Membrane Of Renal Cells Research Articles

SGK1/Nedd4-2 signaling pathway regulates the activity of human organic anion transporters 3.

Human organic anion transporter 3 (hOAT3) is localized at the basolateral membrane of renal proximal tubule cells and facilitates the renal secretion of numerous clinical drugs, including anti-HIV therapeutics, anti-tumor drugs, antibiotics, antihypertension drugs and anti-inflammatories. The present study explored the role of serum and glucocorticoid-inducible kinase 1 (sgk1) in the regulation of hOAT3. It was shown that over-expression of sgk1 in hOAT3-expressing cells stimulated hOAT3 transport activity by enhancing the transporter expression at the plasma membrane, kinetically reflected as an increased maximal transport velocity Vmax without substantial change in the substrate-binding affinity Km . In contrast, treatment of cells with the sgk-specific inhibitor GSK650394 resulted in a dose-dependent inhibition of hOAT3 transport activity. Evidence was further provided that sgk1 regulation of hOAT3 activity was mediated by ubiquitin ligase Nedd4-2, an enzyme previously shown to have an inhibitory effect on hOAT3. It was shown that sgk1 phosphorylated Nedd4-2, weakened the association between Nedd4-2 and hOAT3, and decreased hOAT3 ubiquitination. Functionally, the sgk1-stimulated hOAT3 transport activity was attenuated in the presence of a ligase-dead mutant of Nedd4-2. In summary, the investigation established for the first time that sgk1 stimulates hOAT3 transport activity by interfering with the inhibitory effect of Nedd4-2 on the transporter.

Biophysical characterization of layer-by-layer synthesis of aptamer-drug microparticles for enhanced cell targeting.

Targeted delivery of drug molecules to specific cells in mammalian systems demonstrates a great potential to enhance the efficacy of current pharmaceutical therapies. Conventional strategies for pharmaceutical delivery are often associated with poor therapeutic indices and high systemic cytotoxicity, and this result in poor disease suppression, low surviving rates, and potential contraindication of drug formulation. The emergence of aptamers has elicited new research interests into enhanced targeted drug delivery due to their unique characteristics as targeting elements. Aptamers can be engineered to bind to their cognate cellular targets with high affinity and specificity, and this is important to navigate active drug molecules and deliver sufficient dosage to targeted malignant cells. However, the targeting performance of aptamers can be impacted by several factors including endonuclease-mediated degradation, rapid renal filtration, biochemical complexation, and cell membrane electrostatic repulsion. This has subsequently led to the development of smart aptamer-immobilized biopolymer systems as delivery vehicles for controlled and sustained drug release to specific cells at effective therapeutic dosage and minimal systemic cytotoxicity. This article reports the synthesis and in vitro characterization of a novel multi-layer co-polymeric targeted drug delivery system based on drug-loaded PLGA-Aptamer-PEI (DPAP) formulation with a stage-wise delivery mechanism. A thrombin-specific DNA aptamer was used to develop the DPAP system while Bovine Serum Albumin (BSA) was used as a biopharmaceutical drug in the synthesis process by ultrasonication. Biophysical characterization of the DPAP system showed a spherical shaped particulate formulation with a unimodal particle size distribution of average size ∼0.685 µm and a zeta potential of +0.82 mV. The DPAP formulation showed a high encapsulation efficiency of 89.4 ± 3.6%, a loading capacity of 17.89 ± 0.72 mg BSA protein/100 mg PLGA polymeric particles, low cytotoxicity and a controlled drug release characteristics in 43 days. The results demonstrate a great promise in the development of DPAP formulation for enhanced in vivo cell targeting. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:249-261, 2018.

MRP-1 and BCRP Promote the Externalization of Phosphatidylserine in Oxalate-treated Renal Epithelial Cells: Implications for Calcium Oxalate Urolithiasis

