Basolateral Membrane Of Proximal Tubule Research Articles

Role of Angiotensin II Type 1a Receptor (AT1aR) of Renal Tubules in Regulating Inwardly Rectifying Potassium Channels 4.2 (Kir4.2), Kir4.1, and Epithelial Na+ Channel (ENaC).

Kir4.2 and Kir4.1 play a role in regulating membrane transport in the proximal tubule (PT) and in the distal-convoluted-tubule (DCT), respectively. We generated kidney-tubule-specific-AT1aR-knockout (Ks-AT1aR-KO) mice to examine whether renal AT1aR regulates Kir4.2 and Kir4.1. Ks-AT1aR-KO mice had a lower systolic blood pressure than Agtr1aflox/flox (control) mice. Ks-AT1aR-KO mice had a lower expression of NHE3 (Na+/H+-exchanger 3) and Kir4.2, a major Kir-channel in PT, than Agtr1aflox/flox mice. Whole-cell recording also demonstrated that the membrane potential in PT of Ks-AT1aR-KO mice was lesser negative than Agtr1aflox/flox mice. The expression of Kir4.1 and Kir5.1, Kir4.1/Kir5.1-mediated K+ currents of DCT and DCT membrane potential in Ks-AT1aR-KO mice, were similar to Agtr1aflox/flox mice. However, angiotensin II perfusion for 7 days hyperpolarized the membrane potential in PT and DCT of the control mice but not in Ks-AT1aR-KO mice, while angiotensin II perfusion did not change the expression of Kir4.1, Kir4.2, and Kir5.1. Deletion of AT1aR did not significantly affect the expression of αENaC (epithelial Na+ channel) and βENaC but increased cleaved γENaC expression. Patch-clamp experiments demonstrated that deletion of AT1aR increased amiloride-sensitive Na+-currents in the cortical-collecting duct but not in late-DCT. However, tertiapin-Q sensitive renal outer medullary potassium channel currents were similar in both genotypes. AT1aR determines the baseline membrane potential of PT by controlling Kir4.2 expression/activity but AT1aR is not required for determining the baseline membrane potential of the DCT and Kir4.1/Kir5.1 activity/expression. However, AT1aR is required for angiotensin II-induced hyperpolarization of basolateral membrane of PT and DCT. Deletion of AT1aR had no effect on baseline renal outer medullary potassium channel activity but increased ENaC activity in the CCD.

Adenosine stimulates the basolateral 50pS K+ channel in renal proximal tubule via adenosine-A1 receptor.

Background: The basolateral potassium channels play an important role in maintaining the membrane transport in the renal proximal tubules (PT) and adenosine receptors have been shown to regulate the trans-epithelial Na+ absorption in the PT. The aim of the present study is to explore whether adenosine also regulates the basolateral K+ channel of the PT and to determine the adenosine receptor type and the signaling pathway which mediates the effect of adenosine on the K+ channel. Methods: We have used the single channel recording to examine the basolateral K+ channel activity in the proximal tubules of the mouse kidney. All experiments were performed in cell-attached patches. Results: Single channel recording has detected a 50pS inwardly-rectifying K+ channel with high channel open probability and this 50 pS K+ channel is a predominant type K+ channel in the basolateral membrane of the mouse PT. Adding adenosine increased 50pS K+ channel activity in cell-attached patches, defined by NPo (a product of channel Numbers and Open Probability). The adenosine-induced stimulation of the 50pS K+ channel was absent in the PT pretreated with DPCPX, a selective inhibitor of adenosine A1 receptor. In contrast, adenosine was still able to stimulate the 50pS K+ channel in the PT pretreated with CP-66713, a selective adenosine A2 receptor antagonist. This suggests that the stimulatory effect of adenosine on the 50pS K+ channel of the PT was mediated by adenosine-A1 receptor. Moreover, the effect of adenosine on the 50 pS K+ channel was blocked in the PT pretreated with U-73122 or Calphostin C, suggesting that adenosine-induced stimulation of the 50pS K+ channels of the PT was due to the activation of phospholipase C (PLC) and protein kinase C (PKC) pathway. In contrast, the inhibition of phospholipase A2 (PLA2) with AACOCF3 or inhibition of protein kinase A (PKA) with H8 failed to block the adenosine-induced stimulation of the 50pS K+ channel of the PT. Conclusion: We conclude that adenosine activates the 50pS K+ channels in the basolateral membrane of PT via adenosine-A1 receptor. Furthermore, the effect of adenosine on the 50pS K+ channel is mediated by PLC-PKC signaling pathway.

