Membrane Of Renal Epithelial Cells Research Articles

402-P: ENaC Regulation in Mouse Cortical Collecting Duct Cells by EVs Released during the Inactive and Active Cycles of Salt-Loaded Hypertensive Diabetic db/db Mice

The downregulation of renal epithelial sodium channel (ENaC) activity in response to salt-loading in the diabetic kidney is important to prevent the exacerbation of diabetic kidney disease. An inflammatory response in diabetic db/db mice is associated with constitutively high ENaC activity even after salt-loading. During salt-loading, diabetic db/db mice release extracellular vesicles (EVs) into the urine during the active and inactive cycles that are enriched in bioactive molecules. A vast majority of these EVs are released across the luminal membrane of renal epithelial cells from each segment of the nephron and have the potential to module ENaC activity in recipient collecting duct cells. Our hypothesis was the distinct cargo that is enriched in EVs released during the inactive and active cycles of salt-loaded hypertensive diabetic db/db mice is responsible for increased ENaC membrane protein expression and activity in recipient collecting duct cells. In this study, we measured blood pressure by telemetry during the light (inactive) or dark (active) cycles. In addition, osmolality, electrolytes, and creatinine were measured and EV were isolated from urine samples collected during both cycles. Western blotting and densitometric analysis showed greater ENaC alpha protein expression in membrane fractions of mouse cortical collecting duct (mpkCCD) cells while single-channel patch clamp studies show greater ENaC activity in mpkCCD cells treated with uEVs isolated from the active cycle compared to the inactive cycle of hypertensive diabetic db/db mice. Various proteins and metabolites were found to be enriched in each of the two groups of EVs. Taken together, these data suggest the packaged cargo within EVs released after salt-loading diabetic db/db mice changes over a 24 hour cycle, and various molecules enriched in active cycle EVs contribute to the upregulation of renal ENaC protein expression and activity. Disclosure L.Yu: None. M.F.Gholam: None. A.A.Alli: None. Funding National Institute of Diabetes and Digestive and Kidney Diseases (R01DK123078-01A1 to A.A.A.)

Functional Characterization of a Calcium-Sensing Receptor-Polycystin-2 Channel Complex in the Plasma Membrane of LLC-PK1 Cells

Polycystin-2 (PC2, TRPP2) is a Ca2+-permeable nonselective cation channel from the TRP superfamily. PC2 is encoded by the PKD2 gene, whose mutations are responsible for autosomal dominant polycystic kidney disease (ADPKD). Recent studies from our laboratory (Dai et al, Exp Cell Res, 2017), determined that the PC2 contribution to the whole cell conductance of wild type LLC-PK1 renal epithelial cells is regulated by changes in external Ca2+ concentration, in a mechanism that implicates the Calcium-Sensing Receptor (CaSR). The present study explored the effect of Ca2+ and CaSR agonists in the regulation of PC2 from isolated LLC-PK1 plasma membranes reconstituted in a lipid bilayer system (BLM). Addition of the CaSR agonist R-568 (5 μM) to the “external” side of the bilayer, activated single channel currents consistent with a functional PC2 by 84 ± 12% (n = 4, p < 0.01). These cation-selective currents were subsequently inhibited (91.3 ± 1.1%, p < 0.0001) by addition to the “cytosolic” side of the BLM, of a commercially available anti-PC2 C-terminal antibody to the “cytosolic” or “internal” side of the BLM. Interestingly, similar findings were observed with the material obtained by co-immunoprecipitation of LLC-PK1 cell lysate with anti-CaSR antibody crosslinked in agarose beads. The data indicate the presence of a membrane locate CaSR-PC2 functional complex in LLC-PK1 renal epithelial cells. The presence of the receptor/channel complex is in agreement with the regulatory role external Ca2+ plays in the modulation of PC2 channel function in the plasma membrane of renal epithelial cells. This receptor-channel complex may be part of a regulatory mechanism connecting Ca2+ signals and PC2 function to onset of ADPKD.

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.

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.

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.

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.

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.

Ammonia transport in the kidney by Rhesus glycoproteins.

