Membrane Of Renal Proximal Tubules Research Articles

Characterization of Human Organic Anion Transporter 4 (hOAT4) and Mouse Oat5 (mOat5) As Functional Orthologs for Renal Anion Uptake and Efflux Transport.

Organic anions (OAs) are compounds including drugs or toxicants that are negatively charged at physiologic pH and are typically transported by organic anion transporters (OATs). Human OAT4 (SLC22A11) is expressed in the apical membrane of renal proximal tubules. Although there is no rodent ortholog of hOAT4, rodents express Oat5 (Slc22a19), an anion exchanger that is also localized to the apical membrane of renal proximal tubule cells. The purpose of this study was to determine the functional similarity between mouse Oat5 and human OAT4. Chinese hamster ovary (CHO) cells expressing SLC22A11 or Slc22a19 were used to assess the transport characteristics of radiolabeled ochratoxin (OTA). We determined the kinetics of OTA transport; the resulting Michaelis constant (Kt) and maximal rate of mediated substrate transport (Jmax) values were very similar for both hOAT4 and mOat5: Kt 3.9 and 7.2 μM, respectively, and Jmax 4.4 and 3.9 pmol/cm2, respectively. For the profile of OTA inhibition by OAs, IC50 values were determined for several clinically important drugs and toxicants. The resulting IC50 values ranged from 9 μM for indomethacin to ∼600 μM for the diuretic hydrochlorothiazide. We measured the efflux of OTA from preloaded cells; both hOAT4 and mOat5 supported the efflux of OTA. These data support the hypothesis that OAT4 and Oat5 are functional orthologs and share selectivity for OTA both for reabsorption and secretion. SIGNIFICANCE STATEMENT: This study compares the selectivity profile between human organic anion transporter (OAT4) and mouse Oat5. Our data revealed a similar selectivity profile for ochratoxin A (OTA) reabsorption and secretion by these two transporters, thereby supporting the hypothesis that hOAT4 and mOat5, although not genetic orthologs, behave as functional orthologs for both uptake and efflux. These data will be instrumental in selecting an appropriate animal model when studying the renal disposition of anionic drugs and toxicants.

G Protein-Coupled Receptor 37L1 Modulates Epigenetic Changes in Human Renal Proximal Tubule Cells.

Renal luminal sodium transport is essential for physiological blood pressure control, and abnormalities in this process are strongly implicated in the pathogenesis of essential hypertension. Renal G protein-coupled receptors (GPCRs) are critical for the regulation of the reabsorption of essential nutrients, ions, and water from the glomerular filtrate. Recently, we showed that GPCR 37L1 (GPR37L1) is expressed on the apical membrane of renal proximal tubules (RPT) and regulates luminal sodium transport and blood pressure by modulating the function of the sodium proton exchanger 3 (NHE3). However, little is known about GPR37L1 intracellular signaling. Here, we show that GPR37L1 is localized to the nuclear membrane, in addition to the plasma membrane in human RPT cells. Furthermore, GPR37L1 signals via the PI3K/AKT/mTOR pathway to decrease the expression of DNA (cytosine-5)-methyltransferase 1 (DNMT1) and enhance NHE3 transcription. Overall, we demonstrate the direct role of a nuclear membrane GPCR in the regulation of renal sodium through epigenetic gene regulation.

Fibroblast growth factor 23 leads to endolysosomal routing of the renal phosphate cotransporters NaPi-IIa and NaPi-IIc in vivo.

Na+-dependent phosphate cotransporters NaPi-IIa and NaPi-IIc, located at the brush-border membrane of renal proximal tubules, are regulated by numerous factors, including fibroblast growth factor 23 (FGF23). FGF23 downregulates NaPi-IIa and NaPi-IIc abundance after activating a signaling pathway involving phosphorylation of ERK1/2 (phospho-ERK1/2). FGF23 also downregulates expression of renal 1-α-hydroxylase (Cyp27b1) and upregulates 24-hydroxylase (Cyp24a1), thus reducing plasma calcitriol levels. Here, we examined the time course of FGF23-induced internalization of NaPi-IIa and NaPi-IIc and their intracellular pathway toward degradation in vivo. Mice were injected intraperitoneally with recombinant human (rh)FGF23 in the absence (biochemical analysis) or presence (immunohistochemistry) of leupeptin, an inhibitor of lysosomal proteases. Phosphorylation of ERK1/2 was enhanced 60 min after rhFGF23 administration, and increased phosphorylation was still detected 480 min after injection. Colocalization of phospho-ERK1/2 with NaPi-IIa was seen at 60 and 120 min and partly at 480 min. The abundance of both cotransporters was reduced 240 min after rhFGF23 administration, with a further reduction at 480 min. NaPi-IIa and NaPi-IIc were found to colocalize with clathrin and early endosomal antigen 1 as early as 120 min after rhFGF23 injection. Both cotransporters partially colocalized with cathepsin B and lysosomal-associated membrane protein-1, markers of lysosomes, 120 min after rhFGF23 injection. Thus, NaPi-IIa and NaPi-IIc are internalized within 2 h upon rhFGF23 injection. Both cotransporters share the pathway of clathrin-mediated endocytosis that leads first to early endosomes, finally resulting in trafficking toward the lysosome as early as 120 min after rhFGF23 administration.NEW & NOTEWORTHY The hormone fibroblast growth factor 23 (FGF23) controls phosphate homeostasis by regulating renal phosphate excretion. FGF23 acts on several phosphate transporters in the kidney. Here, we define the time course of this action and demonstrate how phosphate transporters NaPi-IIa and NaPi-IIc are internalized.