To investigate the possible involvement of multidrug resistance-associated protein 1 (MRP-1) and breast cancer resistance protein (BCRP) in the oxalate-induced redistribution of phosphatidylserine (PS) in renal epithelial cell membranes. A western blot analysis was used to examine the MRP-1 and BCRP expression levels. Surface-expressed PS was detected by the annexin V-binding assay. The cell-permeable fluorogenic probe 2,7-dichlorofluorescein diacetate was used to measure the intracellular reactive oxygen species (ROS) level. A rat model of hyperoxaluria was obtained using 0.5% ethylene glycol and 1.0% ammonium chloride. In addition, certain animals received verapamil (50 mg/kg body weight), which is a common inhibitor of MRP-1 and BCRP. The degree of nephrolithiasis was assessed histomorphometrically using sections stained by Pizzolato method and by measuring the calcium oxalate crystal content in the renal tissue. Oxalate produced a concentration-dependent increase in the synthesis of MRP-1 and BCRP. Treatment with MK571 and Ko143 (MRP-1- and BCRP-specific inhibitors, respectively) significantly attenuated the oxalate-induced PS externalization. Adding the antioxidant N-acetyl-l-cysteine significantly reduced MRP-1 and BCRP expression. In vivo, markedly decreased nephrocalcinosis was observed compared with that in the rat model of hyperoxaluria without verapamil treatment. Oxalate induces the upregulation of MRP-1 and BCRP, which act as phospholipid floppases causing PS externalization in the renal epithelial cell membrane. The process is mediated by intracellular ROS production. The ROS-mediated increase in the synthesis of MRP-1 and BCRP can play an important role in hyperoxaluria-promoted calcium oxalate urolithiasis by facilitating phosphatidylserine redistribution in renal epithelial cells.

Dopamine regulates renal osmoregulation during hyposaline stress via DRD1 in the spotted scat (Scatophagus argus).

Dopamine is an important regulator of renal natriuresis and is critical for the adaptation of many animals to changing environmental salinity. However, the molecular mechanisms through which dopamine promotes this adaptation remain poorly understood. We studied the effects of dopamine on renal hypo-osmoregulation in the euryhaline fish Scatophagus argus (S. argus) during abrupt transfer from seawater (SW) to freshwater (FW). Following the transfer, serum dopamine concentration was decreased, and dopamine activated expression of the dopamine receptor 1 (designated SaDRD1) in the kidney, triggering the osmoregulatory signaling cascade. SaDRD1 protein is expressed in the renal proximal tubule cells in vivo, and is localized to the cell membrane of renal primary cells in vitro. Knockdown of SaDRD1 mRNA by siRNA significantly increased Na+/K+-ATPase (NKA) activity in cultured renal primary cells in vitro, suggesting that expression of SaDRD1 may oppose the activity of NKA. We demonstrate that exogenous dopamine enhances the response of NKA to hyposaline stress after transferring primary renal cells from isosmotic medium to hypoosmotic medium. Our results indicate that dopamine regulation via SaDRD1 ignited the renal dopaminergic system to balance the osmotic pressure through inhibiting NKA activity, providing a new perspective on the hyposaline adaptation of fish.

Role of Oxidative Stress in Drug-Induced Kidney Injury.

The kidney plays a primary role in maintaining homeostasis and detoxification of numerous hydrophilic xenobiotics as well as endogenous compounds. Because the kidney is exposed to a larger proportion and higher concentration of drugs and toxins than other organs through the secretion of ionic drugs by tubular organic ion transporters across the luminal membranes of renal tubular epithelial cells, and through the reabsorption of filtered toxins into the lumen of the tubule, these cells are at greater risk for injury. In fact, drug-induced kidney injury is a serious problem in clinical practice and accounts for roughly 20% of cases of acute kidney injury (AKI) among hospitalized patients. Therefore, its early detection is becoming more important. Usually, drug-induced AKI consists of two patterns of renal injury: acute tubular necrosis (ATN) and acute interstitial nephritis (AIN). Whereas AIN develops from medications that incite an allergic reaction, ATN develops from direct toxicity on tubular epithelial cells. Among several cellular mechanisms underlying ATN, oxidative stress plays an important role in progression to ATN by activation of inflammatory response via proinflammatory cytokine release and inflammatory cell accumulation in tissues. This review provides an overview of drugs associated with AKI, the role of oxidative stress in drug-induced AKI, and a biomarker for drug-induced AKI focusing on oxidative stress.

Alpha-enolase on apical surface of renal tubular epithelial cells serves as a calcium oxalate crystal receptor.