Base (HCO3-/CO32-) Transport Properties of SLC4 Proteins: New Insights in Acid-Base Kidney Physiology.

H+ or base transporters and channels in the mammalian genome play important roles in the maintenance of numerous cellular biochemical and physiologic processes throughout the body. Among the known base transporters, those within the SLC4 and SLC26 gene families are involved in cell, transepithelial, and whole organ function. Whether the functional properties of these transporters involve HCO3-, CO32-, or HCO3-/CO32- stimulated H+ (or OH-) transport has not received widespread attention in the literature. Accordingly, "bicarbonate" is the term typically used in most textbooks without greater specificity. Moreover, clinicians and physiologists have historically focused on the blood HCO3- concentration as the base term in the Henderson-Hasselbalch equation in the analysis of clinical acid-base abnormalities, thus, bicarbonate has been assumed to be the species reabsorbed along the nephron as required to maintain the blood [HCO3-] at approximately 25 mM. However, accumulating data in the literature suggest that carbonate, rather than bicarbonate, is the species absorbed across the proximal tubule basolateral membrane, whereas in the collecting duct, bicarbonate is indeed transported. Various experimental approaches leading to this new concept are herein reviewed.

Renal Deletion of LRRC8/VRAC Channels Induces Proximal Tubulopathy.

Volume-regulated anion channels (VRACs) are heterohexamers of LRRC8A with LRRC8B, -C, -D, or -E in various combinations. Depending on the subunit composition, these swelling-activated channels conduct chloride, amino acids, organic osmolytes, and drugs. Despite VRACs' role in cell volume regulation, and large osmolarity changes in the kidney, neither the localization nor the function of VRACs in the kidney is known. Mice expressing epitope-tagged LRRC8 subunits were used to determine the renal localization of all VRAC subunits. Mice carrying constitutive deletions of Lrrc8b-e, or with inducible or cell-specific ablation of Lrrc8a, were analyzed to assess renal functions of VRACs. Analysis included histology, urine and serum parameters in different diuresis states, and metabolomics. The kidney expresses all five VRAC subunits with strikingly distinct localization. Whereas LRRC8C is exclusively found in vascular endothelium, all other subunits are found in the nephron. LRRC8E is specific for intercalated cells, whereas LRRC8A, LRRC8B, and LRRC8D are prominent in basolateral membranes of proximal tubules. Conditional deletion of LRRC8A in proximal but not distal tubules and constitutive deletion of LRRC8D cause proximal tubular injury, increased diuresis, and mild Fanconi-like symptoms. VRAC/LRRC8 channels are crucial for the function and integrity of proximal tubules, but not for more distal nephron segments in spite of their larger need for volume regulation. LRRC8A/D channels may be required for the basolateral exit of many organic compounds, including cellular metabolites, in proximal tubules. Proximal tubular injury likely results from combined accumulation of several transported molecules in the absence of VRAC channels.

Expression Regulation of Water Reabsorption Genes and Transcription Factors in the Kidneys of Lepus yarkandensis.

Lepus yarkandensis is a desert-dwelling animal that has various adaptations to cope with drought. The kidney maintains water and acid-base balance mainly through the vasopressin-regulated water reabsorption pathway and proximal tubular bicarbonate reabsorption pathway. In this study, we compared the differentially expressed genes (DEGs) and transcription factors in the kidneys of L. yarkandensis and Oryctolagus cuniculus to explore the relationship between the DEGs in kidneys and the animals’ adaptations. Transcriptome sequencing data were used to predict the differentially-expressed water reabsorption genes and their transcription factors. Quantitative real-time PCR, immunohistochemistry, and western blotting were used to detect and verify the expression of DEGs in the kidney at mRNA and protein levels. Transcriptome analysis of the kidney of L. yarkandensis and O. cuniculus showed that 6,610 genes were up-regulated and 5,727 genes down-regulated in data shared by both species. According to the data, 232 transcription factors and their corresponding target genes were predicted, from which genes and transcription factors related to renal water reabsorption were screened. Quantitative RT-PCR results showed AQP1, AQP2, ADCY3, HIF1A, CREB3, and NFATc1 had higher expression in the L. yarkandensis kidney; in comparison, FXYD2 mRNA expression levels were lower. In western blotting, transcription factors HIF1A, NFATc1, NF-κB1, and critical genes ADCY3, ATPA1, and SLC4A4, were highly expressed in the kidneys of L. yarkandensis. Immunohistochemical results showed that the ADCY3 protein was in the basolateral membrane of the collecting duct, the ATP1A1 protein was in the basolateral membrane and medulla of proximal tubules, and the SLC4A4 protein was in the basolateral membrane of proximal tubules. According to these results can be inferred that HIF1A, NFATc1, and NF-κB1 play a certain role in regulating the expression of genes related to water reabsorption in the kidney of L. yarkandensis, thus improving the water reclamation efficiency of L. yarkandensis, so as to adapt to the arid desert environment.

