Opossum Kidney Proximal Tubule Cells Research Articles

Distinct and overlapping RGS14 and RGS12 actions regulate NPT2A-mediated phosphate transport

Parathyroid hormone (PTH) and fibroblast growth factor-23 (FGF23) control serum phosphate levels by downregulating the renal Na-phosphate transporter NPT2A, thereby decreasing phosphate absorption and augmenting urinary excretion. This mechanism requires NHERF1, a PDZ scaffold protein, and is governed by the regulator of G protein signaling-14 (RGS14), which harbors a carboxy-terminal PDZ ligand that binds NHERF1. RGS14 is part of a triad of structurally related RGS proteins that includes RGS12 and RGS10. Like RGS14, RGS12 contains a class 1 PDZ ligand. However, unlike RGS14, the larger RGS12 contains an upstream PDZ-binding domain. The studies outlined here examined and characterized the binding of RGS12 with NHERF1 and NPT2A and its function on hormone-regulated phosphate transport. Immunoblotting experiments revealed RGS12 C-terminal PDZ ligand binding to NHERF1. Further structural analysis disclosed that NPT2A engaged full-length RGS12 and the upstream fragment containing the PDZ domain. Neither the downstream RGS12 portion nor RGS14 interacted with NPT2A. PTH and FGF23 profoundly inhibited phosphate uptake in opossum kidney proximal tubule cells. Transfection with human RGS14, or human RGS12, abolished hormone-sensitive phosphate transport as reported for human proximal tubule cells. RGS12 inhibitory activity resides in the downstream region and is comparable to RGS14. The carboxy-terminal RGS12(667–1447) splice variant is prominently expressed in the kidney and may contribute to regulating hormone-sensitive phosphate transport.

Shear stress and oxygen availability drive differential changes in opossum kidney proximal tubule cell metabolism and endocytosis.

Kidney proximal tubule (PT) cells have high-metabolic demands to drive the extraordinary ion and solute transport, water reabsorption, and endocytic uptake that occur in this nephron segment. Increases in renal blood flow alter glomerular filtration rate and lead to rapid mechanosensitive adaptations in PT transport, impacting metabolic demand. Although the PT reabsorbs essentially all of the filtered glucose, PT cells rely primarily on oxidative metabolism rather than glycolysis to meet their energy demands. We lack an understanding of how PT functions are impacted by changes in O2 availability via cortical capillaries and mechanosensitive signaling in response to alterations in luminal flow. Previously, we found that opossum kidney (OK) cells recapitulate key features of PT cells in vivo, including enhanced endocytic uptake and ion transport, when exposed to mechanical stimulation by culture on an orbital shaker. We hypothesized that increased oxygenation resulting from orbital shaking also contributes to this more physiologic phenotype. RNA seq of OK cells maintained under static conditions or exposed to orbital shaking for up to 96 hours showed significant time- and culture-dependent changes in gene expression. Transcriptional and metabolomics data were consistent with a decrease in glycolytic flux and with an increased utilization of aerobic metabolic pathways in cells exposed to orbital shaking. Moreover, we found spatial differences in the pattern of mitogenesis vs development of ion transport and endocytic capacities in our culture system that highlight the complexity of O2 -dependent and mechanosensitive crosstalk to regulate PT cell function.

Orbital shaking drives differential changes in OK proximal tubule cell metabolism and endocytosis

Kidney proximal tubule (PT) cells have high metabolic demands to drive the extraordinary ion and solute transport, water reabsorption, and endocytic uptake that occur in this nephron segment. Increases in renal blood flow alter glomerular filtration rate (GFR) and lead to rapid mechanosensitive adaptations in PT transport, impacting metabolic demand. Although the PT reabsorbs essentially all of the filtered glucose, PT cells rely primarily on oxidative metabolism rather than glycolysis to meet their energy demands. We lack an understanding of how PT metabolism is impacted by changes in O2 availability via cortical capillaries and mechanosensitive signaling in response to alterations in luminal flow. Previously, we found that opossum kidney (OK) cells recapitulate key features of PT cells in vivo, including enhanced endocytic uptake and ion transport, when exposed to mechanical stimulation by culture on an orbital shaker. We hypothesized that increased oxygenation resulting from orbital shaking also contributes to this more physiologic phenotype. RNA Seq of OK cells maintained under static conditions or exposed to orbital shaking for up to 96h revealed significant time‐ and culture‐dependent changes in gene expression. Transcriptional and metabolomics data were consistent with a decrease in glycolytic flux and with an increased utilization of aerobic metabolic pathways in cells exposed to orbital shaking. Moreover, we found spatial differences in the pattern of mitogenesis and endocytic capacity in our culture system that highlight the complexity of O2‐dependent and mechanosensitive crosstalk to regulate PT cell function.Support or Funding InformationTsinghua MD Scholars ProgramNIH R01 DK101484NIH P30 DK079307This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Increased megalin expression in early type 2 diabetes: role of insulin-signaling pathways.