To search for a strategy to prevent kidney stone formation/recurrence, this study addressed the role of α-enolase on apical membrane of renal tubular cells in mediating calcium oxalate monohydrate (COM) crystal adhesion. Its presence on apical membrane and in COM crystal-bound fraction was confirmed by Western blotting and immunofluorescence staining. Pretreating MDCK cells with anti-α-enolase antibody, not isotype-controlled IgG, dramatically reduced cell-crystal adhesion. Immunofluorescence staining also confirmed the direct binding of purified α-enolase to COM crystals at {121} > {100} > {010} crystal faces. Coating COM crystals with urinary proteins diminished the crystal binding capacity to cells and purified α-enolase. Moreover, α-enolase selectively bound to COM, not other crystals. Chemico-protein interactions analysis revealed that α-enolase interacted directly with Ca2+ and Mg2+. Incubating the cells with Mg2+ prior to cell-crystal adhesion assay significantly reduced crystal binding on the cell surface, whereas preincubation with EDTA, a divalent cation chelator, completely abolished Mg2+ effect, indicating that COM and Mg2+ competitively bind to α-enolase. Taken together, we successfully confirmed the role of α-enolase as a COM crystal receptor to mediate COM crystal adhesion at apical membrane of renal tubular cells. It may also serve as a target for stone prevention by blocking cell-crystal adhesion and stone nidus formation.

AT2 Receptors: Potential Therapeutic Targets for Hypertension.

The renin-angiotensin system (RAS) is arguably the most important and best studied hormonal system in the control of blood pressure (BP) and the pathogenesis of hypertension. The RAS features its main effector angiotensin II (Ang II) acting via its 2 major receptors, angiotensin type-1(AT1R) and type-2 (AT2R). In general, AT2Rs oppose the detrimental actions of Ang II via AT1Rs. AT2R activation induces vasodilation and natriuresis, but its effects to lower BP in hypertension have not been as clear as anticipated. Recent studies, however, have demonstrated that acute and chronic AT2R stimulation can induce natriuresis and lower BP in the Ang II infusion model of experimental hypertension. AT2R activation induces receptor recruitment from intracellular sites to the apical plasma membranes of renal proximal tubule cells via a bradykinin, nitric oxide, and cyclic guanosine 3',5' monophosphate signaling pathway that results in internalization and inactivation of sodium (Na+) transporters Na+-H+ exchanger-3 and Na+/K+ATPase. These responses do not require the presence of concurrent AT1R blockade and are effective both in the prevention and reversal of hypertension. This review will address the role of AT2Rs in the control of BP and Na+ excretion and the case for these receptors as potential therapeutic targets for hypertension in humans.

Evaluation and Quantitative Prediction of Renal Transporter-Mediated Drug-Drug Interactions.

With numerous drugs cleared renally, inhibition of uptake transporters localized on the basolateral membrane of renal proximal tubule cells, eg, organic anion transporters (OATs) and organic cation transporters (OCTs), may lead to clinically meaningful drug-drug interactions (DDIs). Additionally, clinical evidence for the possible involvement of efflux transporters, such as P-glycoprotein (P-gp) and multidrug and toxin extrusion protein 1/2-K (MATE1/2-K), in the renal DDIs is emerging. Herein, we review recent progress regarding mechanistic understanding of transporter-mediated renal DDIs as well as the quantitative predictability of renal DDIs using static and physiologically based pharmacokinetic (PBPK) models. Generally, clinical DDI data suggest that the magnitude of plasma exposure changes attributable to renal DDIs is less than 2-fold, unlike the DDIs associated with inhibition of cytochrome P-450s and/or hepatic uptake transporters. It is concluded that although there is a need for risk assessment early in drug development, current available data imply that safety concerns related to the renal DDIs are generally low. Nevertheless, consideration must be given to the therapeutic index of the victim drug and potential risk in a specific patient population (eg, renal impairment). Finally, in vitro transporter data and clinical pharmacokinetic parameters obtained from the first-in-human studies have proven useful in support of quantitative prediction of DDIs associated with inhibition of renal secretory transporters, OATs or OCTs.

Surface heat shock protein 90 serves as a potential receptor for calcium oxalate crystal on apical membrane of renal tubular epithelial cells.