Quantitation of Plasma Membrane Drug Transporters in Kidney Tissue and Cell Lines Using a Novel Proteomic Approach Enabled a Prospective Prediction of Metformin Disposition.

The successful prospective incorporation of in vitro transporter kinetics in physiologically based pharmacokinetic (PBPK) models to describe drug disposition remains challenging. Although determination of scaling factors to extrapolate in vitro to in vivo transporter kinetics has been facilitated by quantitative proteomics, no robust assessment comparing membrane recoveries between different cells/tissues has been made. HEK293 cells overexpressing OCT2, MATE1, and MATE2K or human kidney cortex were homogenized and centrifuged to obtain the total membrane fractions, which were subsequently subjected to liquid-liquid extraction followed by centrifugation and precipitation to isolate plasma membrane fractions. Plasma membrane recoveries determined by quantitation of the marker Na+/K+-ATPase in lysate and plasma membrane fractions were ≤20% but within 3-fold across different cells and tissues. A separate study demonstrated that recoveries are comparable between basolateral and apical membranes of renal proximal tubules, as measured by Na+/K+-ATPase and γ-glutamyl transpeptidase 1, respectively. The plasma membrane expression of OCT2, MATE1, and MATE2K was quantified and relative expression factors (REFs) were determined as the ratio between the tissue and cell concentrations. Corrections using plasma membrane recovery had minimal impact on REF values (<2-fold). In vitro transporter kinetics of metformin were extrapolated to in vivo using the corresponding REFs in a PBPK model. The simulated metformin exposures were within 2-fold of clinical exposure. These results demonstrate that transporter REFs based on plasma membrane expression enable a prediction of transporter-mediated drug disposition. Such REFs may be estimated without the correction of plasma membrane recovery when the same procedure is applied between different matrices. SIGNIFICANCE STATEMENT: Transporter REFs based on plasma membrane expression enable in vitro-in vivo extrapolation of transporter kinetics. Plasma membrane recoveries as determined by the quantification of sodium-potassium adenosine triphosphatase were comparable between the in vitro and in vivo systems used in the present study, and therefore had minimal impact on the transporter REF values.

The Effects of Co‐Expression of Rhbg with Human CA‐IV on NH 3 /NH 4 + and CO 2 Transport

Background Rhbg is a member of the mammalian glycosylated rhesus (Rh) protein family expressed in multiple tissues including the basolateral membrane of the renal collecting duct. Rhbg was found to transport NH4+ and NH3 in the collecting duct. Carbonic anhydrase IV (CA-IV) is one of several carbonic anhydrases expressed in the mammalian kidney where it catalyzes the reversible hydration of CO2. It is anchored at both apical and basolateral membranes of renal proximal tubules and the collecting duct. Objective and Hypothesis Our previous studies showed that Rhbg and CA-IV proteins co-immunoprecipitated, suggesting that there is a physical interaction between Rhbg and CA-IV. This study is to detect if there is any functional effect related to this interaction. Methods We measured transport of NH4+, NH3, and CO2 in 4 groups of Xenopus oocytes expressing the following: In the first group, we co-expressed the mouse Rhbg with human CA-IV (by injecting oocytes with Rhbg and CA-IV mRNA) and compared the measurements to 3 groups of oocytes expressing either Rhbg or CA-IV, or injected with H2O. We used ion-selective microelectrodes to measure changes in surface pH (pHs), to monitor NH3 transport, and changes in intracellular pH (pHi) to monitor NH4+ and CO2 transport. We also used two-electrode voltage-clamp to measure current change (ΔI) caused by electrogenic NH4+ transport. In each oocyte, we monitored one of the three parameters indicated above (pHs, pHi or ΔI), in response to five experimental conditions: 1) increased CO2 (1.5%CO2/10mM HCO3-); 2) NH3/NH4+ (5mM NH4Cl in HEPES); 3) Methylamine/Methylammonium, often used as a substitute for ammonium (MA/MA+, 5mM in HEPES); 4) 5mM NH3/NH4+ in 1.5%CO2; 5) 5mM MA/MA+ in 1.5% CO2. Results Our results indicate that co-expressing CA-IV with Rhbg: 1) inhibited electrogenic NH4+ transport by Rhbg in the presence and absence of CO2; 2) reduced MA and NH3 transport by Rhbg only in the presence of CO2; 3) had no detectable effect on CO2 transport by Rhbg. Conclusions We demonstrated for the first time that co-expression of Rhbg and CA-IV inhibited electrogenic transport of NH4+ by Rhbg and reduced NH3 and MA transport in the presence of CO2. The function of Rhbg as a CO2 gas channel was demonstrated as an increase in the rate of CO2-induced pHiacidification in Rhbg expressing oocytes and was not altered by expression of CA-IV. The interaction of Rhbg and CA-IV is important to explain their role in renal acid-base homeostasis. We propose that NH3/NH4+ transport by Rhbg is modified in the presence of CO2.