The megalin/cubilin complex is responsible for the majority of serum protein reclamation in the proximal tubules. The current study examined if decreases in their renal expression, along with the albumin recycling protein neonatal Fc receptor (FcRn) could account for proteinuria/albuminuria in the Zucker diabetic fatty rat model of type 2 diabetes. Immunoblots of renal cortex samples obtained at worsening disease stages demonstrated no loss in megalin, cubilin, or FcRn, even when proteinuria was measured. Additionally, early diabetic rats exhibited significantly increased renal megalin expression when compared with controls (adjusted P < 0.01). Based on these results, the ability of insulin to increase megalin was examined in a clonal subpopulation of the opossum kidney proximal tubule cell line. Insulin treatments (24 h, 100 nM) under high glucose conditions significantly increased megalin protein ( P < 0.0001), mRNA ( P < 0.0001), and albumin endocytosis. The effect on megalin expression was prevented with inhibitors against key effectors of insulin intracellular signaling, phosphatidylinositide 3-kinase and Akt. Studies using rapamycin to inhibit the mechanistic target of rapamycin complex 1 (mTORC1) resulted in a loss of insulin-induced megalin expression. However, subsequent evaluation demonstrated these effects were independent of initial mTORC1 suppression. The presented results provide insight into the expression of megalin, cubilin, and FcRn in type 2 diabetes, which may be impacted by elevated insulin and glucose. Furthermore, proximal tubule endocytic activity in early diabetics may be enhanced, a process that could have a significant role in proteinuria-induced renal damage.

Localization of the calcium-regulated citrate transport process in proximal tubule cells

Urinary citrate is an important inhibitor of calcium-stone formation. Most of the citrate reabsorption in the proximal tubule is thought to occur via a dicarboxylate transporter NaDC1 located in the apical membrane. OK cells, an established opossum kidney proximal tubule cell line, transport citrate but the characteristics change with extracellular calcium such that low calcium solutions stimulate total citrate transport as well as increase the apparent affinity for transport. The present studies address several fundamental properties of this novel process: the polarity of the transport process, the location of the calcium-sensitivity and whether NaDC1 is present in OK cells. OK cells grown on permeable supports exhibited apical >basolateral citrate transport. Apical transport of both citrate and succinate was sensitive to extracellular calcium whereas basolateral transport was not. Apical calcium, rather than basolateral, was the predominant determinant of changes in transport. Also 2,3-dimethylsuccinate, previously identified as an inhibitor of basolateral dicarboxylate transport, inhibited apical citrate uptake. Although the calcium-sensitive transport process in OK cells is functionally not typical NaDC1, NaDC1 is present in OK cells by Western blot and PCR. By immunolocalization studies, NaDC1 was predominantly located in discrete apical membrane or subapical areas. However, by biotinylation, apical NaDC1 decreases in the apical membrane with lowering calcium. In sum, OK cells express a calcium-sensitive/regulated dicarboxylate process at the apical membrane which responds to variations in apical calcium. Despite the functional differences of this process compared to NaDC1, NaDC1 is present in these cells, but predominantly in subapical vesicles.

Parathyroid hormone (PTH) decreases sodium-phosphate cotransporter type IIa (NpT2a) mRNA stability

The acute inhibitory effects of parathyroid hormone (PTH) on proximal tubule Na(+)-K(+)-ATPase (Na-K) and sodium-dependent phosphate (NaPi) transport have been extensively studied, while little is known about the chronic effects of PTH. Patients with primary hyperparathyroidism, a condition characterized by chronic elevations in PTH, exhibit persistent hypophosphatemia but not significant evidence of salt wasting. We postulate that chronic PTH stimulation results in differential desensitization of PTH responses. To address this hypothesis, we compared the effects of chronic PTH stimulation on Na-P(i) cotransporter (Npt2a) expression and Na-K activity and expression in Sprague Dawley rats, transgenic mice featuring parathyroid-specific cyclin D1 overexpression (PTH-D1), and proximal tubule cell culture models. We demonstrated a progressive decrease in brush-border membrane (BBM) expression of Npt2a from rats treated with PTH for 6 h or 4 days, while Na-K expression and activity in the basolateral membranes (BLM) exhibited an initial decrease followed by recovery to control levels by 4 days. Npt2a protein expression in PTH-D1 mice was decreased relative to control animals, whereas levels of Na-K, NHERF-1, and PTH receptor remained unchanged. In PTH-D1 mice, NpT2a mRNA expression was reduced by 50% relative to control mice. In opossum kidney proximal tubule cells, PTH decreased Npt2a mRNA levels. Both actinomycin D and cycloheximide treatment prevented the PTH-mediated decrease in Npt2a mRNA, suggesting that the PTH response requires transcription and translation. These findings suggest that responses to chronic PTH exposure are selectively regulated at a posttranscriptional level. The persistence of the phosphaturic response to PTH occurs through posttranscriptional mechanisms.