The Defense against Whole‐Animal Respiratory Acidosis in AT 1A knockout mice

Numerous diseases cause acid-base disturbances, and severe alterations in arterial pH (pHa) can have devastating consequences for the patient. The renal proximal tubule (PT) normally handles ~80% of renal H⁺ secretion, and appropriately adjusts its rate of acid secretion (JH), most of which represents HCO3− reabsorption, in response to respiratory acidosis (RAc: increased [CO2] → decreased pH) and metabolic acidosis(MAc: decreased [HCO3−] → decreased pH). Rather than responding to changes in pH at the extracellular side of the basolateral (BL) membrane, the PT raises JHwhen [CO2]BL rises or [HCO₃⁻]BL falls. How exactly the PT senses Δ[CO2]BL and Δ[HCO3−]BL and transduces the signal throughout the cellis poorly understood. However, our laboratory has made three observations. First, the knockout (KO) of receptor protein tyrosine phosphatase γ(RPTPγ), a novel extracellular CO2/HCO3− sensor localized in the PT BL membrane, eliminates the ΔJHproduced by Δ[CO2]BL or Δ[HCO3−]BL, and substantially reduces the ability of the mouse to regulate pHa during MAc. Second, inhibitors of ErbB receptor tyrosine kinases at the BL membrane likewise eliminate the ΔJH response. And third, the ΔJH response requires active ACE to produce ANG II that, upon secretion, binds to apical AT1A receptors. We hypothesize that RPTPγ and its downstream effectors (ErbB1, ErbB2, ACE, and AT1A) are also essential in the whole-body responses to RAc. Here we describe a novel assay in which we subject WT vs AT1A⁻/⁻ mice to 8% inspired CO2 to induce RAc. We cannulate the left carotid artery and, after recovery, sequentially sample arterial blood from conscious mice under control (Ctrl = ~0% CO2) vs RAc conditions at 5 min, 1 h, 24 h, 48 h and 7 days post-initiation to determine the ability of the mouse to defend pHa against the acid-base disturbance. After 5 min of 8% CO2, in both WT and AT1A⁻/⁻ pHa decreases from ~7.41 to ~7.24, arterial p CO2increases from ~29 to ~64 mmHg, and [HCO3−]a increases from ~18 to ~28 mM. 4 h post hypercapnia onset, pHa recovers to ~7.28 in both WT and AT1A-/- mice, arterial pCO2 increases to ~73 mmHg, and [HCO₃⁻]a increases to ~34 mM. At 48 h, pHa plateaus at ~7.34 (i.e. without returning to baseline values). These data indicate that the partial pHa recovery during RAc is similar in both WT and AT1A⁻/⁻ mice. Previous preliminary data showed that AT1A⁻/⁻ mice have a limited ability to defend pHa during MAc. However, AT1A does not seem to be essential to defend pHa during RAc.

Potassium channels in control of renal function

Potassium channels are important to control membrane potential and drive epithelial transport processes. In this issue of Kidney International, Bignon etal. report the role of the Kir4.2 K+-channel, localized at the basolateral membrane of proximal tubules, in the reabsorption of bicarbonate and the modulation of renal ammoniagenesis. The findings have implications for our understanding of how the kidney reacts to hypokalemia, an acid load, and the metabolic acidosis of patients with advanced stages of chronic kidney disease.