G protein‐coupled receptor 37L1 participates in epigenetic gene regulation in human renal proximal tubule cells

G protein‐coupled receptors (GPCRs) in the kidney regulate the reabsorption of essential nutrients, ions, and water from the glomerular filtrate. Abnormality in the renal luminal sodium transport plays an important role in the pathogenesis of essential hypertension. There are a growing number of GPCRs and their interacting protein components that are localized on the nuclear membrane or within the nucleus, where they retain their signaling functions. In most cases, nuclear GPCRs are constitutively expressed on the nuclear membrane. The functions of these nuclear GPCRs are not well understood. Recently we showed that G protein‐coupled receptor 37L1 (GPR37L1), expressed on the apical membrane of renal proximal tubules (RPT), regulates luminal sodium transport and blood pressure via modulating NHE3 function. However, the GPR37L1 signaling pathway in the RPT has not been studied. Co‐immunoprecipitation (Co‐IP), combined with mass spectrometry analyses, revealed the association of GPR37L1 with nuclear import proteins: IPO7, IPO8, KPNA2, KPNB1; nuclear export proteins: XPO1 and XPO7; nuclear pore complex proteins: NUP210; kinases involved in signaling: mTOR, PRKDC, IRS4; and a DNA (Cytosine‐5‐)‐Methyltransferase 1 (DNMT1) in the GPR37L1 protein complex. Immunoblot analyses of nuclear preparations from the human RPT cells (hRPTCs) confirmed the presence of GPR37L1 in the nucleus. Confocal fluorescence imaging of hRPTCs expressing GPR37L1 tagged with green fluorescence protein (GPR37L1‐GFP) showed distinct nuclear membrane expression of GPR37L1. Association of GPR37L1 with mTOR, PRKDC, and DNMT1 proteins was confirmed by Co‐IP followed by immunoblot analyses. Overexpression of GPR37L1 in hRPTCs increased the phosphorylation of PI3, and AKT and decreased the expression of DNMT1, and NHE3 promoter methylation. Our data show that GPR37L1 signals via the PI3K/AKT/mTOR pathway to decrease the expression of DNMT1. Decreased DNMT1 function reduces CpG methylation on the NHE3 promoter that in turn increases NHE3 transcription. This is the first report showing the direct role of a nuclear GPCR in the regulation of renal sodium transport through epigenetic gene regulation.Support or Funding InformationR56DK116828: Prasad Konkalmatt

Meprin β expression associated with decreased proximal tubule PKA Cβ levels in ischemia/reperfusion‐induced acute kidney injury