Renal ammonia metabolism is a fundamental element of acid-base homeostasis, comprising a major component of both basal and physiologically altered renal net acid excretion. Over the past several years, a fundamental change in our understanding of the mechanisms of renal epithelial cell ammonia transport has occurred, replacing the previous model which was based upon diffusion equilibrium for NH3 and trapping of NH4(+) with a new model in which specific and regulated transport of both NH3 and NH4(+) across renal epithelial cell membranes via specific membrane proteins is required for normal ammonia metabolism. A major advance has been the recognition that members of a recently recognized transporter family, the Rhesus glycoprotein family, mediate critical roles in renal and extrarenal ammonia transport. The erythroid-specific Rhesus glycoprotein, Rh A Glycoprotein (Rhag), was the first Rhesus glycoprotein recognized as an ammonia-specific transporter. Subsequently, the nonerythroid Rh glycoproteins, Rh B Glycoprotein (Rhbg) and Rh C Glycoprotein (Rhcg), were cloned and identified as ammonia transporters. They are expressed in specific cell populations and membrane domains in distal renal epithelial cells, where they facilitate ammonia secretion. In this review, we discuss the distribution of Rhbg and Rhcg in the kidney, the regulation of their expression and activity in physiological disturbances, the effects of genetic deletion on renal ammonia metabolism, and the molecular mechanisms of Rh glycoprotein-mediated ammonia transport.

Progesterone induced mesenchymal differentiation and rescued cystic dilation of renal tubules of Pkd1−/− mice

Autosomal dominant polycystic kidney disease (ADPKD), the most common hereditary disease affecting the kidneys, is caused in 85% of cases by mutations in the PKD1 gene. The protein encoded by this gene, polycystin-1, is a renal epithelial cell membrane mechanoreceptor, sensing morphogenetic cues in the extracellular environment, which regulate the tissue architecture and differentiation. However, how such mutations result in the formation of cysts is still unclear. We performed a precise characterization of mesenchymal differentiation using PAX2, WNT4 and WT1 as a marker, which revealed that impairment of the differentiation process preceded the development of cysts in Pkd1−/− mice. We performed an in vitro organ culture and found that progesterone and a derivative thereof facilitated mesenchymal differentiation, and partially prevented the formation of cysts in Pkd1−/− kidneys. An injection of progesterone or this derivative into the intraperitoneal space of pregnant females also improved the survival of Pkd1−/− embryos. Our findings suggest that compounds which enhance mesenchymal differentiation in the nephrogenesis might be useful for the therapeutic approach to prevent the formation of cysts in ADPKD patients.

AS160: a new Na,K‐ATPase partner that regulates the trafficking of the sodium pump in response to energy depletion and renal ischemia

The Na,K‐ATPase appears to be located in two different cellular pools. The major sodium pump pool is located at the plasma membrane, while the other pool resides in cytoplasmic vesicular compartments which can translocate to the plasma membrane in response to physiological stimuli. We have identified AS160 as a new Na,K‐ATPase partner. We also demonstrated that the knockdown of AS160 increases the level of cellular Na,K‐ATPase activity, suggesting that AS160 modulates the activity of the enzyme. In the present study we characterized aspects of the physiological role of the interaction between AS160 and Na,K‐ATPase. We find that AS160 is required for the intracellular accumulation of the Na, K‐ATPase that occurs in response to energy depletion in cultured epithelial cells. In intact animals ischemic kidney injury has been shown to induce internalization of the Na,K‐ATPase from the plasma membranes of renal epithelial cells. We assessed the distribution of the Na,K‐ATPase following renal ischemia and reperfusion in kidneys from wild type and AS160 knockout mice. We observed a substantial decrease in the injury‐induced cytoplasmic accumulation of the Na,K‐ATPase in the renal epithelial cells of the AS160 knockout mice as compared to those of the wild type controls. We propose that AS160 plays a key role in regulating the redistribution of the Na,K‐ATPase that occurs in response to energy depletion and renal ischemia.

Low efficiency of functional translation of ouabain-resistant α2-Na+,K+-ATPase mRNA in C7-MDCK epithelial cells

