Membrane Of Kidney Proximal Tubule Research Articles

Lysophosphatidic acid (LPA) as a modulator of plasma membrane Ca2+-ATPase from basolateral membranes of kidney proximal tubules.

Lysophosphatidic acid (LPA) acts through the activation of G protein-coupled receptors, in a Ca2+-dependent manner. We show the effects of LPA on the plasma membrane Ca2+-ATPase (PMCA) from kidney proximal tubule cells. The Ca2+-ATPase activity was inhibited by nanomolar concentrations of LPA, with maximal inhibition (~50%) obtained with 20 nM LPA. This inhibitory action on PMCA activity was blocked by Ki16425, an antagonist for LPA receptors, indicating that this lipid acts via LPA1 and/or LPA3 receptor. This effect is PKC-dependent, since it is abolished by calphostin C and U73122, PKC, and PLC inhibitors, respectively. Furthermore, the addition of 10-8 M PMA, a well-known PKC activator, mimicked PMCA modulation by LPA. We also demonstrated that the PKC activation leads to an increase in PMCA phosphorylation. These results indicate that LPA triggers LPA1 and/or LPA3 receptors at the BLM, inducing PKC-dependent phosphorylation with further inhibition of PMCA. Thus, LPA is part of the regulatory lipid network present at the BLM and plays an important role in the regulation of intracellular Ca2+ concentration that may result in significant physiological alterations in other Ca2+-dependent events ascribed to the renal tissue.

Oral Proteasomal Inhibitors Ixazomib, Oprozomib, and Delanzomib Upregulate the Function of Organic Anion Transporter 3 (OAT3): Implications in OAT3-Mediated Drug-Drug Interactions.

Organic anion transporter 3 (OAT3) is mainly expressed at the basolateral membrane of kidney proximal tubules, and is involved in the renal elimination of various kinds of important drugs, potentially affecting drug efficacy or toxicity. Our laboratory previously reported that ubiquitin modification of OAT3 triggers the endocytosis of OAT3 from the plasma membrane to intracellular endosomes, followed by degradation. Oral anticancer drugs ixazomib, oprozomib, and delanzomib, as proteasomal inhibitors, target the ubiquitin–proteasome system in clinics. Therefore, this study investigated the effects of ixazomib, oprozomib, and delanzomib on the expression and transport activity of OAT3 and elucidated the underlying mechanisms. We showed that all three drugs significantly increased the accumulation of ubiquitinated OAT3, which was consistent with decreased intracellular 20S proteasomal activity; stimulated OAT3-mediated transport of estrone sulfate and p-aminohippuric acid; and increased OAT3 surface expression. The enhanced transport activity and OAT3 expression following drug treatment resulted from an increase in maximum transport velocity of OAT3 without altering the substrate binding affinity, and from a decreased OAT3 degradation. Together, our study discovered a novel role of anticancer agents ixazomib, oprozomib, and delanzomib in upregulating OAT3 function, unveiled the proteasome as a promising target for OAT3 regulation, and provided implication of OAT3-mediated drug–drug interactions, which should be warned against during combination therapies with proteasome inhibitor drugs.

Meprin \u03b2 metalloproteases associated with differential metabolite profiles in the plasma and urine of mice with type 1 diabetes and diabetic nephropathy