Ischemia/ reperfusion (IR) is a leading cause of acute kidney injury (AKI). Meprins are metalloproteases that are expressed abundantly in the brush border membranes of renal proximal tubules (PTs). The levels of expression and the localization of meprins were previously shown to associate with IR‐induced AKI. Studies from our group showed that meprin B proteolytically cleaves three isoforms of the catalytic subunit of PKA, namely, PKA Cα1, Cβ1, and Cβ2. The cleavage by meprin B resulted in decreased kinase activity of these PKA C isoforms. Furthermore, the total levels of PKA C were significantly decreased in the meprin expressing PTs at 3h post‐IR when compared to distal tubules which are deficient in meprins. The objective of the current study was to determine which of the PKA C isoforms is impacted by meprin B and thus involved in the IR‐response mediated by meprin β. To this end, surgical procedures were used to induce unilateral IR (with contralateral nephrectomy) in 12‐week old wild‐type (WT) and meprin β knockout (βKO) mice on a C57BL/6 background. Kidney tissues were harvested at 6h post IR. Kidney proteins were extracted and fractionated into BBM‐, cytosolic‐, and nuclear‐enriched fractions. Western blot analysis was used to determine the levels of Cα and Cβ in the cytosolic‐ and nuclear‐enriched fractions. Immunofluorescence staining was used to determine the localization of PKA C (total), Cβ, and Cα. Statistical analysis utilized 2‐way ANOVA. The total levels of PKA C decreased in kidneys from WT mice subjected to IR but not in βKO counterparts. However, use of isoform‐specific antibodies demonstrated increases in the levels of Cβ but not Cα in the cytosolic‐ and nuclear‐enriched fractions. Two Cβ protein bands (41 and 37 kDa) were detected in cytosolic‐ and one band (41 kDa) in nuclear‐enriched fractions. The levels of the 41 kDa Cβ protein species were higher in lysates from WT kidneys at 6h post‐IR (P=0.0001). In contrast, the 37 kDa species, which was detected in nuclear‐enriched fractions only, decreased in βKO (P=0.002) but not in WT kidneys subjected to IR. A closer histological analysis of kidney sections showed that the level of damage to the brush borders was not uniform in all proximal tubules. More importantly, immunohistochemical analysis showed that the PKA Cβ levels decreased in damaged/injured meprin β expressing PTs but appeared to increase in intact PTs. These changes were not observed in PTs from meprin βKO kidneys. Individual interstitial cells (presumed to be resident macrophages) also stained positively for Cβ in WT but not in βKO kidney sections. Taken together, the data suggest that the PKA Cβ isoform is involved in the response to IR‐induced renal injury. The Cβ levels are known to be abundant in lymphoid tissue but our data indicate expression in immune cells resident in kidney tissue as well as epithelial tubular cells in response to IR‐induced AKI. More studies are needed to determine the response genes mediated by PKA Cβ signaling in IR to increase our understanding of how meprins modulate kidney injury in mice.Support or Funding InformationThis study was supported by the National Institute of Health/National Institutes of General Medical Sciences (NIGMS) Grant #s SC3GM102049 and SC1GM118271

Expression of NaPi-IIb in rodent and human kidney and upregulation in a model of chronic kidney disease.

Na+-coupled phosphate cotransporters from the SLC34 and SLC20 families of solute carriers mediate transepithelial transport of inorganic phosphate (Pi). NaPi-IIa/Slc34a1, NaPi-IIc/Slc34a3, and Pit-2/Slc20a2 are all expressed at the apical membrane of renal proximal tubules and therefore contribute to renal Pi reabsorption. Unlike NaPi-IIa and NaPi-IIc, which are rather kidney-specific, NaPi-IIb/Slc34a2 is expressed in several epithelial tissues, including the intestine, lung, testis, and mammary glands. Recently, the expression of NaPi-IIb was also reported in kidneys from rats fed on high Pi. Here, we systematically quantified the mRNA expression of SLC34 and SLC20 cotransporters in kidneys from mice, rats, and humans. In all three species, NaPi-IIa mRNA was by far the most abundant renal transcript. Low and comparable mRNA levels of the other four transporters, including NaPi-IIb, were detected in kidneys from rodents and humans. In mice, the renal expression of NaPi-IIa transcripts was restricted to the cortex, whereas NaPi-IIb mRNA was observed in medullary segments. Consistently, NaPi-IIb protein colocalized with uromodulin at the luminal membrane of thick ascending limbs of the loop of Henle segments. The abundance of NaPi-IIb transcripts in kidneys from mice was neither affected by dietary Pi, the absence of renal NaPi-IIc, nor the depletion of intestinal NaPi-IIb. In contrast, it was highly upregulated in a model of oxalate-induced kidney disease where all other SLC34 phosphate transporters were downregulated. Thus, NaPi-IIb may contribute to renal phosphate reabsorption, and its upregulation in kidney disease might promote hyperphosphatemia.

SUN-138 Roles of Na+/HCO3- cotransporter NBCn2 in acid-base transport in the apical membrane of renal proximal tubules

Proximal tubules (PTs) reclaim approximately 80% of filtered HCO3- Acid-base transport in PTs is mainly Na dependent and conducted coordinately by apical Na+/H+ exchanger (NHE)3 and basolateral Na+/HCO3- cotransporter (NBC)e1. It is believed that the apical NHE3 and the vacuolar H+ ATPase (V-ATPase) are mainly responsible for the reabsorption in the apical membrane of PTs. However, an NHE inhibitor, S3226 or amiloride inhibited less than 50% of Na or HCO3- reabsorption in vivo. A recent study reported that devastating mutation of NHE3 did not cause proximal renal tubular acidosis in human.

