and approval times. More studies are needed to determine factors resulting in higher likelihood of denial and to evaluate approval rates and times after implementation of restrictive prior authorization guidelines.

Anterograde trafficking of KCa3.1 in polarized epithelia is Rab1- and Rab8-dependent and recycling endosome-independent.

The intermediate conductance, Ca2+-activated K+ channel (KCa3.1) targets to the basolateral (BL) membrane in polarized epithelia where it plays a key role in transepithelial ion transport. However, there are no studies defining the anterograde and retrograde trafficking of KCa3.1 in polarized epithelia. Herein, we utilize Biotin Ligase Acceptor Peptide (BLAP)-tagged KCa3.1 to address these trafficking steps in polarized epithelia, using MDCK, Caco-2 and FRT cells. We demonstrate that KCa3.1 is exclusively targeted to the BL membrane in these cells when grown on filter supports. Following endocytosis, KCa3.1 degradation is prevented by inhibition of lysosomal/proteosomal pathways. Further, the ubiquitylation of KCa3.1 is increased following endocytosis from the BL membrane and PR-619, a deubiquitylase inhibitor, prevents degradation, indicating KCa3.1 is targeted for degradation by ubiquitylation. We demonstrate that KCa3.1 is targeted to the BL membrane in polarized LLC-PK1 cells which lack the µ1B subunit of the AP-1 complex, indicating BL targeting of KCa3.1 is independent of µ1B. As Rabs 1, 2, 6 and 8 play roles in ER/Golgi exit and trafficking of proteins to the BL membrane, we evaluated the role of these Rabs in the trafficking of KCa3.1. In the presence of dominant negative Rab1 or Rab8, KCa3.1 cell surface expression was significantly reduced, whereas Rabs 2 and 6 had no effect. We also co-immunoprecipitated KCa3.1 with both Rab1 and Rab8. These results suggest these Rabs are necessary for the anterograde trafficking of KCa3.1. Finally, we determined whether KCa3.1 traffics directly to the BL membrane or through recycling endosomes in MDCK cells. For these studies, we used either recycling endosome ablation or dominant negative RME-1 constructs and determined that KCa3.1 is trafficked directly to the BL membrane rather than via recycling endosomes. These results are the first to describe the anterograde and retrograde trafficking of KCa3.1 in polarized epithelia cells.

Polycystin-1 cleavage and the regulation of transcriptional pathways.

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.

Polycystin-1C terminus cleavage and its relation with polycystin-2, two proteins involved in polycystic kidney disease.

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.

Polycystin-1 C terminus cleavage and its relation with polycystin-2, two proteins involved in polycystic kidney disease / Clivaje del C-terminal de policistina-1 y su relación con policistina-2, dos proteínas involucradas en la poliquistosis renal

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.(AU)

La poliquistosis renal autosómica dominante (ADPKD por sus siglas en inglés) es una causa genética muy común de falla renal crónica que se caracteriza por el progresivo desarrollo y agrandamiento de quistes en los riñones y en otros órganos. El proceso de cistogénesis comprende incrementos en la proliferación y muerte celular por apoptosis, así como alteraciones en la distribución intracelular de proteínas, el movimiento transcelular de solutos y organización de la matriz extracelular. ADPKD es causada por mutaciones en los genes que codifican para policistina-1 (PC-1) o policistina-2 (PC-2). PC-1 puede sufrir múltiples clivajes y los fragmentos generados intervienen en diferentes cascadas de señalización involucradas en mecanismos de proliferación y diferenciación celular. Uno de estos clivajes libera el extremo C-terminal citoplasmático de la PC-1. Se ha demostrado que los extremos C-terminal citoplasmático de PC-1 y PC-2 pueden interactuar tanto in vitro como in vivo. El propósito de esta revisión es resumir la literatura más reciente que sugiere que PC-1 y PC-2 pueden funcionar a través de una cascada de señalización común necesaria para la tubulogénesis normal. Creemos que una mejor comprensión de los mecanismos moleculares de acción de PC-1 y PC-2 contribuirán al progreso en el diagnóstico y tratamiento de ADPKD.(AU)

Polycystin-1 C terminus cleavage and its relation with polycystin-2, two proteins involved in polycystic kidney disease / Clivaje del C-terminal de policistina-1 y su relación con policistina-2, dos proteínas involucradas en la poliquistosis renal

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.