BackgroundMeprin metalloproteases are abundantly expressed in the brush border membranes of kidney proximal tubules and small intestines. Meprins are also expressed in podocytes and leukocytes (monocytes and macrophages). Meprins are implicated in the pathophysiology of diabetic nephropathy (DN) but underlying mechanisms are not fully understood. Single nucleotide polymophisms (SNPs) in the meprin β gene were associated with DKD in human subjects. Furthermore, meprin α and β double deficiency resulted in more severe kidney injury and higher mortality rates in mice with Streptozotocin (STZ)-induced type 1 diabetes. Identification of meprin substrates has provided insights on how meprins could modulate kidney injury. Meprin targets in the kidney include extracellular matrix (ECM) proteins, modulators of inflammation, and proteins involved in the protein kinase A (PKA) and PKC signaling pathways. The current study used a global metabolomics approach to determine how meprin β expression impacts the metabolite milieu in diabetes and DKD.MethodsLow dose STZ was used to induce type 1 diabetes in 8-week old wild-type (WT) and meprin β knockout (βKO) mice. Blood and urine samples were obtained at 4 and 8 weeks post-STZ injection. Assays for albumin, creatinine, neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule − 1 (KIM-1), and cystatin C were used for biochemical assessment of kidney injury. Data for biomarkers of kidney injury utilized two-way ANOVA. Metabolomics data analysis utilized UPLC-QTOF MS and multivariate statistics.ResultsThe number of metabolites with diabetes-associated changes in levels were significantly higher in the WT mice when compared to meprin βKO counterparts. Annotated meprin β expression-associated metabolites with strong variable importance in projection (VIP) scores play roles in lipid metabolism (LysoPC(16:1(9Z)), taurocholic acid), amino acid metabolism (indoxyl sulfate, hippuric acid), and neurotransmitter/stress hormone synthesis (cortisol, 3-methoxy-4-hydroxyphenylethylene glycolsulfate, homovanillic acid sulfate). Metabolites that associated with meprin β deficiency include; 3,5-dihydroxy-3′,4′-dimethoxy-6,7-methylenedioxyflavone 3-glucuronide, pantothenic acid, and indoxyl glucuronide (all decreased in plasma).ConclusionTaken together, the annotated metabolites suggest that meprin β impacts complications of diabetes such as DKD by altering distinct metabolite profiles.

Isoform-specific interactions between meprin metalloproteases and the catalytic subunit of protein kinase A: significance in acute and chronic kidney injury

Meprin metalloproteases are abundantly expressed in the brush-border membranes of kidney proximal tubules. Meprins are implicated in ischemia-reperfusion (IR)-induced renal injury and diabetic nephropathy. The protein kinase A (PKA) signaling pathway modulates extracellular matrix metabolism in diabetic kidneys. The present study evaluated isoform-specific interactions between the catalytic subunit of PKA (PKA C) and meprins. To this end, cytosolic-enriched kidney proteins from meprin αβ double knockout mice, and purified forms of recombinant mouse PKA Cα, Cβ1, and Cβ2, were incubated with activated forms of either homomeric meprin A or meprin B. The cleaved protein products were subjected to SDS-PAGE and analyzed by Coomassie staining and Western blot analysis. While meprin A only cleaved PKA Cβ1, meprin B cleaved all three PKA C isoforms. Analysis of the proteolytic fragments by mass spectrometry revealed that meprin A and B cleave the PKA C isoforms at defined sites, resulting in unique cleavage products. Michaelis-Menten enzyme kinetics demonstrated that meprin B-mediated cleavage of PKA Cα occurs at a rate consistent with that of other physiologically relevant meprin substrates. Meprin cleavage decreased the kinase activity of PKA Cα, Cβ1, and Cβ2. PKA C levels were higher in diabetic kidneys, with evidence of in vivo fragmentation in wild-type diabetic kidneys. Confocal microscopy showed localization of meprin A in the glomeruli of diabetic kidneys. At 3 h post-IR, PKA C levels in proximal tubules decreased compared with distal tubules, which lack meprins. These data suggest that meprins may impact kidney injury, in part, via modulation of PKA signaling pathways.

Isoform‐specific interactions between meprin metalloproteases and the catalytic subunit of protein kinase A (690.13)