Adhesion of calcium oxalate monohydrate (COM) crystals on renal tubular epithelial cells is a crucial step in kidney stone formation. Finding potential crystal receptors on the apical membrane of the cells may lead to a novel approach to prevent kidney stone disease. Our previous study identified a large number of crystal-binding proteins on the apical membrane of MDCK cells. However, their functional role as potential crystal receptors had not been validated. The present study aimed to address the potential role of heat shock protein 90 (HSP90) as a COM crystal receptor. The apical membrane was isolated from polarized MDCK cells by the peeling method and recovered proteins were incubated with COM crystals. Western blot analysis confirmed the presence of HSP90 in the apical membrane and the crystal-bound fraction. Immunofluorescence staining without permeabilization and laser-scanning confocal microscopy confirmed the surface HSP90 expression on the apical membrane of the intact cells. Crystal adhesion assay showed that blocking surface HSP90 by specific anti-HSP90 antibody and knockdown of HSP90 by small interfering RNA (siRNA) dramatically reduced crystal binding on the apical surface of MDCK cells (by approximately 1/2 and 2/3, respectively). Additionally, crystal internalization assay revealed the presence of HSP90 on the membrane of endocytic vesicle containing the internalized COM crystal. Moreover, pretreatment of MDCK cells with anti-HSP90 antibody significantly reduced crystal internalization (by approximately 1/3). Taken together, our data indicate that HSP90 serves as a potential receptor for COM crystals on the apical membrane of renal tubular epithelial cells and is involved in endocytosis/internalization of the crystals into the cells.

Externalization of phosphatidylserine via multidrug resistance 1 (MDR1)/P-glycoprotein in oxalate-treated renal epithelial cells: implications for calcium oxalate urolithiasis.

We investigated the possible involvement of multidrug resistance protein 1 P-glycoprotein (MDR1 P-gp) in the oxalate-induced redistribution of phosphatidylserine in renal epithelial cell membranes. Real-time PCR and western blotting were used to examine MDR1 expression in Madin-Darby canine kidney cells at the mRNA and protein levels, respectively, whereas surface-expressed phosphatidylserine was detected by the annexin V-binding assay. Oxalate treatment resulted in increased synthesis of MDR1, which resulted in phosphatidylserine (PS) externalization in the renal epithelial cell membrane. Treatment with the MDR1 inhibitor PSC833 significantly attenuated phosphatidylserine externalization. Transfection of the human MDR1 gene into renal epithelial cells significantly increased PS externalization. To our knowledge, this study is the first to show that oxalate increases the synthesis of MDR1 P-gp, which plays a key role in hyperoxaluria-promoted calcium oxalate urolithiasis by facilitating phosphatidylserine redistribution in renal epithelial cells.

Human oligopeptide transporter 2 (PEPT2) mediates cellular uptake of polymyxins.

Polymyxins are a last-line therapy to treat MDR Gram-negative bacterial infections. Nephrotoxicity is the dose-limiting factor for polymyxins and recent studies demonstrated significant accumulation of polymyxins in renal tubular cells. However, little is known about the mechanism of polymyxin uptake into these cells. Oligopeptide transporter 2 (PEPT2) is a solute carrier transporter (SLC) expressed at the apical membrane of renal proximal tubular cells and facilitates drug reabsorption in the kidney. In this study, we examined the role of PEPT2 in polymyxin uptake into renal tubular cells. We investigated the inhibitory effects of colistin and polymyxin B on the substrate uptake mediated through 15 essential SLCs in overexpressing HEK293 cells. The inhibitory potency of both polymyxins on PEPT2-mediated substrate uptake was measured. Fluorescence imaging was employed to investigate PEPT2-mediated uptake of the polymyxin fluorescent probe MIPS-9541 and a transport assay was conducted with MIPS-9541 and [(3)H]polymyxin B1. Colistin and polymyxin B potently inhibited PEPT2-mediated [(3)H]glycyl-sarcosine uptake (IC50 11.4 ± 3.1 and 18.3 ± 4.2 μM, respectively). In contrast, they had no or only mild inhibitory effects on the transport activity of the other 14 SLCs evaluated. MIPS-9541 potently inhibited PEPT2-mediated [(3)H]glycyl-sarcosine uptake (IC50 15.9 μM) and is also a substrate of PEPT2 (Km 74.9 μM). [(3)H]polymyxin B1 was also significantly taken up by PEPT2-expressing cells (Km 87.3 μM). Our study provides the first evidence of PEPT2-mediated uptake of polymyxins and contributes to a better understanding of the accumulation of polymyxins in renal tubular cells.

Multidrug resistance-associated protein 4 is a determinant of arsenite resistance.