Na(+),K(+)-ATPase is a heterodimer consisting of catalytic α1-α4 and regulatory β1-β3 subunits. Recently, we reported that transfection with ouabain-resistant α1R-Na(+),K(+)-ATPase rescues renal epithelial C7-MDCK cells exclusively expressing the ouabain-sensitive α1S-isoform from the cytotoxic action of ouabain. To explore the role of α2 subunit in ion transport and cytotoxic action of ouabain, we compared the effect of ouabain on K(+) ((86)Rb) influx and the survival of ouabain-treated C7-MDCK cells stably transfected with α1R- and α2R-Na(+),K(+)-ATPase. α2R mRNA in transfected cells was ∼8-fold more abundant than α1R mRNA, whereas immunoreactive α2R protein content was 5-fold lower than endogenous α1S protein. A concentration of 10µmol/L ouabain led to complete inhibition of (86)Rb influx both in mock- and α2R-transfected cells, whereas maximal inhibition of (86)Rb influx in α1R-transfectd cells was observed at 1000µmol/L ouabain. In contrast to the massive death of mock- and α2R-transfected cells exposed to 3µmol/L ouabain , α1R-cells survived after 24h incubation with 1000µmol/L ouabain. Thus, our results show that unlike α1R, the presence of α2R-Na(+),K(+)-ATPase subunit mRNA and immunoreactive protein does not contribute to Na(+)/K(+) pump activity, and does not rescue C7-MDCK cells from the cytotoxic action of ouabain. Our results also suggest that the lack of impact of transfected α2-Na(+),K(+)-ATPase on Na(+)/K(+) pump activity and cell survival can be attributed to the low efficiency of its translation and (or) delivery to the plasma membrane of renal epithelial cells.

Moment Analysis of Drug Disposition in Rat Kidney: Role of Basolateral Membrane Transport in Renal Transepithelial Transport of p-Aminohippurate

The determining step in transepithelial transport of p-aminohippurate (PAH) in renal tubular secretion has been elucidated in the rat isolated perfused kidney. The method was based upon a multiple indicator dilution experiment and non-compartmental moment analysis. Single-pass dilution curves were obtained from venous and urinary effluents after simultaneous intra-arterial injections of Evans blue with albumin, [3H]inulin and [14C]PAH. Probenecid was used as a transport inhibitor and dissolved in both perfusate and injection solution. The urinary excretion of PAH decreased depending on the probenecid concentration, while that of inulin was not affected. No correlation was observed between the amount of secretion and the mean residence time of secreted PAH in renal epithelial cells (Tcell). Since Tcell ought to be affected by the rate of secretion from cells to lumen, it was suggested that the secretion rate was independent of the amount secreted. In contrast, the amount of PAH excreted via tubular secretion showed a linear correlation with the volume of distribution in the kidney and the apparent rate constant for tubular uptake of PAH. Since these kinetic parameters reflect the transport from blood into cells across the basolateral membranes of renal epithelial cells, the present results demonstrated that basolateral membrane transport is a determining step in the transcellular transport of PAH and that the major effect of probenecid is the inhibition of transport from blood into cells.

Synergistic Interaction between Ciliary Genes Reflects the Importance of Mutational Load in Ciliopathies

The formation of renal cysts occurs in roughly 10 to 15% of the population,1 and although simple cysts are largely asymptomatic, the formation of multiple cysts can be extremely detrimental. The two major multicystic syndromes, autosomal recessive polycystic kidney disease (PKD) and autosomal dominant PKD, are primarily linked to disruptions in the pkhd1 and pkd1 / pkd2 genes, respectively2; however, several additional syndromes are also characterized by kidney cyst development, including nephronophthisis (NPHP), Joubert syndrome, Bardet-Biedl syndrome, Orofaciodigital syndrome 1, and Meckel syndrome (MKS). During the past decade, there has been a great upsurgence in identifying genes affected in these syndromes. One of the most striking discoveries is that protein products of these genes localize to the cilium and/or the associated basal body and affect ciliogenesis and cilia morphology or function when mutated,3 collectively identifying these syndromes as ciliopathies. Why do kidney cysts form? An increasing body of evidence indicates that mutations affecting ciliogenesis or cilia function can give rise to cysts. Primary cilia extend from the apical membrane of renal epithelial cells into the lumen, where they act as mechanosensors. One model suggests that fluid flow within the kidney bends the cilia and triggers intracellular calcium signaling, which, in turn, regulates epithelial tubule morphology.2 Polycystin 1 and polycystin 2, the protein products of the pkd1 and pkd2 genes, respectively, localize to renal cilia4 and may act as the mediators of mechanosensation in these structures.5 Mutations in pkd1 or pkd2 do not result in morphologic cilia defects but rather affect calcium influx, which leads to cyst formation. In addition, mutations that result in aberrant cilia …

Polycystin-dependent fluid flow sensing targets histone deacetylase 5 to prevent the development of renal cysts