NBCe1-A Regulates Proximal Tubule Ammonia Metabolism under Basal Conditions and in Response to Metabolic Acidosis.

Renal ammonia metabolism is the primary mechanism through which the kidneys maintain acid-base homeostasis, but the molecular mechanisms regulating renal ammonia generation are unclear. In these studies, we evaluated the role of the proximal tubule basolateral plasma membrane electrogenic sodium bicarbonate cotransporter 1 variant A (NBCe1-A) in this process. Deletion of the NBCe1-A gene caused severe spontaneous metabolic acidosis in mice. Despite this metabolic acidosis, which normally causes a dramatic increase in ammonia excretion, absolute urinary ammonia concentration was unaltered. Additionally, NBCe1-A deletion almost completely blocked the ability to increase ammonia excretion after exogenous acid loading. Under basal conditions and during acid loading, urine pH was more acidic in mice with NBCe1-A deletion than in wild-type controls, indicating that the abnormal ammonia excretion was not caused by a primary failure of urine acidification. Instead, NBCe1-A deletion altered the expression levels of multiple enzymes involved in proximal tubule ammonia generation, including phosphate-dependent glutaminase, phosphoenolpyruvate carboxykinase, and glutamine synthetase, under basal conditions and after exogenous acid loading. Deletion of NBCe1-A did not impair expression of key proteins involved in collecting duct ammonia secretion. These studies demonstrate that the integral membrane protein NBCe1-A has a critical role in basal and acidosis-stimulated ammonia metabolism through the regulation of proximal tubule ammonia-metabolizing enzymes.

Distribution of organic anion transporters NaDC3 and OAT1-3 along the human nephron.

The initial step in renal secretion of organic anions (OAs) is mediated by transporters in the basolateral membrane (BLM). Contributors to this process are primary active Na(+)-K(+)-ATPase (EC 3.6.3.9), secondary active Na(+)-dicarboxylate cotransporter 3 (NaDC3/SLC13A3), and tertiary active OA transporters (OATs) OAT1/SLC22A6, OAT2/SLC22A7, and OAT3/SLC22A8. In human kidneys, we analyzed the localization of these transporters by immunochemical methods in tissue cryosections and isolated membranes. The specificity of antibodies was validated with human embryonic kidney-293 cells stably transfected with functional OATs. Na(+)-K(+)-ATPase was immunolocalized to the BLM along the entire human nephron. NaDC3-related immunostaining was detected in the BLM of proximal tubules and in the BLM and/or luminal membrane of principal cells in connecting segments and collecting ducts. The thin and thick ascending limbs, macula densa, and distal tubules exhibited no reactivity with the anti-NaDC3 antibody. OAT1-OAT3-related immunostaining in human kidneys was detected only in the BLM of cortical proximal tubules; all three OATs were stained more intensely in S1/S2 segments compared with S3 segment in medullary rays, whereas the S3 segment in the outer stripe remained unstained. Expression of NaDC3, OAT1, OAT2, and OAT3 proteins exhibited considerable interindividual variability in both male and female kidneys, and sex differences in their expression could not be detected. Our experiments provide a side-by-side comparison of basolateral transporters cooperating in renal OA secretion in the human kidney.

Mutation in the Monocarboxylate Transporter 12 Gene Affects Guanidinoacetate Excretion but Does Not Cause Glucosuria.

A heterozygous mutation (c.643C>A; p.Q215X) in the monocarboxylate transporter 12-encoding gene MCT12 (also known as SLC16A12) that mediates creatine transport was recently identified as the cause of a syndrome with juvenile cataracts, microcornea, and glucosuria in a single family. Whereas the MCT12 mutation cosegregated with the eye phenotype, poor correlation with the glucosuria phenotype did not support a pathogenic role of the mutation in the kidney. Here, we examined MCT12 in the kidney and found that it resides on basolateral membranes of proximal tubules. Patients with MCT12 mutation exhibited reduced plasma levels and increased fractional excretion of guanidinoacetate, but normal creatine levels, suggesting that MCT12 may function as a guanidinoacetate transporter in vivo However, functional studies in Xenopus oocytes revealed that MCT12 transports creatine but not its precursor, guanidinoacetate. Genetic analysis revealed a separate, undescribed heterozygous mutation (c.265G>A; p.A89T) in the sodium/glucose cotransporter 2-encoding gene SGLT2 (also known as SLC5A2) in the family that segregated with the renal glucosuria phenotype. When overexpressed in HEK293 cells, the mutant SGLT2 transporter did not efficiently translocate to the plasma membrane, and displayed greatly reduced transport activity. In summary, our data indicate that MCT12 functions as a basolateral exit pathway for creatine in the proximal tubule. Heterozygous mutation of MCT12 affects systemic levels and renal handling of guanidinoacetate, possibly through an indirect mechanism. Furthermore, our data reveal a digenic syndrome in the index family, with simultaneous MCT12 and SGLT2 mutation. Thus, glucosuria is not part of the MCT12 mutation syndrome.