Although arsenic trioxide (arsenite, AsIII) has shown a remarkable efficacy in the treatment of acute promyelocytic leukemia patients, multidrug resistance is still a major concern for its clinical use. Multidrug resistance-associated protein 4 (MRP4), which belongs to the ATP-binding cassette (ABC) superfamily of transporters, is localized to the basolateral membrane of hepatocytes and the apical membrane of renal proximal tubule cells. Due to its characteristic localization, MRP4 is proposed as a candidate in the elimination of arsenic and may contribute to resistance to AsIII. To test this hypothesis, stable HEK293 cells overexpressing MRP4 or MRP2 were used to establish the role of these two transporters in AsIII resistance. The IC50 values of AsIII in MRP4 cells were approximately 6-fold higher than those in MRP2 cells, supporting an important role for MRP4 in resistance to AsIII. The capacity of MRP4 to confer resistance to AsIII was further confirmed by a dramatic decrease in the IC50 values with the addition of MK571, an MRP4 inhibitor, and cyclosporine A, a well-known broad-spectrum inhibitor of ABC transporters. Surprisingly, the sensitivity of the MRP2 cells to AsIII was similar to that of the parent cells, although insufficient formation of glutathione and/or Se conjugated arsenic compounds in the MRP2 cells might limit transport. Given that MRP4 is a major contributor to arsenic resistance in vitro, further investigation into the correlation between MRP4 expression and treatment outcome of leukemia patients treated with arsenic-based regimens is warranted.

Monitoring Diabetic Nephropathy by Circulating Gangliosides

Gangliosides are multifunctional molecules, abundantly expressed in renal cell membrane but also in sera of patients with renal disease. The aim of this study was to quantify the serum levels of sialic acid-ganglioside in patients diagnosed with diabetes for an eventual biomarker stratification of patients with renal complications. We included 35 diabetic patients without metabolic complications, 35 patients with diabetic nephropathy, 35 non-diabetic individuals. We found that sialic acid ganglioside serum level was significantly increased in patients with diabetic nephropathy compared to the level obtained in patients with uncomplicated diabetes and to non-diabetic controls. A statistically significant positive correlation was obtained between serum levels of sialic acid gangliosides, HbA1c, and serum creatinine in patients with diabetes without complications. Moreover positive correlation was found between sialic acid ganglioside and blood glucose, HbA1c, urea, creatinine, microalbuminuria in patients with diabetic nephropathy. We can conclude that serum sialic acid-gangliosides are statistically increased in diabetic nephropathy positively correlated with microalbuminuria.

Ecto-5'-nucleotidase expression is associated with the progression of renal cell carcinoma.

Renal cell carcinoma (RCC) is a common tissue tumor that occurs across all age groups and has become one of the types of cancer with the fastest increasing incidence. Due to the resistance of RCC chemo- and radiotherapy, surgery is the only currently effective treatment. Therefore, specific markers for the diagnosis and prognosis of RCC are expected to result in novel methods of treatment. Ecto-5'-nucleotidase, also termed cluster of differentiation (CD)73, is a protein that is activated in several types of aggressive cancer and may promote cancer progression. CD73 was examined in the present study to determine the association between the protein and RCC. The expression levels of CD73 in 159 RCC tissue sections and 30 paratumorous normal renal tissue samples obtained from 235 patients that underwent nephrectomy were examined by immunohistochemical staining. By contrast, the expression level of P-glycoprotein (P-gp), a potential prognostic factor in RCC, was also examined in 85 RCC and 13 normal tissue samples. Intense CD73 expression was identified in 75 out of 159 RCC cell membranes compared with normal renal tissues. In contrast, there was high P-gp expression in the blood vessels of 42 out of 85 RCC tissues and there was no significant difference between the P-gp expression identified in RCC cells (34 out of 85) and the cell membrane of normal renal cells (2 out of 13). The expression level of CD73 in RCC cells was significantly associated with tumor type, tumor node metastasis (TNM) stage, and tumor grade. However, the expression of P-gp in RCC cells was only associated with the TNM stage and tumor grade. Using a multivariable Cox regression analysis, it was found that the median survival rate of RCC patients with intense CD73 expression in RCC cells was 62.06±5.35 months, which was drastically shorter compared with rare CD73 expression (103.72±3.67 months). In conclusion, the expression level of CD73 is significantly associated with RCC tumor progression and may serve as a favorable marker for the diagnosis and prognosis of RCC, in addition to being a therapeutic target for the treatment of RCC.

Akt Substrate of 160 kD Regulates Na+,K+-ATPase Trafficking in Response to Energy Depletion and Renal Ischemia.