La poliquistosis renal autosómica dominante (ADPKD por sus siglas en inglés) es una causa genética muy común de falla renal crónica que se caracteriza por el progresivo desarrollo y agrandamiento de quistes en los riñones y en otros órganos. El proceso de cistogénesis comprende incrementos en la proliferación y muerte celular por apoptosis, así como alteraciones en la distribución intracelular de proteínas, el movimiento transcelular de solutos y organización de la matriz extracelular. ADPKD es causada por mutaciones en los genes que codifican para policistina-1 (PC-1) o policistina-2 (PC-2). PC-1 puede sufrir múltiples clivajes y los fragmentos generados intervienen en diferentes cascadas de señalización involucradas en mecanismos de proliferación y diferenciación celular. Uno de estos clivajes libera el extremo C-terminal citoplasmático de la PC-1. Se ha demostrado que los extremos C-terminal citoplasmático de PC-1 y PC-2 pueden interactuar tanto in vitro como in vivo. El propósito de esta revisión es resumir la literatura más reciente que sugiere que PC-1 y PC-2 pueden funcionar a través de una cascada de señalización común necesaria para la tubulogénesis normal. Creemos que una mejor comprensión de los mecanismos moleculares de acción de PC-1 y PC-2 contribuirán al progreso en el diagnóstico y tratamiento de ADPKD.

Polycystin-1C terminus cleavage and its relation with polycystin-2, two proteins involved in polycystic kidney disease. / Polycystin-1C terminus cleavage and its relation with polycystin-2, two proteins involved in polycystic kidney disease.

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.

Dynamin- and Rab5-dependent endocytosis of a Ca2+ -activated K+ channel, KCa2.3.

Regulation of the number of ion channels at the plasma membrane is a critical component of the physiological response. We recently demonstrated that the Ca(2+)-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.

Acute podocyte vascular endothelial growth factor (VEGF-A) knockdown disrupts alphaVbeta3 integrin signaling in the glomerulus.

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 (VEGF(KD)) 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 VEGF-A 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 alpha(V)beta(3) integrin in vivo. VEGF receptor-2 (VEGFR2) interacts with beta(3) integrin and neuropilin-1 in the kidney in vivo and in VEGF(KD) podocytes. Podocyte VEGF knockdown disrupts alpha(V)beta(3) integrin activation in glomeruli, detected by WOW1-Fab. VEGF silencing in cultured VEGF(KD) podocytes downregulates fibronectin and disrupts alpha(V)beta(3) integrin activation cell-autonomously. Collectively, these studies indicate that podocyte VEGF-A regulates alpha(V)beta(3) integrin signaling in the glomerulus, and that podocyte VEGF knockdown disrupts alpha(V)beta(3) integrin activity via decreased VEGFR2 signaling, thereby damaging the three layers of the glomerular filtration barrier, causing proteinuria and acute renal failure.

Vascular endothelial growth factor receptor 2 direct interaction with nephrin links VEGF-A signals to actin in kidney podocytes.

The transmembrane protein nephrin is an essential component of slit diaphragms, the specialized cell junctions that link podocyte foot processes. Podocytes are epithelial cells that surround the glomerular capillaries in the kidney and are necessary for the organ-filtering function. Nephrin signaling complex transduces extracellular cues to the podocyte cytoskeleton and regulates podocyte shape and function. Vascular endothelial growth factor A (VEGF-A) is a required growth factor produced and secreted by podocytes. Accumulating evidence suggests a cross-talk between VEGF-A and nephrin signaling pathways. We previously showed that in vivo nephrin associates with VEGF receptor-2 (VEGFR2), the signaling receptor for VEGF-A. In the present work, we characterized the interaction between nephrin and VEGFR2 in cultured cells and in vitro. We demonstrate that nephrin-VEGFR2 interaction is direct using mass spectrometry, immunoprecipitation, GST-binding assays, and blot overlay experiments. This interaction occurs through VEGFR2 and nephrin cytoplasmic domains. Nephrin-VEGFR2 interaction is modulated by tyrosine phosphorylation of both cytoplasmic domains. Furthermore, the nephrin-VEGFR2 complex involves Nck and actin. VEGF-A signaling via this complex results in decreased cell size. We provide evidence that this multiprotein interaction occurs in cultured podocytes. We propose that the nephrin-VEGFR2 complex acts as a key mediator to transduce local VEGF-A signals to the podocyte actin cytoskeleton, regulating the foot process structure and glomerular filter integrity.