Meprin metalloproteases are abundantly expressed in the brush border membranes of kidney proximal tubules. Meprins are also expressed in podocytes, skin, and leukocytes. Meprins have been implicated in the pathology of acute and chronic kidney injury. To understand how meprins impact kidney disease, it is important to characterize interactions between meprins and their substrates. This study evaluated isoform‐specific interactions between the catalytic subunit of protein kinase A (PKA C) and meprins. Kidney proteins from meprin αβ double knockout mice, as well as purified recombinant mouse PKA Cα, Cβ1, and Cβ2, were incubated with activated forms of either meprin A (α‐α) or meprin B (β‐β). Cleavage products were analyzed by Coomassie staining or Western blot analysis. To identify meprin cleavage sites on PKA C isoforms, full length PKA C and meprin cleaved fragments were trypsin digested, separated by C18 nanoflow liquid chromatography‐MALDI, and analyzed by ProteinPilot. To characterize the kinetics of meprin B cleavage of PKA Cα, the Km and kcat were determined using Michaelis‐Menten enzyme kinetics. Meprin A only cleaved PKA Cβ1 while meprin B cleaved all three PKA C isoforms. Moreover, meprin cleavage of PKA Cα and Cβ2 significantly decreased their kinase activity, suggesting that the impact of meprins on kidney injury may be due, in part, to modulation of PKA signaling pathways.Grant Funding Source: Supported by NIH grants SC3GM102049, GM19301, GM19301, F31GM099415, and DK19691

Expression of SLC2A9 Isoforms in the Kidney and Their Localization in Polarized Epithelial Cells

BackgroundMany genome-wide association studies pointed out that SLC2A9 gene, which encodes a voltage-driven urate transporter, SLC2A9/GLUT9 (a.k.a. URATv1), as one of the most influential genes for serum urate levels. SLC2A9 is reported to encode two splice variants: SLC2A9-S (512 amino acids) and SLC2A9-L (540 amino acids), only difference being at their N-termini. We investigated isoform-specific localization of SLC2A9 in the human kidney and role of N-terminal amino acids in differential sorting in vitro.Methodology/Principal FindingsIsoform specific antibodies against SLC2A9 were developed and human kidney sections were stained. SLC2A9-S was expressed in the apical side of the collecting duct while SLC2A9-L was expressed in the basolateral side of the proximal tubule. GFP fused SLC2A9s were expressed in MDCK cells and intracellular localization was observed. SLC2A9-S was expressed at both apical and basolateral membranes, whereas SLC2A9-L was expressed only at the basolateral membrane. Although SLC2A9-L has a putative di-leucine motif at 33th and 34th leucine, deletion of the motif or replacement of leucine did not affect its subcellular localization. When up to 16 amino acids were removed from the N-terminal of SLC2A9-S or when up to 25 amino acids were removed from the N-terminal of SLC2A9-L, there was no change in their sorting. Deletion of 20 amino acids from SLC2A9-S was not expressed in the cell. More than 30 amino acids deletion from SLC2A9-L resulted in expression at both apical and basolateral membranes as well as in the lysosome. When amino acids from 25th and 30th were changed to alanine in SLC2A9-L, expression pattern was the same as wild-type.Conclusions/SignificanceSLC2A9-L was expressed in the basolateral membrane of kidney proximal tubules in humans and this isoform is likely to responsible for urate reabsorption. N-terminal amino acids unique to each isoform played an important role in protein stability and trafficking.

Potent Inhibitors of Human Organic Anion Transporters 1 and 3 from Clinical Drug Libraries: Discovery and Molecular Characterization

Transporter-mediated drug-drug interactions in the kidney dramatically influence the pharmacokinetics and other clinical effects of drugs. Human organic anion transporters 1 (hOAT1) and 3 (hOAT3) are the major transporters in the basolateral membrane of kidney proximal tubules, mediating the rate-limiting step in the elimination of a broad spectrum of drugs. In the present study, we screened two clinical drug libraries against hOAT1 and hOAT3. Of the 727 compounds screened, 92 compounds inhibited hOAT1 and 262 compounds inhibited hOAT3. When prioritized based on the peak unbound plasma concentrations of these compounds, three inhibitors for hOAT1 and seven inhibitors for hOAT3 were subsequently identified with high inhibitory potency (>95%). Computational analyses revealed that inhibitors and noninhibitors can be differentiated from each other on the basis of several physicochemical features, including number of hydrogen-bond donors, number of rotatable bonds, and topological polar surface area (TPSA) for hOAT1; and molecular weight, number of hydrogen-bond donors and acceptors, TPSA, partition coefficient (log P(7.4)), and polarizability for hOAT3. Pharmacophore modeling identified two common structural features associated with inhibitors for hOAT1 and hOAT3, viz., an anionic hydrogen-bond acceptor atom, and an aromatic center separated by ∼5.7 Å. Such model provides mechanistic insights for predicting new OAT inhibitors.