Clinical Extrapolation of the Effects of Dolutegravir and Other HIV Integrase Inhibitors on Folate Transport Pathways.

Preliminary analysis of ongoing birth surveillance study identified evidence of potential increased risk for neural tube defects (NTDs) in newborns associated with exposure to dolutegravir at the time of conception. Folate deficiency is a common cause of NTDs. Dolutegravir and other HIV integrase inhibitor drugs were evaluated in vitro for inhibition of folate transport pathways: proton-coupled folate transporter (PCFT), reduced folate carrier (RFC), and folate receptor α (FRα)-mediated endocytosis. Inhibition of folate transport was extrapolated to the clinic by using established approaches for transporters in intestine, distribution tissues, and basolateral and apical membranes of renal proximal tubules (2017 FDA Guidance). The positive controls, methotrexate and pemetrexed, demonstrated clinically relevant inhibition of PCFT, RFC, and FRα in folate absorption, distribution, and renal sparing. Valproic acid was used as a negative control that elicits folate-independent NTDs; valproic acid did not inhibit PCFT, RFC, or FRα At clinical doses and exposures, the observed in vitro inhibition of FRα by dolutegravir and cabotegravir was not flagged as clinically relevant; PCFT and RFC inhibition was not observed in vitro. Bictegravir inhibited both PCFT and FRα, but the observed inhibition did not reach the criteria for clinical relevance. Elvitegravir and raltegravir inhibited PCFT, but only raltegravir inhibition of intestinal PCFT was flagged as potentially clinically relevant at the highest 1.2-g dose (not the 400-mg dose). These studies showed that dolutegravir is not a clinical inhibitor of folate transport pathways, and it is not predicted to elicit clinical decreases in maternal and fetal folate levels. Clinically relevant HIV integrase inhibitor drug class effect on folate transport pathways was not observed. SIGNIFICANCE STATEMENT: Preliminary analysis of ongoing birth surveillance study identified evidence of potential increased risk for neural tube defects (NTDs) in newborns associated with exposure to the HIV integrase inhibitor dolutegravir at the time of conception; folate deficiency is a common cause of NTDs. Dolutegravir and other HIV integrase inhibitor drugs were evaluated in vitro for inhibition of the major folate transport pathways: proton-coupled folate transporter, reduced folate carrier, and folate receptor α-mediated endocytosis. The present studies showed that dolutegravir is not a clinical inhibitor of folate transport pathways, and it is not predicted to elicit clinical decreases in maternal and fetal folate levels. Furthermore, clinically relevant HIV integrase inhibitor drug class effect on folate transport pathways was not observed.

Tandem Requirement for Renal D1R and D5R Functionality

The peripheral dopaminergic system assists in maintaining blood pressure homeostasis by engendering natriuresis mainly through the D1R and D5R receptors. These receptors share structural features and pharmacological profiles; there is no specific agonist that can discriminate between these two receptors. Genetic ablation of one receptor in the kidney results in the loss of activity of the other. Therefore, we evaluated the mechanisms for the cooperative and co‐dependent interaction between these two receptors. Both receptors share the same anatomical distribution, specifically at the brush border and luminal membranes of renal proximal tubules and collecting ducts, respectively. These receptors dimerized (~110 kDa) and coimmunoprecipitated each other, basally and after fenoldopam (D1R/D5R agonist, 1 μM/15 min) treatment. Bimolecular fluorescence complementation showed that heterologously expressed D1R and D5R tagged with the C and N termini of the fluorescent protein EYFP, respectively, heterodimerized basally in HEK‐293 cells. This was corroborated by confocal microscopy of HEK‐293 cells that were transiently transfected with D1R::RFP and D5R::GFP, which showed colocalization basally at the plasma membrane, most abundantly at the apical membrane in polarized cells. The D1R and D5R partitioned to lipid and non‐lipid rafts; agonist treatment enhanced the lipid raft segregation of D1R and D5R. Both receptors were abundantly expressed in renal proximal tubules of human and C57Bl/6 mouse kidneys, where they colocalized basally that was increased by fenoldopam (1 μM/15 min). D1R/D5R stimulation is known to increase cAMP production and inhibit renal sodium transport. Receptor gene silencing via antisense technology in human renal proximal tubule cells (hRPTCs) decreased the cAMP response to fenoldopam (−71±2% for D1R, vs. −38±7% for D5R, vs. −90±2% for both, *P&lt;0.05). In hRPTCs grown in Transwells, fenoldopam or ouabain (500 nM/30 min) given at the basolateral compartment inhibited Na+/K+‐ATPase (NKA) activity and increased intracellular Na+. siRNA‐mediated gene silencing of DRD1/DRD5 prevented the inhibition of NKA activity by fenoldopam (*P&lt;0.05). Our observations demonstrate the requirement for D1R/D5R to form heterodimers to achieve their full functionality.Support or Funding InformationR01DK039308, R01HL092196, and P01HL074940This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cloning, function, and localization of human, canine, and Drosophila ZIP10 (SLC39A10), a Zn2+ transporter.