Relevance of VEGF and nephrin expression in glomerular diseases.

The glomerular filtration barrier is affected in a large number of acquired and inherited diseases resulting in extensive leakage of plasma albumin and larger proteins, leading to nephrotic syndrome and end-stage renal disease. Unfortunately, the molecular mechanisms governing the development of the nephrotic syndrome remain poorly understood. Here, I give an overview of recent investigations that have focused on characterizing the interrelationships between the slit diaphragm components and podocytes-secreted VEGF, which have a significant role for maintaining the normal podocyte structure and the integrity of the filtering barrier.

Induction of podocyte VEGF164 overexpression at different stages of development causes congenital nephrosis or steroid-resistant nephrotic syndrome.

The tight regulation of vascular endothelial growth factor-A (VEGF-A) signaling is required for both the development and maintenance of the glomerular filtration barrier, but the pathogenic role of excessive amounts of VEGF-A detected in multiple renal diseases remains poorly defined. We generated inducible transgenic mice that overexpress podocyte VEGF164 at any chosen stage of development. In this study, we report the phenotypes that result from podocyte VEGF164 excess during organogenesis and after birth. On doxycycline induction, podocin-rtTA:tet-O-VEGF164 mice express twofold higher kidney VEGF164 levels than single transgenic mice, localized to podocytes. Podocyte VEGF164 overexpression during organogenesis resulted in albuminuria at birth and was associated with glomerulomegaly, uniform podocyte effacement, very few and wide foot processes joined by occluding junctions, almost complete absence of slit diaphragms, and swollen endothelial cells with few fenestrae as revealed by transmission electron microscopy. Podocyte VEGF164 overexpression after birth caused massive albuminuria in 70% of 2-week-old mice, glomerulomegaly, and minimal changes on light microscopy. Transmission electron microscopy showed podocyte effacement and fusion and morphologically normal endothelial cells. Podocyte VEGF164 overexpression induced nephrin down-regulation without podocyte loss. VEGF164-induced abnormalities were reversible on removal of doxycycline and were unresponsive to methylprednisolone. Collectively, the data suggest that moderate podocyte VEGF164 overexpression during organogenesis results in congenital nephrotic syndrome, whereas VEGF164 overexpression after birth induces a steroid-resistant minimal change like-disease in mice.

Overexpression of VEGF-A in podocytes of adult mice causes glomerular disease.

We sought to examine the pathogenic role of excessive VEGF-A expression in podocytes, since it has been reported that diabetic nephropathy and other glomerular diseases are associated with increased VEGF-A expression. The induction of podocyte-specific VEGF164 overexpression in adult transgenic mice led to proteinuria, glomerulomegaly, glomerular basement membrane thickening, mesangial expansion, loss of slit diaphragms, and podocyte effacement. When doxycycline-mediated VEGF164 was stopped, these abnormalities reversed. These findings were associated with reversible downregulation of metalloproteinase 9 and nephrin expression. Using transmission electron microscopy, we established that VEGF-A receptor-2 (VEGFR2) was expressed in podocytes and glomerular endothelial cells. We also found that VEGF164 induced VEGFR2 phosphorylation in podocytes. Further, we were able to co-immunoprecipitate VEGFR2 and nephrin using whole kidney lysates, confirming interaction in vivo. This implies that autocrine and paracrine VEGF-A signaling through VEGFR2 occurs in podocytes and may mediate the glomerular phenotype caused by VEGF164 overexpression. Thus, we suggest that podocyte VEGF164 overexpression in adult mice is sufficient to induce glomerular filtration barrier structural and functional abnormalities similar to those present in murine diabetic nephropathy.

Polycystin-1 C-terminal cleavage is modulated by polycystin-2 expression.