Reduced cholesterol levels in renal membranes of undernourished rats may account for urinary Na+ loss

It has been demonstrated that reabsorption of Na⁺ in the thick ascending limb is reduced and the ability to concentrate urine can be compromised in undernourished individuals. Alterations in phospholipid and cholesterol content in renal membranes, leading to Na⁺ loss and the inability to concentrate urine, were investigated in undernourished rats. Sixty-day-old male Wistar rats were utilized to evaluate (1) phospholipid and cholesterol content in the membrane fraction of whole kidneys, (2) cholesterol content and the levels of active Na⁺ transporters, (Na⁺ + K⁺)ATPase and Na⁺-ATPase, in basolateral membranes of kidney proximal tubules, and (3) functional indicators of medullary urine concentration. Body weight in the undernourished group was 73 % lower than in control. Undernourishment did not affect the levels of cholesterol in serum or in renal homogenates. However, membranes of whole kidneys revealed 56 and 66 % reduction in the levels of total phospholipids and cholesterol, respectively. Furthermore, cholesterol and (Na⁺ + K⁺)ATPase activity in proximal tubule membranes were reduced by 55 and 68 %, respectively. Oxidative stress remained unaltered in the kidneys of undernourished rats. In contrast, Na⁺-ATPase activity, an enzyme with all regulatory components in membrane, was increased in the proximal tubules of undernourished rats. Free water clearance and fractional Na⁺ excretion were increased by 86 and 24 %, respectively, and urinary osmolal concentration was 21 % lower in undernourished rats than controls. Life-long undernutrition reduces the levels of total phospholipids and cholesterol in membranes of renal tubular cells. This alteration in membrane integrity could diminish (Na⁺ + K⁺)ATPase activity resulting in reduced Na⁺ reabsorption and urinary concentrating ability.

Apical membrane segregation of phosphatidylinositol-4,5-bisphosphate influences parathyroid hormone 1 receptor compartmental signaling and localization via direct regulation of ezrin in LLC-PK1 cells

The parathyroid hormone 1 receptor (PTH1R), a primary regulator of mineral ion homeostasis, is expressed on both the apical and basolateral membranes of kidney proximal tubules and in the LLC-PK1 kidney cell line. In LLC-PK1 cells, apical PTH1R subpopulations are far more effective at signaling via phospholipase (PLC) than basolateral counterparts, revealing the presence of compartmental signaling. Apical PTH1R localization is dependent upon direct interactions with ezrin, an actin-membrane cross-linking scaffold protein. Ezrin undergoes an activation process that is dependent upon phosphorylation and binding to phosphatidylinositol-4,5-bisphosphate (PIP2), a lipid that is selectively concentrated to apical surfaces of polarized epithelia. Consistently, the intracellular probe for PIP2, GFP-PLCδ1-PH, localizes to the apical membranes of LLC-PK1 cells, directly overlapping ezrin and PTH1R expression. Activation of the apical PTH1R shifts the GFP-PLCδ1-PH probe from the apical membrane to the cytosol and basolateral membranes, reflecting domain-specific activation of PLC and hydrolysis of PIP2. This compartmental signaling is likely due to the polarized localization of PIP2, the substrate for PLC. PIP2 degradation using a membrane-directed phosphatase shifts ezrin localization to the cytosol and induces ezrin de-phosphorylation, processes consistent with inactivation. PIP2 degradation also shifts PTH1R expression from brush border microvilli to basolateral membranes and markedly blunts PTH-elicited activation of the MAPK pathway. Transient expression of ezrin in HEK293 cells shifts PTH1R expression from the plasma membrane to microvilli-like surface projections that also contain PIP2. As a result, ezrin enhances PTH mediated activation of the PLC pathway in this cell model with increasing total receptor surface expression. Collectively, these findings demonstrate that the apical segregation of PIP2 to the apical domains not only promotes the activation of ezrin and the subsequent formation of the PTH1R containing scaffold, but also ensures the presence of ample substrate for propagating the PLC pathway.