Expression of basolateral organic anion and cation transporters in experimental cadmium nephrotoxicity in rat kidney.

Cadmium (Cd)-intoxicated experimental animals exhibit impaired renal secretion of organic anions (OA) and cations (OC), indicating their transporters (Oats and Octs) in the proximal tubule (PT) basolateral membrane as possible targets of Cd. To correlate transport data from the literature with the expression of relevant transporters, we performed immunochemical and RT-PCR studies of renal Oats and Octs in the subchronic (treatment with CdCl2; 2 mg Cd/kg b.m./day, for 2 weeks) and acute (treatment with Cd-metallothionein (CdMT); 0.4 mg Cd/kg b.m., 6 or 12 h before killing) models of Cd nephrotoxicity. In the subchronic model, PT exhibited a minor loss of basolateral invaginations and overall unchanged expression of Na(+)/K(+)-ATPase and GAPDH proteins and mRNAs, while the expression of Oat and Oct proteins and their mRNAs was strongly downregulated. In the acute model, a time-related redistribution of basolateral transporters to the intracellular vesicular compartment was a major finding. However, 6 h following CdMT treatment, the total abundance of Oat and Oct proteins in the renal tissue remained unchanged, the expression of mRNAs decreased only for Oats, while a limited Oat1 and Na(+)/K(+)-ATPase immunoreactivity in the PT apical membrane indicated loss of cell polarity. As tested in rats treated with colchicine, the observed loss/redistribution of basolateral transporters in both models may be independent on microtubules. Therefore, the diminished renal secretion of OA and OC via PT in Cd nephrotoxicity may result from (a) limited loss of secretory surface (basolateral invaginations), (b) selective loss of Oats and Octs, and

Mouse organic cation transporter 1 determines properties and regulation of basolateral organic cation transport in renal proximal tubules

The proximal tubule of mouse kidney expresses mouse organic cation transporter 1 (mOCT1), mOCT2, and much less mOCT3. Therefore, mOCT-mediated transport across the basolateral membrane of proximal tubules reflects properties of at least mOCT1 and mOCT2. Here, we unraveled substrate affinities and modulation of transport activity by acute regulation by protein kinases on mOCT1 and mOCT2 separately and compared these findings with those from isolated proximal tubules of male and female mOCT2−/− mice. These data are also compared to our recent reports on isolated tubules from wild-type and mOCT1/2 double knockout (mOCT1/2−/−) mice. OCT-mediated transport in proximal tubules of mOCT2−/− mice was only 20 % lower compared to those isolated from wild-type mice. While mOCT1 was regulated by all five pathways examined [protein kinase A (PKA), protein kinase C (PKC), p56lck, phosphoinositide 3-kinase (PI3K), and calmodulin (CaM)], mOCT2 activity was modulated by PKA, p56lck, and CaM only, however, in the same direction. As mOCT-mediated transport across the basolateral membrane of mOCT2−/− mice expressing only mOCT1 and to a small amount mOCT3 was identical to that observed for tubules isolated from wild-type mice and to that observed for human embryonic kidney 293 (HEK293) cells stably expressing mOCT1, mOCT1 represents the relevant paralog for OCT-dependent organic cation transport in the mouse kidney. Gender does not play a major role in expression and activity of renal OCT-mediated transport in the mouse. Properties of mouse OCT considerably differ from those of rat or human origin, and thus, observations made in these rodents cannot directly be transferred to the human situation

Glucocorticoid mediates the transcription of OAT-PG, a kidney-specific prostaglandin transporter