Polycystin 1 and polycystin 2 are large transmembrane proteins, which, when mutated, cause autosomal dominant polycystic kidney disease (ADPKD), a highly prevalent human genetic disease. The polycystins are thought to form a receptor-calcium channel complex in the plasma membrane of renal epithelial cells and elicit a calcium influx in response to mechanical stimulation, such as fluid flow across the apical surface of renal epithelial cells. The functional role of the polycystins in mechanosensation remains largely unknown. Here, we found that myocyte enhancer factor 2C (MEF2C) and histone deacetylase 5 (HDAC5), two key regulators of cardiac hypertrophy, are targets of polycystin-dependent fluid stress sensing in renal epithelial cells in mice. We show that fluid flow stimulation of polarized epithelial monolayers induced phosphorylation and nuclear export of HDAC5, which are crucial events in the activation of MEF2C-based transcription. Kidney-specific knockout of Mef2c, or genetrap-inactivation of a MEF2C transcriptional target, MIM, resulted in extensive renal tubule dilation and cysts, whereas Hdac5 heterozygosity or treatment with TSA, an HDAC inhibitor, reduced cyst formation in Pkd2(-/-) mouse embryos. These findings suggest a common signaling motif between myocardial hypertrophy and maintenance of renal epithelial architecture, and a potential therapeutic approach to treat ADPKD.

Effect of lamivudine on uptake of organic cations by rat renal brush-border and basolateral membrane vesicles.

The effect of lamivudine on uptake of a representative organic cation, tetraethylammonium (TEA), by rat renal brush-border membrane vesicles (BBMV) and basolateral membrane vesicles (BLMV) has been investigated. The pH-driven uptake of TEA by BBMV (pHin = 6.0, pHout = 7.5) was inhibited by lamivudine. The IC50 value (concentration resulting in 50% inhibition) for the concentration-dependent effect of lamivudine on TEA uptake by BBMV after 30 s was 2668 microM whereas IC50 values for cimetidine and trimethoprim were < 2.5 microM and < 25 microM, respectively. The early uptake of TEA by BLMV was also reduced significantly by lamivudine. The IC50 value for the concentration-dependent effect of lamivudine on uptake of TEA by BLMV at 30 s was > 25 mM, whereas the IC50 values for cimetidine and trimethoprim were 2116 microM and 445 microM, respectively. These findings suggest that compared with other cationic drugs, such as trimethoprim and cimetidine, lamivudine is a weak inhibitor of organic cation transport into the tubules by the brush-border and basolateral membranes of renal epithelial cells. It is unlikely lamivudine will have any significant effect on the excretion of co-administered cationic drugs by the renal tubules.

Acute Hypertonicity Alters Aquaporin-2 Trafficking and Induces a MAPK-dependent Accumulation at the Plasma Membrane of Renal Epithelial Cells

The unique phenotype of renal medullary cells allows them to survive and functionally adapt to changes of interstitial osmolality/tonicity. We investigated the effects of acute hypertonic challenge on AQP2 (aquaporin-2) water channel trafficking. In the absence of vasopressin, hypertonicity alone induced rapid (<10 min) plasma membrane accumulation of AQP2 in rat kidney collecting duct principal cells in situ, and in several kidney epithelial lines. Confocal microscopy revealed that AQP2 also accumulated in the trans-Golgi network (TGN) following hypertonic challenge. AQP2 mutants that mimic the Ser(256)-phosphorylated and -nonphosphorylated state accumulated at the cell surface and TGN, respectively. Hypertonicity did not induce a change in cytosolic cAMP concentration, but inhibition of either calmodulin or cAMP-dependent protein kinase A activity blunted the hypertonicity-induced increase of AQP2 cell surface expression. Hypertonicity increased p38, ERK1/2, and JNK MAPK activity. Inhibiting MAPK activity abolished hypertonicity-induced accumulation of AQP2 at the cell surface but did not affect either vasopressin-dependent AQP2 trafficking or hypertonicity-induced AQP2 accumulation in the TGN. Finally, increased AQP2 cell surface expression induced by hypertonicity largely resulted from a reduction in endocytosis but not from an increase in exocytosis. These data indicate that acute hypertonicity profoundly alters AQP2 trafficking and that hypertonicity-induced AQP2 accumulation at the cell surface depends on MAP kinase activity. This may have important implications on adaptational processes governing transcellular water flux and/or cell survival under extreme conditions of hypertonicity.