Dietary fructose, salt absorption and hypertension in metabolic syndrome: towards a new paradigm.

The worldwide increase in the incidence of metabolic syndrome correlates with marked increase in total fructose intake in the form of high-fructose corn syrup, beverage and table sugar. Increased dietary fructose intake in rodents has been shown to recapitulate many aspects of metabolic syndrome by causing hypertension, insulin resistance and hyperlipidaemia. Recent studies demonstrated that increased dietary fructose intake stimulates salt absorption in the small intestine and kidney tubules, resulting in a state of salt overload and thus causing hypertension. The absorption of salt (sodium and chloride) in the small intestine is predominantly mediated via the chloride/base exchangers DRA (Down Regulated in Adenoma) (SLC26A3) and PAT1 (Putative Anion Transporter 1) (SLC26A6), and the Na(+) /H(+) exchanger NHE3 (Sodium Hydrogen Exchanger3) (SLC9A3). PAT1 and NHE3 also co-localize on the apical membrane of kidney proximal tubule. Luminal fructose stimulated salt absorption in the jejunum and kidney tubules, responses that were significantly diminished in PAT1 null mice. These studies further demonstrated that Glut5 (SLC2A5) is the major fructose-absorbing transporter in the small intestine (and kidney proximal tubule) and plays an essential role in the systemic homeostasis of fructose. Increased dietary fructose intake for several weeks upregulated the expression of NHE3, PAT1 and Glut5 in the intestine and resulted in hypertension in wild-type mice, a response that was almost abolished in PAT1 null mice and abrogated in Glut5 null mice. This article will discuss the interaction of Glut5 with salt-absorbing transporters and review the role of dietary fructose in enhanced salt absorption in intestine and kidney as it relates to the pathogenesis of hypertension in metabolic syndrome.

Role of Isoleucine-554 in Lithium Binding by the Na+/Dicarboxylate Cotransporter NaDC1

Sodium-coupled transport of citric acid cycle intermediates, such as succinate and citrate, is mediated by the NaDC1 transporter located on the apical membrane of kidney proximal tubule and small intestine cells. Our previous study showed that transmembrane helix (TM) 11 of NaDC1 is important for sodium and lithium binding, as well as for determining citrate affinity [Kahn and Pajor (1999) Biochemistry 38, 6151]. In the present study, 21 amino acids in TM11 and the extracellular loop of NaDC1 were mutated one at a time to cysteine. All of the mutants were well expressed on the plasma membrane, but many of them had decreased transport activity. The G550C, W561C, and L568C mutants were inactive, suggesting that these residues may be critical for function. None of the cysteine mutants was sensitive to inhibition by the membrane-impermeant cysteine reagents, MTSET or MTSES, suggesting that the helix is inaccessible to the extracellular solvent. Although NaDC1 is inhibited by low concentrations of lithium in the presence of sodium, the I554C mutant was stimulated by lithium with a K(0.5) of 4.8 mM. The I554C mutant also had decreased affinity for sodium. We conclude that TM11 is likely to be an outer helix in NaDC1 that contains several residues critical for transport. Ile-554 in the middle of the helix may be an important determinant of cation affinity and selectivity, in particular the high affinity cation binding site that recognizes lithium.