OAT-PG is a kidney-specific prostaglandin transporter and exclusively expressed at the basolateral membrane of proximal tubules in rodent kidneys. We previously reported that OAT-PG was dominantly expressed in the male kidney similar to the other SLC22 family proteins as organic anion transporter (OAT) 1 and OAT3. Recently, Wegner et al. revealed that a transcription factor, B-cell CLL/lymphoma 6 (BCL6), is associated with the male-dominant expressions of OAT1 and OAT3 in the rat kidney. Here, we performed the luciferase assay to investigate whether OAT-PG is also transcriptionally regulated by BCL6. However, the promoter activity of OAT-PG was not directly affected by BCL6 overexpression nor the testosterone treatment, suggesting that different regulatory mechanisms underlie the male-dominant transcriptional regulation of OAT-PG compared to those of OAT1 and OAT3. We newly found that adrenalectomy (Adx) of male rat caused a significant reduction of OAT-PG expression without any significant changes in the OAT1 and OAT3 expressions, and it was recovered by the dexamethasone administration. Furthermore, the renocortical PGE2 concentration was markedly increased in Adx male rat, concomitant with the downregulation of OAT-PG, and it was reduced to the basal level by dexamethasone treatment. In the luciferase assay, dexamethasone stimulated OAT-PG promoter activity but not OAT1. The luciferase activity responsiveness to dexamethasone was significantly reduced by the deletion of glucocorticoid response elements in the OAT-PG promoter region. These results suggest that glucocorticoid plays an important role in the regulation of the renocortical PGE2 concentration by the transcriptional regulation of OAT-PG in the rat kidney.

Precise comparison of protein localization among OCT, OAT, and MATE in human kidney

Organic anion transporters (OATs) and organic cation transporters (OCT) play pivotal roles in the uptake of drugs into epithelial cells at the basolateral membranes, and multidrug and toxin extrusion (MATE) mediates drug secretion into urine at the brush-border membranes. In this study, the expression and distribution of apical MATE1 and MATE2-K, and basolateral OAT1, OAT3, and OCT2 were compared using serial sections of human kidney cortex. First, mRNA expression in the proximal tubules was evaluated using laser microdissection. Levels of OAT, OCT2, and MATE mRNA in the proximal tubules were greatly higher compared with glomerulus. The results quantitatively indicated that these transporters were localized to proximal tubules in the renal cortex. Second, MATE1 and MATE2-K protein were detected in proximal epithelial cells in which OCT2 protein was expressed at the basolateral membranes. In addition, MATE1 was expressed at the brush-border membranes of tubular epithelial cells in which OAT1 and OAT3 were expressed. The results confirmed that OAT1, OAT3, OCT2, MATE1, and MATE2-K were coexpressed in tubular epithelial cells. The cooperation among OAT, OCT, and MATE in renal drug secretion was consistent with their distribution.

Abstract 471: Mechanism of Pressure-Natriuresis: Evidence that Extracellular cGMP Signals through Renal Basolateral Na+ K+-ATP-ase

Pressure-Natriuresis (P-N) is a major compensatory mechanism that prevents an increase in blood pressure by increasing renal sodium excretion (U Na V). Our group showed that cGMP, at least in part, mediates P-N. cGMP signals from the extracellular space through a yet unidentified receptor in the renal proximal tubule basolateral membrane. We hypothesize that cGMP binds to Na + K + ATP-ase (NKA), activating both the Src kinase and the EGFR-Erk-Akt signaling cascades. In uninephrectomized rats, the renal perfusion pressure (RPP) was increased from 100 to 135 mmHg. During high RPP, U Na V increased 6.3-fold and the ratio of phosphorylated Src (Tyr 416 )/total Src (pSrc) increased from 0.07±0.02 to 0.71±0.12 μmol/min (P<0.001, N=5 for both). Blocking the NKA ouabain (OUA) binding site by infusing rostafuroxin (RF, a specific OUA antagonist without effect on NKA pump activity) directly into the renal interstitium reduced U Na V 6.4-fold and pSrc to 0.22±0.05 (N=8, P<0.05). Both phosphorylation of Erk 1/2 (p44/42 MAPK) and Akt (Ser 473 ) were increased during the high RPP period (0.35±0.07 to 2.08±0.21; N=6 for both, P<10 -4 and 0.39±0.06 to 1.04±0.15; N=8 for both, P<0.01, respectively). However, RF did not decrease pErk and pAkt, likely due to in vivo compensatory processes following NKA blockade. To delineate the role of cGMP in P-N, we infused cGMP into the renal interstitium. U Na V increased 5.7-fold and pSrc increased from 0.09±0.02 (N=4) to 0.35±0.1 (N=6, P<0.05). OUA infusion was used as a positive control and increased pSrc to 0.77±0.01 (N=4, P<10 -6 ). Coinfusion of cGMP and RF reduced pSrc to 0.15±0.03 (P=0.07, N=6). Surprisingly, RF infusion also decreased pErk from 0.71±0.17 (N=6, in cGMP-infused rats) to 0.33±0.09 (N=6, in cGMP and RF-coinfused rats, P=0.085). In summary, renal interstitial infusion of an NKA signaling antagonist RF blocks Src activation during both P-N and cGMP-induced natriuresis. P-N-induced Erk and Akt activation is not affected by RF. However, Erk and Akt phosphorylation is reduced during cGMP-induced natriuresis, suggesting a possible competition between cGMP and RF for the same binding site on NKA. The data support the hypothesis that P-N is mediated by extracellular cGMP binding to basolateral NKA, which serves as a receptor.