Zinc (Zn2+) is the second most abundant trace element, but is considered a micronutrient, as it is a cofactor for many enzymes and transcription factors. Whereas Zn2+ deficiency can cause cognitive immune or metabolic dysfunction and infertility, excess Zn2+ is nephrotoxic. As for other ions and solutes, Zn2+ is moved into and out of cells by specific membrane transporters: ZnT, Zip, and NRAMP/DMT proteins. ZIP10 is reported to be localized at the apical membrane of renal proximal tubules in rats, where it is believed to play a role in Zn2+ import. Renal regulation of Zn2+ is of particular interest in light of growing evidence that Zn2+ may play a role in kidney stone formation. The objective of this study was to show that ZIP10 homologs transport Zn2+, as well as ZIP10, kidney localization across species. We cloned ZIP10 from dog, human, and Drosophila ( CG10006), tested clones for Zn2+ uptake in Xenopus oocytes and localized the protein in renal structures. CG10006, rather than foi (fear-of-intimacy, CG6817) is the primary ZIP10 homolog found in Drosophila Malpighian tubules. The ZIP10 antibody recognizes recombinant dog, human, and Drosophila ZIP10 proteins. Immunohistochemistry reveals that ZIP10 in higher mammals is found not only in the proximal tubule, but also in the collecting duct system. These ZIP10 proteins show Zn2+ transport. Together, these studies reveal ZIP10 kidney localization, a role in renal Zn2+ transport, and indicates that CG10006 is a Drosophila homolog of ZIP10.

The Clinical Implication of Compounds which could induce the function of human organic cation transporter 2 (hOCT2), while inhibit the function of human Multidrug and Toxin Extrusion Proteins 1 (hMATE1)

The human organic cation transporter 2 and human multidrug and toxin extrusion protein 1 (hOCT2/hMATE1) transport system is a pair of transporter proteins highly expressed at the basolateral and apical membrane of renal proximal tubules respectively. They function collaboratively to facilitate elimination of a variety of compounds into urine, thus playing a crucial role in drug disposition and response. Preliminary data from our lab showed that acute exposure (20 minutes) of Cd2+ could significantly upregulate the activities of human hOCT2, while inhibit the function of hMATE1, both in a dose‐dependent manner (Fig. 1A&amp;1B). Beside Cd2+, we identified imatinib, an anticancer drug, could induce the function of hOCT2 while inhibit the function of hMATE1 (Fig. 2). In vivo study also showed that pre‐treatment of imatinib could significantly increase the accumulation of metformin in mice kidney while had no impact on the plasma exposure of metformin (Fig. 3A&amp;3B). It implicated that the index of plasma drug exposure will not be able to correctly reflect drug‐drug interaction when a drug could induce hOCT2 while inhibit hMATE1 at the same time, which potentially could cause significant drug accumulation in kidney tubular cells.Support or Funding InformationNIH: R01GM099742FDA: U01FD004320This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Identifying the localization and exploring a functional role for Gprc5c in the kidney

The investigation of orphan GPCRs (GPRs) has the potential to uncover novel insights into whole animal physiology. In this study, our goal was to determine the renal localization of Gprc5c, a receptor that we previously reported to be highly expressed in murine whole kidney, and to examine physiologic parameters in Gprc5c knockout (KO) mice to gain insight into function. Gprc5c localized to the apical membrane of renal proximal tubules (PTs) in mice, rats, and humans. With the comparison of Gprc5c wild-type (WT) and KO mice, we found that Gprc5c KO mice have altered acid-base homeostasis. Specifically, Gprc5c KO mice have lower blood pH and higher urine pH compared with WT mice, with a reduced level of titratable acids in their urine. In an in vitro GPCR internalization assay, we observed that Gprc5c internalization (an index of activation) was triggered by alkaline extracellular pH. Furthermore, with the use of an in vitro BCECF assay, we observed that Gprc5c increases Na+/H+ exchanger 3 (NHE3) activity at alkaline pH. We also find that the NHE3 activity is reduced in Gprc5c KO mice by 2 photon imaging in seminaphthorhodafluors (SNARF)-4F-loaded kidney sections. NHE3 is a primary contributor to apical transport of H+ in the renal PT. Together, these data imply that Gprc5c modulates the renal contribution to systemic pH homeostasis, at least in part, by taking part in the regulation of NHE3.-Rajkumar, P., Cha, B., Yin, J., Arend, L. J., Păunescu, T. G., Hirabayashi, Y., Donowitz, M., Pluznick, J. L. Identifying the localization and exploring a functional role for Gprc5c in the kidney.