Orphan Transporter SLC6A18 Is Renal Neutral Amino Acid Transporter B0AT3

The orphan transporter Slc6a18 (XT2) is highly expressed at the luminal membrane of kidney proximal tubules and displays approximately 50% identity with Slc6a19 (B(0)AT1), which is the main neutral amino acid transporter in both kidney and small intestine. As yet, the amino acid transport function of XT2 has only been experimentally supported by the urinary glycine loss observed in xt2 null mice. We report here that in Xenopus laevis oocytes, co-expressed ACE2 (angiotensin-converting enzyme 2) associates with XT2 and reveals its function as a Na(+)- and Cl(-)-de pend ent neutral amino acid transporter. In contrast to its association with ACE2 observed in Xenopus laevis oocytes, our experiments with ace2 and collectrin null mice demonstrate that in vivo it is Collectrin, a smaller homologue of ACE2, that is required for functional expression of XT2 in kidney. To assess the function of XT2 in vivo, we reanalyzed its knock-out mouse model after more than 10 generations of backcrossing into C57BL/6 background. In addition to the previously published glycinuria, we observed a urinary loss of several other amino acids, in particular beta-branched and small neutral ones. Using telemetry, we confirmed the previously described link of XT2 absence with hypertension but only in physically restrained animals. Taken together, our data indicate that the formerly orphan transporter XT2 functions as a sodium and chloride-de pend ent neutral amino acid transporter that we propose to rename B(0)AT3.

The role of SLC26A6‐mediated chloride/oxalate exchange in causing susceptibility to nephrolithiasis

The role of SLC26A6‐mediated chloride/oxalate exchange in causing susceptibility to nephrolithiasis

Mouse organic anion transporter 2 and 3 (mOAT2/3[ Slc22a7/8]) mediates the renal transport of bumetanide

Multispecific organic anion transporters play an important role in the excretion and the elimination of a wide variety of endogenous and exogenous substrates. To date, five murine OAT homologs such as mouse organic anion transporters 1–3, 5, and 6 (mOAT1–3, 5 and 6) have been isolated and well characterized. With the exception of mOAT6, other mOAT isoforms are predominantly expressed in the kidney. The aim of this study was to examine whether mOAT2/3, as well as hOAT2/3, transports the diuretic bumetanide using a Xenopus laevis oocyte expression system. When expressed in Xenopus oocytes, mOAT2/3 mediated the high affinity transport of bumetanide. The apparent K m values for the uptake of bumetanide via mOAT2 and mOAT3 were 9.12 ± 2.42 μM and 1.01 ± 0.27 μM, respectively. Immunohistochemical analysis revealed that mOAT2 is expressed on the luminal membrane site of the proximal tubule. Our results indicate that mOAT2 and 3, as well as human homologs, are molecules for the transport of bumetanide on the luminal membranes of kidney proximal tubules.

Glucose-Stimulated Upregulation of GLUT2 Gene Is Mediated by Sterol Response Element–Binding Protein-1c in the Hepatocytes

GLUT2 is mainly expressed in the liver, beta-cells of the pancreas, and the basolateral membrane of kidney proximal tubules and plays an important role in glucose homeostasis in living organisms. The transcription of the GLUT2 gene is known to be upregulated in the liver during postprandial hyperglycemic states or in type 2 diabetes. However, a molecular mechanism by which glucose activates GLUT2 gene expression is not known. In this study, we report evidence that sterol response element-binding protein (SREBP)-1c plays a key role in glucose-stimulated GLUT2 gene expression. The GLUT2 promoter reporter is activated by SREBP-1c, and the activation is inhibited by a dominant-negative form of SREBP-1c (SREBP-1c DN). Adenoviral expression of SREBP-1c DN suppressed glucose-stimulated GLUT2 mRNA level in primary hepatocytes. An electrophoretic mobility shift assay and mutational analysis of the GLUT2 promoter revealed that SREBP-1c binds to the -84/-76 region of the GLUT2 promoter. Chromatin immunoprecipitation revealed that the binding of SREBP-1c to the -84/-76 region was increased by glucose concentration in a dose-dependent manner. These results indicate that SREBP-1c mediates glucose-stimulated GLUT2 gene expression in hepatocytes.