Kidney Transplantation Down-Regulates Expression of Organic Cation Transporters, Which Translocate β-Blockers and Fluoroquinolones

Kidney transplanted patients are often treated with immunosuppressive, antihypertensive, and antibiotic drugs such as cyclosporine A (CsA), β-blockers, and fluoroquinolones, respectively. Organic cation transporters (OCT) expressed in the basolateral membrane of proximal tubules represent an important drug excretion route. In this work, the renal expression of OCT after syngeneic and allogeneic kidney transplantation in rats with or without CsA immunosuppression was studied. Moreover, the interactions of CsA, β-blockers (pindolol/atenolol), and fluoroquinolones (ofloxacin/norfloxacin) with rOCT1, rOCT2, hOCT1, and hOCT2 in stably transfected HEK293-cells were studied. Kidney transplantation was associated with reduced expression of rOCT1, while rOCT2 showed only reduced expression after allogeneic transplantation. All drugs interacted subtype- and species-dependently with OCT. However, only atenolol, pindolol, and ofloxacin were transported by hOCT2, the main OCT in human kidneys. While CsA is not an OCT substrate, it exerts a short-term effect on OCT activity, changing their affinity for some substrates. In conclusion, appropriate drug dosing in transplanted patients is difficult partly because OCT are down-regulated and because concomitant CsA treatment may influence the affinity of the transporters. Moreover, drug-drug competition at the transporter can also alter drug excretion rate.

Sex-dependent expression of Oat3 (Slc22a8) and Oat1 (Slc22a6) proteins in murine kidneys

In the mouse kidney, organic anion transporter 3 (mOat3, Slc22a8) was previously localized to the basolateral membrane (BLM) of proximal tubule (PT), thick ascending limb of Henle, macula densa, distal tubule, and cortical collecting duct. However, the specificity of anti-Oat3 antibodies (Oat3-Ab) used in these studies was not properly verified. Moreover, the sex-dependent expression of mOat3, and of the functionally similar transporter mOat1 (Slc22a6), in the mouse kidney has been studied at mRNA level, whereas their protein expression is poorly documented. Here we investigated 1) specificity of Oat3-Abs by using Oat3 knockout (KO) mice, 2) cell localization of renal mOat3 with a specific mOat3-Ab, 3) sex-dependent expression of renal mOat3 and mOat1 proteins, and 4) hormone(s) responsible for observed sex differences. As previously shown, an Oat3-Ab against the rat protein stained the BLM of various nephron segments in wild-type (WT) mice, but the same staining pattern was noted along the nephron of Oat3 KO mice. However, the mOat3-Ab exclusively stained the BLM of PT in WT mice, where it colocalized with the mOat1 protein, whereas no staining of Oat3 protein was noted in the kidney of Oat3 KO mice. The expression of mOat3 protein was lower in male mice, upregulated by castration, and downregulated by testosterone treatment. The expression of mOat1 protein was stronger in males, downregulated by castration, and upregulated by testosterone treatment. Thus, at the protein level, mOat3 and mOat1 exhibit sex-dependent expression with an opposite pattern; mOat3 is female dominant due to androgen inhibition, while mOat1 is male dominant due to androgen stimulation.