Effects of phenylpropanoids on human organic anion transporters hOAT1 and hOAT3

Human organic anion transporters hOAT1/SLC22A6 and hOAT3/SLC22A8 are highly expressed on the basolateral membrane of renal proximal tubules and mediate tubular uptake of anionic drugs from blood. They play an important role for drug disposition, and therefore close studies of their ligand recognition are important for drug therapy and development. In this study, we performed uptake experiments using HEK293 and fluorescent anion 6-carboxyfluorescein to asses the effects of phenylpropanoids on hOAT1 and hOAT3. We found that phenylpropanoids, 3-(4′-isopentenyloxyphenyl)-benzoic acid (IBA), 3-(4′-isopentenyloxy-3′-methoxyphenyl)-benzoic acid (IMBA), and 3-(4′-geranyloxy-3′-methoxy phenyl)-benzoic acid (GMBA) inhibited hOAT1 and hOAT3. The Ki values for hOAT1 were comparable to that of probenecid, a strong inhibitor of hOAT1 and hOAT3. While IBA demonstrated competitive inhibition, IMBA and GMBA showed mixed-type inhibition. After preincubation and washout, the inhibitory effects remained with IMBA and GMBA but not IBA, suggesting that the functional group at 3’-position is responsible for these differences. In conclusion, IBA, IMBA, and GMBA are inhibitors of hOAT1 and hOAT3.

Cellular Uptake of Levocetirizine by Organic Anion Transporter 4

The pharmacokinetics of cetirizine, a nonsedating antihistamine, is profoundly affected by transporter-mediated membrane transport in the kidney. In this study, we aimed to investigate the transport mechanism of levocetirizine, the pharmacologically active enantiomer of cetirizine, via human organic anion transporter 4 (OAT4) expressed in the apical membrane of renal proximal tubules and the basal plasma membrane of placental syncytiotrophoblasts. In cells expressing human OAT4 under the control of tetracycline, levocetirizine uptake was increased by tetracycline treatment. On the other hand, OAT4 expression did not facilitate efflux of preloaded levocetirizine from the cells, either in the presence or absence of extracellular Cl−. The OAT4-mediated levocetirizine uptake was concentration-dependent with a Km of 38 μM. The uptake rate of levocetirizine via OAT4 was approximately twice that of racemic cetirizine, indicating stereoselective uptake of levocetirizine. On the other hand, OAT4-mediated [3H]dehydroepiandrosterone sulfate uptake was inhibited by dextrocetirizine and levocetirizine. Overall, our findings indicate that OAT4 mediates levocetirizine uptake but is unlikely to mediate the efflux.

Insulin stimulates SGLT2-mediated tubular glucose absorption via oxidative stress generation.

BackgroundNinety percent of glucose filtered by the glomerulus is reabsorbed by a sodium-glucose cotransporter 2 (SGLT2), which is expressed mainly on the apical membrane of renal proximal tubules. Since SGLT-2-mediated glucose reabsorption is enhanced under diabetic conditions, selective inhibition of SGLT2 has been proposed as a potential therapeutic target for the treatment of patients with diabetes. However, it remains unclear which diabetes-associated factors are involved in overexpression of SGLT2.MethodsTherefore, in this study, we examined whether insulin, high glucose, advanced glycation end products (AGEs), or H2O2 stimulated SGLT2 expression in human cultured proximal tubular cells, and then investigated the underlying molecular mechanisms.ResultsHigh glucose or AGEs did not affect SGLT2 expression in tubular cells. Insulin significantly increased tubular SGLT2 level in a dose-dependent manner, whereas bell-shaped dose-response curves were observed for H2O2-treated cells. An anti-oxidant, N-acetylcysteine completely blocked insulin-induced up-regulation of SGLT2 as well as increase in glucose absorption by tubular cells. Furthermore, insulin dose-dependently increased reactive oxygen species generation in tubular cells.ConclusionsOur present study demonstrated that insulin could stimulate SGLT-2-mediated glucose entry into cultured proximal tubular cells via oxidative stress generation. Suppression of the insulin-induced overexpression of SGLT2 in tubular cells might be a novel therapeutic strategy for the treatment of diabetic nephropathy.