Protein kinase C-mediated inhibition of renal Ca 2+ ATPase by physiological concentrations of angiotensin II is reversed by AT 1- and AT 2-receptor antagonists

Angiotensin II (Ang II) increases the cytosolic Ca 2+ concentration in different cell types. In this study, we investigate the effect of Ang II on the Ca 2+ ATPase of purified basolateral membranes of kidney proximal tubules. This enzyme pumps Ca 2+ out of the cytosol in a reaction coupled to ATP hydrolysis, and it is responsible for the fine-tuned regulation of cytosolic Ca 2+ activity. Ca 2+-ATPase activity is inhibited by picomolar concentrations of Ang II, with maximal inhibition being attained at ≈50% of the control values. The presence of raising concentrations (10 −11 to 10 −7 M) of losartan (an AT 1-receptor antagonist) or PD123319 (an AT 2-receptor antagonist) gradually reverts inhibition by Ang II. Both the phospholipase C (PLC) inhibitor U-73122 (10 −6 M) and the inhibitor of protein kinase C (PKC) staurosporine (10 −7 M) prevent inhibition of the Ca 2+ pump by Ang II. Incubation of the previously isolated membranes with a PKC activator—the phorbol ester 12- O-tetradecanoylphorbol-13-acetate (10 −8 M)—mimics the inhibition found with Ang II, and the effects of the compounds are not additive. Taken as a whole, these results indicate the Ang II inhibits Ca 2+-ATPase by activation of a PKC system present in primed state in these membranes after binding of the hormone to losartan- and PD123319-sensitive receptors coupled to a PLC. Therefore, inhibition of the basolateral membrane Ca 2+-ATPase by kinase-mediated phosphorylation appears to be one of the pathways by which Ang II promotes an increase in the cytosolic Ca 2+ concentration of proximal tubule cells.

Angiotensin-(1–7) modulates the ouabain-insensitive Na +-ATPase activity from basolateral membrane of the proximal tubule

Angiotensin-(1–7) (Ang-(1–7)) modulates the Na +-ATPase, but not the Na +,K +-ATPase activity present in pig kidney proximal tubules. The Na +-ATPase, insensitive to ouabain, but sensitive to furosemide, is stimulated by Ang-(1–7) (68% by 10 −9 M), in a dose-dependent manner. This effect is due to an increase in V max, while the apparent affinity of the enzyme for Na + is not modified. Saralasin, a general angiotensin receptor antagonist, abolishes the stimulation, demonstrating that the Ang-(1–7) effect is mediated by receptor. The Ang-(1–7) stimulatory effect is not changed by either PD 123319, an AT 2 receptor antagonist, or A779, an Ang-(1–7) receptor antagonist. On the other hand, increasing the concentration of the AT 1 receptor antagonist losartan from 10 −11 to 10 −9 M, reverses the Ang(1–7) stimulation completely. A further increase to 10 −3 M losartan reverses the Na +-ATPase activity to a level similar to that obtained with Ang-(1–7) (10 −9 M) alone. The stimulatory effect of Ang-(1–7) at 10 −9 M is similar to the effect of angiotensin II (AG II) alone. However, when the two peptides are both present, Na +-ATPase activity is restored to control values. These data suggest that Ang-(1–7) selectively modulates the Na +-ATPase activity present in basolateral membranes of kidney proximal tubules through a losartan-sensitive receptor. This receptor is probably different from the receptor involved in the stimulation of the Na +-ATPase activity by angiotensin II.

LAT2, a New Basolateral 4F2hc/CD98-associated Amino Acid Transporter of Kidney and Intestine

Glycoprotein-associated amino acid transporters (gpaAT) are permease-related proteins that require heterodimerization to express their function. So far, four vertebrate gpaATs have been shown to associate with 4F2hc/CD98 for functional expression, whereas one gpaAT specifically associates with rBAT. In this study, we characterized a novel gpaAT, LAT2, for which mouse and human cDNAs were identified by expressed sequence tag data base searches. The encoded ortholog proteins are 531 and 535 amino acids long and 92% identical. They share 52 and 48% residues with the gpaATs LAT1 and y(+)LAT1, respectively. When mouse LAT2 and human 4F2hc cRNAs were co-injected into Xenopus oocytes, disulfide-linked heterodimers were formed, and an L-type amino acid uptake was induced, which differed slightly from that produced by LAT1-4F2hc: the apparent affinity for L-phenylalanine was higher, and L-alanine was transported at physiological concentrations. In the presence of an external amino acid substrate, LAT2-4F2hc also mediated amino acid efflux. LAT2 mRNA is expressed mainly in kidney and intestine, whereas LAT1 mRNA is expressed widely. Immunofluorescence experiments showed colocalization of 4F2hc and LAT2 at the basolateral membrane of kidney proximal tubules and small intestine epithelia. In conclusion, LAT2 forms with LAT1 a subfamily of L-type gpaATs. We propose that LAT1 is involved in cellular amino acid uptake, whereas LAT2 plays a role in epithelial amino acid (re)absorption.