Renal ischemia and reperfusion injury causes loss of renal epithelial cell polarity and perturbations in tubular solute and fluid transport. Na(+),K(+)-ATPase, which is normally found at the basolateral plasma membrane of renal epithelial cells, is internalized and accumulates in intracellular compartments after renal ischemic injury. We previously reported that the subcellular distribution of Na(+),K(+)-ATPase is modulated by direct binding to Akt substrate of 160 kD (AS160), a Rab GTPase-activating protein that regulates the trafficking of glucose transporter 4 in response to insulin and muscle contraction. Here, we investigated the effect of AS160 on Na(+),K(+)-ATPase trafficking in response to energy depletion. We found that AS160 is required for the intracellular accumulation of Na(+),K(+)-ATPase that occurs in response to energy depletion in cultured epithelial cells. Energy depletion led to dephosphorylation of AS160 at S588, which was required for the energy depletion-induced accumulation of Na,K-ATPase in intracellular compartments. In AS160-knockout mice, the effects of renal ischemia on the distribution of Na(+),K(+)-ATPase were substantially reduced in the epithelial cells of distal segments of the renal tubules. These data demonstrate that AS160 has a direct role in linking the trafficking of Na(+),K(+)-ATPase to the energy state of renal epithelial cells.

English

ABSTRACT Pharmacodynamical studies showed that most drugs elicit their effects by acting on 3 kinds of protein molecules known as receptors, enzymes or transporters. Although their detail properties had not been explained for decades the roles of transporters in drug kinetics and dynamics has been well understood, even have been applied in the therapy. Transporters are classified into 2 major classes, the solute carriers (SLC) and ATP-binding cassette (ABC) families. SLC transporters do not possess ATP binding site property as those of ABC transporters. SLC transporters consist of 3 SLC subfamilies i.e. organic cation transporters (OCTs), organic anion transporters (OATs) and organic anion transporting polypeptides (OATPs). In contrast, ABC transporters require ATP hydrolysis to transport substrate across cell membrane. Human ABC-transporters consist of ABCA1- 13, ABCB1-11, ABCC1-12, ABCD1-4, ABCE1, ABCF1-3 and ABCG1-8 subfamily. Although the originally funtion of transporter is to transport specific physiological substrate such as nutrient, hormone, cytokines, neurotransmitters and other physiological subtances across cell membrane the specificity is not restricted to each substrate. Drugs and other xenobiotics which have structural similarity to the physiological substrates are recognized and transported by the related transporters. The competition of them on transporters therefore may lead to the occurence of drug-drug interactions (DDI) or drugphysiological substrate interaction in the drug-kinetics phase. Many transporters located in the liver, intestinal and renal epithelial cell membranes involve in the transport of endogenous substance or xenobiotics including drugs play important roles as protective barrier. Since transporters also serve as the targets of drug action it is understood that transporters play important role in the pathogenesis of diseases as well as in the drug therapy of diseases.

Rosiglitazone Promotes AQP2 Plasma Membrane Expression In Renal Cells Via a Ca2+-Dependent/cAMP-Independent Mechanism

Background/Aims: Thiazolidinediones are highly beneficial in the treatment of type II diabetes. However, they are also associated with edema and increased risk of congestive heart failure. Several studies demonstrated that rosiglitazone (RGZ) increases the abundance of aquaporin-2 (AQP2) at the plasma membrane of renal cells. The aim of this study was to investigate whether RGZ might activate a transduction pathway facilitating AQP2 membrane accumulation in renal cells. Methods: We analyzed the effect of RGZ on renal AQP2 intracellular trafficking in MCD4 renal cells by confocal microscopy and apical surface biotinylation. Cytosolic Ca²⁺ dynamics were measured by a video-imaging approach in single cell. Transient Receptor Potential (TRP) channels expression was determined by RT-PCR. Results: We showed that in MCD4 cells, short-term exposure to RGZ dramatically increases the amount of apically expressed AQP2 independently on cAMP production, PKA activation and AQP2 phosphorylation. RGZ elicited a cytosolic Ca²⁺ transient due to Ca²⁺ influx prevented by ruthenium red, suggesting the involvement of TRP plasma membrane channels. We identified TRPV6 as the possible candidate mediating this effect. Conclusions: Taken together these results provide a possible molecular mechanism explaining the increased AQP2 membrane expression under RGZ treatment: in renal cells RGZ elicits Ca²⁺ transients facilitating AQP2 exposure at the apical plasma membrane, thus increasing collecting duct water permeability. Importantly, this effect suggests an unexplored application of RGZ in the treatment of pathological states characterized by impaired AQP2 trafficking at the plasma membrane.