Circular patterns of calcium oxalate crystals induced by defective Langmuir-Blodgett film

The injury of the renal epithelial cell membrane can promote the nucleation of nascent crystals, as well as adhesion of crystals on it. It thus accelerates the formation of renal calculi. In this paper, the defective Langmuir-Blodgett (LB) films were used as a model system to simulate the injured renal epithelial cell membrane. The microcosmic structure of the defective LB film and the molecular mechanism of the effect of this film on nucleation, growth, deposited patterns and adhesion of calcium oxalate monohydrate (COM) were investigated. The circular defective domains were formed in dipalmitoylphosphatidylcholine (DPPC) LB film after the film was treated by potassium oxalate. These domains could induce ring-shaped patterns of COM crystals. In comparison, the LB film without pretreatment by potassium oxalate only induced random growth of hexagonal COM crystals. As the crystallization time increased, the size of COM crystals in the patterns increased, the crystal patterns changed from empty circles to solid circles, and the number of the circular patterns with small size (5–20 μm) increased. The results would shed light on the molecular mechanism of urolithiasis induced by injury of the renal epithelial membrane at the molecular and supramolecular level.

Profiling at mRNA, protein, and metabolite levels reveals alterations in renal amino acid handling and glutathione metabolism in kidney tissue ofPept2−/−mice

PEPT2 is an integral membrane protein in the apical membrane of renal epithelial cells that operates as a rheogenic transporter for di- and tripeptides and structurally related drugs. Its prime role is thought to be the reabsorption of filtered di- and tripeptides contributing to amino acid homeostasis. To elucidate the role of PEPT2 in renal amino acid metabolism we submitted kidney tissues of wild-type and a Pept2(-/-) mouse line to a comprehensive transcriptome, proteome and metabolome profiling and analyzed urinary amino acids and dipeptides. cDNA microarray analysis identified 147 differentially expressed transcripts in transporter-deficient animals, and proteome analysis by 2D-PAGE and MALDI-TOF-MS identified 37 differentially expressed proteins. Metabolite profiling by GC-MS revealed predominantly altered concentrations of amino acids and derivatives. Urinary excretion of amino acids demonstrated increased glycine and cysteine/cystine concentrations and dipeptides in urine were assessed by amino acid analysis of urine samples before and after in vitro dipeptidase digestion. Dipeptides constituted a noticeable fraction of urinary amino acids in Pept2(-/-) animals, only, and dipeptide-bound glycine and cystine were selectively increased in Pept2(-/-) urine samples. These findings were confirmed by a drastically increased excretion of cysteinyl-glycine (cys-gly). Urinary loss of cys-gly together with lower concentrations of cysteine, glycine, and oxoproline in kidney tissue and altered expression of mRNA and proteins involved in glutathione (GSH) metabolism suggests that PEPT2 is predominantly a system for reabsorption of cys-gly originating from GSH break-down, thus contributing to resynthesis of GSH.

Membrane localization and pH-dependent transport of a newly cloned organic cation transporter (PMAT) in kidney cells

Plasma membrane monoamine transporter (PMAT) is a novel membrane transporter recently cloned and characterized in our laboratory. We previously demonstrated that PMAT functions as a polyspecific organic cation transporter and efficiently transports many organic cations such as monoamine neurotransmitters and 1-methyl-4-phenylpyridinium (MPP(+)). In this study, we explored the role of PMAT in the renal handling of organic cations. Using a polyclonal antibody generated toward the NH(2)-terminal 66 amino acid residues of human PMAT, we showed that the PMAT protein (approximately 55 kDa) is expressed in the human kidney and is primarily targeted to the apical membranes when expressed in polarized Madin-Darby canine kidney (MDCK) cells. Using MDCK cells stably expressing human PMAT, we showed that PMAT-mediated MPP(+) uptake is strongly dependent on extracellular pH. Lowering extracellular pH from 7.4 to 6.6 greatly stimulated PMAT-mediated MPP(+) uptake, whereas elevating extracellular pH to 8.2 abolished transporter activity. Kinetic analysis revealed that the apparent V(max) at pH 6.6 is about fourfold higher than that at pH 7.4, whereas the apparent K(m) values were not statistically different at these two conditions. Under acidic conditions (pH 6.6), the proton ionophore, carbonyl cyanide p-trifluormethoxyphenylhydrazone, drastically reduced PMAT-mediated MPP(+) uptake, suggesting that the stimulatory effect of proton may be due to transporter coupling with a proton gradient. Taken together, our data suggest that PMAT is expressed on the apical membranes of renal epithelial cells and may use luminal proton gradient to drive organic cation reabsorption in the kidney.