Basement membrane protein nidogen-1 is a target of meprin β in cisplatin nephrotoxicity

Meprins are oligomeric metalloproteinases that are abundantly expressed in the brush-border membranes of renal proximal tubules. During acute kidney injury (AKI) induced by cisplatin or ischemia-reperfusion, membrane-bound meprins are shed and their localization is altered from the apical membranes toward the basolateral surface of the proximal tubules. Meprins are capable of cleaving basement membrane proteins in vitro, however, it is not known whether meprins are able to degrade extracellular matrix proteins under pathophysiological conditions in vivo. The present study demonstrates that a basement membrane protein, nidogen-1, is cleaved and excreted in the urine of mice subjected to cisplatin-induced nephrotoxicity, a model of AKI. Cleaved nidogen-1 was not detected in the urine of untreated mice, but during the progression of cisplatin nephrotoxicity, the excretion of cleaved nidogen-1 increased in a time-dependent manner. The meprin inhibitor actinonin markedly prevented urinary excretion of the cleaved nidogen-1. In addition, meprin β-deficient mice, but not meprin α-deficient mice, subjected to cisplatin nephrotoxicity significantly suppressed excretion of cleaved nidogen-1, further suggesting that meprin β is involved in the cleavage of nidogen-1. These studies provide strong evidence for a pathophysiological link between meprin β and urinary excretion of cleaved nidogen-1 during cisplatin-induced AKI.

Na+/H+ Exchanger Regulatory Factor 3 Is Critical for Multidrug Resistance Protein 4–Mediated Drug Efflux in the Kidney

Na(+)/H(+) exchanger regulatory factor 3 (NHERF3) is a PSD-95/discs large/ZO-1 (PDZ)-based adaptor protein that regulates several membrane-transporting proteins in epithelia. However, the in vivo physiologic role of NHERF3 in transepithelial transport remains poorly understood. Multidrug resistance protein 4 (MRP4) is an ATP binding cassette transporter that mediates the efflux of organic molecules, such as nucleoside analogs, in the gastrointestinal and renal epithelia. Here, we report that Nherf3 knockout (Nherf3(-/-)) mice exhibit profound reductions in Mrp4 expression and Mrp4-mediated drug transport in the kidney. A search for the binding partners of the COOH-terminal PDZ binding motif of MRP4 among several epithelial PDZ proteins indicated that MRP4 associated most strongly with NHERF3. When expressed in HEK293 cells, NHERF3 increased membrane expression of MRP4 by reducing internalization of cell surface MRP4 and consequently, augmented MRP4-mediated efflux of adefovir, a nucleoside-based antiviral agent and well known substrate of MRP4. Examination of wild-type and Nherf3(-/-) mice revealed that Nherf3 is most abundantly expressed in the kidney and has a prominent role in modulating Mrp4 levels. Deletion of Nherf3 in mice caused a profound reduction in Mrp4 expression at the apical membrane of renal proximal tubules and evoked a significant increase in the plasma and kidney concentrations of adefovir, with a corresponding decrease in the systemic clearance of this drug. These results suggest that NHERF3 is a key regulator of organic transport in the kidney, particularly MRP4-mediated clearance of drug molecules.

Functional Coupling of a Urate-Anion Exchanger URAT1 and Sodium-Dependent Anion Transporter SMCT2 on a PDZK1 Scaffold; Proposal of “Transportsome” for Urate-Transport

Transporters utilize electrochemical gradients across membranes to translocate their substrates through the membranes. Many eukaryotic plasma membrane symporters transport substrates in a Na+-dependent manner by using an electrochemical Na+ gradient across plasma membranes, while exchangers couple the movement of a substrate against its electrochemical gradient with the movement of another substrate down its electrochemical gradient in the opposite direction. Although the Na+ gradient is ubiquitous and stable, the electrochemical gradients of substrates for exchangers usually depend on cells and are affected by cellular conditions. The concentrations of intracellular substrates limit the accumulation of extracellular substrates thorough exchangers. To overcome the constraint, exchangers need to couple with other proteins that help accumulation of substrates around exchangers. We have been proposing that such functional coupling of transporters is general and constitutes the functional unit of transport that we call “transportsome”. Here, we show one of the examples. Proteomics analysis following co-immunoprecipitation revealed that urate-anion exchanger URAT1 and Na+-dependent anion transporter SMCT2 formed a complex through scaffold protein PDZK1 in the apical membrane of renal proximal tubules. FRET experiment clearly indicated that PDZK1 assembled URAT1 and SMCT2 in living cells. To elucidate functional coupling of the transporters, URAT1 and SMCT2 were overexpressed in insect cells, purified and reconstituted into proteoliposomes in the presence or absence of PDZK1. The initial rate of urate-uptake was increased in the presence of Na+ when co-reconstituted with PDZK1 while Na+ did not alter the rate without PDZK1. These results suggest that SMCT2 generate an electrochemical gradient of the substrate which drives the urate-uptake via URAT1 located in close proximity to SMCT2 with PDZK1 scaffold. Taken together, the transporters and the scaffold protein formed Transportsome for urate-transport.