Proximal renal tubular acidosis mediated by mutations in NBCe1-A: unraveling the transporter's structure-functional properties

NBCe1 belongs to the SLC4 family of base transporting membrane proteins that plays a significant role in renal, extrarenal, and systemic acid-base homeostasis. Recent progress has been made in characterizing the structure-function properties of NBCe1 (encoded by the SLC4A4 gene), and those factors that regulate its function. In the kidney, the NBCe1-A variant that is expressed on the basolateral membrane of proximal tubule is the key transporter responsible for overall transepithelial bicarbonate absorption in this nephron segment. NBCe1 mutations impair transepithelial bicarbonate absorption causing the syndrome of proximal renal tubular acidosis (pRTA). Studies of naturally occurring NBCe1 mutant proteins in heterologous expression systems have been very helpful in elucidation the structure-functional properties of the transporter. NBCe1 mutations are now known to cause pRTA by various mechanisms including the alteration of the transporter function (substrate ion interaction, electrogenicity), abnormal processing to the plasma membrane, and a perturbation in its structural properties. The elucidation of how NBCe1 mutations cause pRTA in addition to the recent studies which have provided further insight into the topology of the transporter have played an important role in uncovering its critically important structural-function properties.

Deletion of the γ-Aminobutyric Acid Transporter 2 (GAT2 and SLC6A13) Gene in Mice Leads to Changes in Liver and Brain Taurine Contents

The GABA transporters (GAT1, GAT2, GAT3, and BGT1) have mostly been discussed in relation to their potential roles in controlling the action of transmitter GABA in the nervous system. We have generated the first mice lacking the GAT2 (slc6a13) gene. Deletion of GAT2 (both mRNA and protein) neither affected growth, fertility, nor life span under nonchallenging rearing conditions. Immunocytochemistry showed that the GAT2 protein was predominantly expressed in the plasma membranes of periportal hepatocytes and in the basolateral membranes of proximal tubules in the renal cortex. This was validated by processing tissue from wild-type and knockout mice in parallel. Deletion of GAT2 reduced liver taurine levels by 50%, without affecting the expression of the taurine transporter TAUT. These results suggest an important role for GAT2 in taurine uptake from portal blood into liver. In support of this notion, GAT2-transfected HEK293 cells transported [(3)H]taurine. Furthermore, most of the uptake of [(3)H]GABA by cultured rat hepatocytes was due to GAT2, and this uptake was inhibited by taurine. GAT2 was not detected in brain parenchyma proper, excluding a role in GABA inactivation. It was, however, expressed in the leptomeninges and in a subpopulation of brain blood vessels. Deletion of GAT2 increased brain taurine levels by 20%, suggesting a taurine-exporting role for GAT2 in the brain.

Hepatic and renal sat‐1 and CFEX in ethylene glycol‐induced oxalate nephrolithiasis in rats

The epidemiological incidence of oxalate (OX) nephrolithiasis is higher in men than in women. More than 85% of the body OX is produced in liver and then released into the systemic circulation by the action of sulfate‐OX exchanger sat1 in the hepatocyte sinusoidal membrane. In kidneys, sat1 in the proximal tubule basolateral membrane mediates OX uptake, whereas the chloride‐formate‐ oxalate exchanger CFEX in the brush‐border membrane finally extrudes OX into the urine. Here we used the model of ethylene glycol (EG)‐induced OX nephrolithiasis in adult male (M) and female (F) rats to study sat1, CFEX, and rate‐limiting enzymes of OX synthesis, alcohol dehydrogenase (Adh1) and hydroxy acid oxidase (Hao1). Compared to controls, EG‐treated animals exhibited higher concentrations of OX in plasma and urine and higher abundance of OX crystals in urine, with a M dominance. Sat1 protein in liver and kidneys, which was M‐dominant in control animals, in EG‐treated animals increased only in F rats, while sat1 mRNA stayed unchanged. CFEX mRNA in both organs was sex‐independent and unaffected by EG treatment. Adh1 and Hao1 mRNA in both organs remained also unchanged upon EG treatment. In conclusion, despite hyperoxaluria in EG‐treated animals, the expression of sat1 in M and of CFEX in both sexes was sufficient to handle the EG‐induced production and secretion of OX.