Autosomal dominant polycystic kidney disease is caused by mutations in the genes encoding polycystin-1 (PC-1) and polycystin-2 (PC-2). PC-1 cleavage releases its cytoplasmic C-terminal tail (CTT), which enters the nucleus. To determine whether PC-1 CTT cleavage is influenced by PC-2, a quantitative cleavage assay was utilized, in which the DNA binding and activation domains of Gal4 and VP16, respectively, were appended to PC-1 downstream of its CTT domain (PKDgalvp). Cells cotransfected with the resultant PKDgalvp fusion protein and PC-2 showed an increase in luciferase activity and in CTT expression, indicating that the C-terminal tail of PC-1 is cleaved and enters the nucleus. To assess whether CTT cleavage depends upon Ca2+ signaling, cells transfected with PKDgalvp alone or together with PC-2 were incubated with several agents that alter intracellular Ca2+ concentrations. PC-2 enhancement of luciferase activity was not altered by any of these treatments. Using a series of PC-2 C-terminal truncated mutations, we identified a portion of the PC-2 protein that is required to stimulate PC-1 CTT accumulation. These data demonstrate that release of the CTT from PC-1 is influenced and stabilized by PC-2. This effect is independent of Ca2+ but is regulated by sequences contained within the PC-2 C-terminal tail, suggesting a mechanism through which PC-1 and PC-2 may modulate a novel signaling pathway.

Mechanisms of PKC-dependent Na+ K+ ATPase phosphorylation in the rat kidney with chronic renal failure.

The present work was designed to study Na+ K+ ATPase alpha1-subunit phosphorylation in rats with chronic renal failure (CRF) in comparison with normal rats. Na+ K+ ATPase alpha1-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 alpha1-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 micromol 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 alpha1-subunit phosphorylation/total Na+ K+ ATPase alpha1-subunit expression per microg 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(-6) M 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(-6) M and dopamine 10(-6) M decreased immunosignal in CRF rats, corresponding to a higher Na+ K+ ATPase alpha1-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(-6) M did not modify phosphorylation degree at Ser-23 of Na+ K+ ATPase alpha1-subunit (100.21 +/- 3.00% compared with basal CRF). In control rats, FK 506 10(-6) M decreased the immunosignal, which corresponds to a higher Na+ K+ ATPase alpha1-subunit phosphorylation degree at Ser-23. The data suggest that the regulation of basal Na+ K+ ATPase alpha1-subunit phosphorylation degree at Ser-23 in mTAL segments of CRF rats was primarily dependent on PKC activation rather than calcineurin dependent mechanisms.

Mechanisms of Na+-K+-ATPase phosphorylation by PKC in the medullary thick ascending limb of Henle in the rat.

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 alpha1 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 alpha1 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.

Endogenous vasopressin regulates Na-K-ATPase and Na(+)-K(+)-Cl(-) cotransporter rbsc-1 in rat outer medulla.

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(2)-receptor antagonist. OPC-31260 decreased Na-K-ATPase activity from 11.2 +/- 1.5 to 3.7 +/- 0.8 in controls (P < 0.05) and from 19.0 +/- 0.8 to 2.9 +/- 0.5 micromol P(i). mg protein(-1) x h(-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 +/- 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 +/- 9.5 and 105.3 +/- 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 +/- 6.5 and 30.0 +/- 6.9%, respectively (P < 0.05), and was not influenced by CRF (95.7 +/- 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.

Cellular adaptation of the rat medullary thick ascending limb to chronic renal failure

Chronic renal failure (CRF) is accompanied by adaptive changes in renal and extrarrenal epithelial ionic transport. Fluid reabsorption in the thick ascending limb of Henle is increased and the capacity to lower the urine osmolality in water diuresis is preserved. To study the cellular mechanism of this adaptation, we measured intracellular cAMP in microdissected medullary thick ascending limb (mTAL) segments in rats with CRF. mTAL exhibited in CRF nephrons an increase of basal cAMP from 6.0 +/- 1.5 in controls to 47.0 + 10.3 fmol. mm-1 tubule in CRF (P < 0.05). Maximally stimulated cAMP levels (10(-3) M IBMX plus 10(-5) M Forskolin) were different from basal levels in controls (6.0 + 1.5 vs 63.1 +/- 18.8, P < 0.05) but not from basal levels in CRF (47.0 +/- 10.3 vs 63.0 +/- 16.0, P = N.S.). Preincubation with the adenylate cyclase inhibitor 2'5' -dideoxyadenosine (DDA) 10(-4) M produced no changes in cAMP in controls (93.7 +/- 10.3 percent of DDA untreated samples) whereas it decreased to 76.2 +/- 8.8 percent (24 percent inhibition) in CRF (P < 0.05). No differences between controls and CRF groups were found in basal and stimulated cAMP in red blood cells and distal colon. The data would suggest that the cAMP pathway is an intracellular signal for mTAL adaptation in epithelial transport and that the adenylate-cyclase system is specifically activated in CRF.