Novel functional complexity of polycystin-1 by GPS cleavage in vivo: role in polycystic kidney disease.

Polycystin-1 (Pc1) cleavage at the G protein-coupled receptor (GPCR) proteolytic site (GPS) is required for normal kidney morphology in humans and mice. We found a complex pattern of endogenous Pc1 forms by GPS cleavage. GPS cleavage generates not only the heterodimeric cleaved full-length Pc1 (Pc1(cFL)) in which the N-terminal fragment (NTF) remains noncovalently associated with the C-terminal fragment (CTF) but also a novel (Pc1) form (Pc1(deN)) in which NTF becomes detached from CTF. Uncleaved Pc1 (Pc1(U)) resides primarily in the endoplasmic reticulum (ER), whereas both Pc1(cFL) and Pc1(deN) traffic through the secretory pathway in vivo. GPS cleavage is not a prerequisite, however, for Pc1 trafficking in vivo. Importantly, Pc1(deN) is predominantly found at the plasma membrane of renal epithelial cells. By functional genetic complementation with five Pkd1 mouse models, we discovered that CTF plays a crucial role in Pc1(deN) trafficking. Our studies support GPS cleavage as a critical regulatory mechanism of Pc1 biogenesis and trafficking for proper kidney development and homeostasis.

Vesicle-associated Membrane Protein 2 (VAMP2) but Not VAMP3 Mediates cAMP-stimulated Trafficking of the Renal Na+-K+-2Cl− Co-transporter NKCC2 in Thick Ascending Limbs

In the kidney, epithelial cells of the thick ascending limb (TAL) reabsorb NaCl via the apical Na(+)/K(+)/2Cl(-) co-transporter NKCC2. Steady-state surface NKCC2 levels in the apical membrane are maintained by a balance between exocytic delivery, endocytosis, and recycling. cAMP is the second messenger of hormones that enhance NaCl absorption. cAMP stimulates NKCC2 exocytic delivery via protein kinase A (PKA), increasing steady-state surface NKCC2. However, the molecular mechanism involved has not been studied. We found that several members of the SNARE family of membrane fusion proteins are expressed in TALs. Here we report that NKCC2 co-immunoprecipitates with VAMP2 in rat TALs, and they co-localize in discrete domains at the apical surface. cAMP stimulation enhanced VAMP2 exocytic delivery to the plasma membrane of renal cells, and stimulation of PKA enhanced VAMP2-NKCC2 co-immunoprecipitation in TALs. In vivo silencing of VAMP2 but not VAMP3 in TALs blunted cAMP-stimulated steady-state surface NKCC2 expression and completely blocked cAMP-stimulated NKCC2 exocytic delivery. VAMP2 was not involved in constitutive NKCC2 delivery. We concluded that VAMP2 but not VAMP3 selectively mediates cAMP-stimulated NKCC2 exocytic delivery and surface expression in TALs. We also demonstrated that cAMP stimulation enhances VAMP2 exocytosis and promotes VAMP2 interaction with NKCC2.

Degradation mechanism of a Golgi-retained distal renal tubular acidosis mutant of the kidney anion exchanger 1 in renal cells.

Distal renal tubular acidosis (dRTA) can be caused by mutations in the SLC4A1 gene encoding the anion exchanger 1 (AE1). Both recessive and dominant mutations result in mistrafficking of proteins, preventing them from reaching the basolateral membrane of renal epithelial cells, where their function is needed. In this study, we show that two dRTA mutants are prematurely degraded. Therefore, we investigated the degradation pathway of the kidney AE1 G701D mutant that is retained in the Golgi. Little is known about degradation of nonnative membrane proteins from the Golgi compartments in mammalian cells. We show that the kidney AE1 G701D mutant is polyubiquitylated and degraded by the lysosome and the proteosome. This mutant reaches the plasma membrane, where it is endocytosed and degraded by the lysosome via a mechanism dependent on the peripheral quality control machinery. Furthermore, we show that the function of the mutant is rescued at the cell surface upon inhibition of the lysosome and incubation with a chemical chaperone. We conclude that modulating the peripheral quality control machinery may provide a novel therapeutic option for treatment of patients with dRTA due to a Golgi-retained mutant.