Adenosine inhibits the renal plasma-membrane (Ca2+ + Mg2+)-ATPase through a pathway sensitive to cholera toxin and sphingosine.

Adenosine, a potent autacoid produced and released in kidneys, affects nearly all aspects of renal function, and an increase in cytosolic calcium has been implicated in adenosine effects. The aim of this work was to investigate whether adenosine modifies the calcium pump present in basolateral membranes of kidney proximal tubule cells. Adenosine exerts a biphasic influence on (Ca2+ + Mg2+)-ATPase activity. Inhibition occurs up to 0.1 microM and then gradually disappears as the adenosine concentration increases to 100 microM, an effect mimicked by the adenosine analog N6-cyclohexyladenosine, which preferentially binds to A1-type receptors. In contrast, the A2 receptor agonist 5', N-ethylcarboxamideadenosine is ineffective. The A1 receptor antagonist 8-cyclopentyl-1,3-dimethylxanthine blocks the inhibitory effect of 0.1 microM adenosine and stimulates (Ca2+ + Mg2+)-ATPase activity in the presence of 1 mM adenosine, a concentration high enough to occupy the low-affinity A2 receptors. Inhibition by adenosine increases as medium ATP is lowered to micromolar concentrations, is maintained in the presence of pertussis toxin, and is completely abolished with 0.1 microM cholera toxin or 1 microM sphingosine. The inhibitory effect of adenosine can be reproduced by guanosine 5'-[gamma-thio]triphosphate, inositol 1,4, 5-trisphosphate or the diacylglycerol analog 12-O-tetradecanoylphorbol 13-acetate. In conjunction with the selectivity for its analogs and for its receptor agonist, the concentration profile of adenosine effects indicates that both inhibitory (A1) and stimulatory (A2) receptors are involved. The results obtained with the toxins indicate that a pathway that is modulated by G-proteins, involves a phospholipase C and a protein kinase C, and is affected by local variations in adenosine concentrations participates in the regulation of the (Ca2+ + Mg2+)-ATPase resident in basolateral membranes of kidney proximal tubules.

Expression of the conjugate export pump encoded by the mrp2 gene in the apical membrane of kidney proximal tubules.

A novel ATP-dependent export pump for amphiphilic anionic conjugates, which has been cloned recently from liver, was identified in rat kidney and localized to the apical membrane domain of proximal tubule epithelia. This 190-kD membrane glycoprotein (Mrp2) has been described previously as the hepatocyte canalicular isoform of the multidrug resistance protein and as the canalicular multispecific organic anion transporter. Mrp2 was identified in kidney by reverse transcription PCR followed by sequencing of the amplified 786-bp fragment and by immunoblotting, using an antibody specifically reacting with the carboxy terminus of rat Mrp2. Double immunofluorescence and confocal laser-scanning microscopy showed the presence of Mrp2 in the brush-border membrane domain of segments S1, S2, and S3 of proximal tubule epithelia. Mrp2 was not detectable in other segments of the nephron. The onset of Mrp2 expression during development occurred in a very early stage of nephron development. Mrp2 represents the first cloned ATP-dependent export pump for amphiphilic organic anions identified in kidney and localized to the apical membrane domain of proximal tubule epithelia. Mrp2 may contribute to cellular detoxification and to the secretion of endogenous and xenobiotic anionic substances, most of which are conjugates, from the blood into urine.