Calcium sensitivity of dicarboxylate transport in cultured proximal tubule cells

Urinary citrate is an important inhibitor of calcium nephrolithiasis and is primarily determined by proximal tubule reabsorption. The major transporter to reabsorb citrate is Na(+)-dicarboxylate cotransporter (NaDC1), which transports dicarboxylates, including the divalent form of citrate. We previously found that opossum kidney (OK) proximal tubule cells variably express either divalent or trivalent citrate transport, depending on extracellular calcium. The present studies were performed to delineate the mechanism of the effect of calcium on citrate and succinate transport in these cells. Transport was measured using isotope uptake assays. In some studies, NaDC1 transport was studied in Xenopus oocytes, expressing either the rabbit or opossum ortholog. In the OK cell culture model, lowering extracellular calcium increased both citrate and succinate transport by more than twofold; the effect was specific in that glucose transport was not altered. Citrate and succinate were found to reciprocally inhibit transport at low extracellular calcium (<60 μM), but not at normal calcium (1.2 mM); this mutual inhibition is consistent with dicarboxylate transport. The inhibition varied progressively at intermediate levels of extracellular calcium. In addition to changing the relative magnitude and interaction of citrate and succinate transport, decreasing calcium also increased the affinity of the transport process for various other dicarboxylates. Also, the affinity for succinate, at low concentrations of substrate, was increased by calcium removal. In contrast, in oocytes expressing NaDC1, calcium did not have a similar effect on transport, indicating that NaDC1 could not likely account for the findings in OK cells. In summary, extracellular calcium regulates constitutive citrate and succinate transport in OK proximal tubule cells, probably via a novel transport process that is not NaDC1. The calcium effect on citrate transport parallels in vivo studies that demonstrate the regulation of urinary citrate excretion with urinary calcium excretion, a process that may be important in decreasing urinary calcium stone formation.

A third mode of ouabain signaling. Focus on “Regulation of ERK1/2 by ouabain and Na-K-ATPase-dependent energy utilization and AMPK activation in parotid acinar cells”

in recent years evidence has emerged that the Na-K-ATPase, a canonical active ion pump, has parallel roles as a platform for cell signaling. Na-K-ATPase has been shown to associate directly with other proteins in signaling complexes in cardiac myocytes, renal cells, and several other cell types ([4

The COOH terminus of megalin regulates gene expression in opossum kidney proximal tubule cells

We recently reported that megalin is subjected to regulated intramembrane proteolysis (RIP) and includes 1) protein kinase C (PKC)-regulated, metalloprotease-mediated ectodomain shedding producing a membrane-bound megalin COOH-terminal fragment (MCTF) and 2) gamma-secretase-mediated cleavage of the MCTF producing a soluble megalin intracellular domain (MICD). Based on studies of RIP of other receptors, the MICD is predicted to target to the nucleus and regulate gene expression. To determine whether RIP of megalin regulates proximal tubule gene expression, we stably expressed the transfected MCTF (tMCTF) or transfected MICD (tMICD) in opossum kidney proximal tubule (OKP) cells and examined the resulting phenotype. Immunoblotting and immunocytochemical analysis of tMCTF cells showed the tMCTF was expressed and constitutively processed by gamma-secretase. Analysis of specific protein expression in tMCTF- and tMICD-transfected cells using Western blot showed endogenous megalin and Na(+)/H(+) exchanger 3 (NHE3) protein expression to be dramatically lower than that of control cells. Expression of other proteins including myosin VI, beta-adaptin, and the Na-K-ATPase appeared unchanged. Analysis of specific mRNA expression using quantitative real-time PCR showed megalin and NHE3 mRNA levels were significantly lower in tMCTF- and tMICD-transfected cells compared with controls. Inhibition of gamma-secretase activity in tMCTF cells resulted in an 8- to 10-fold recovery of megalin mRNA within 4 h. These data show that the COOH-terminal domain of megalin regulates expression of specific proteins in OKP cells and provides the first evidence that RIP of megalin may be part of a signaling pathway linking protein absorption and gene expression in proximal tubule.

Constitutive Activity of Inwardly Rectifying K+ Channel at Physiological [Ca]i Is Mediated by Ca2+/CaMK II Pathway in Opossum Kidney Proximal Tubule Cells

Using patch-clamp technique, we studied the role of the Ca2+/calmodulin kinase II (CaMK II)-mediated phosphorylation process on the K+ channel with an inward conductance of 90 pS in opossum kidney proximal tubule cells (OKPCs). The intracellular Ca2+ concentration ([Ca]i) was measured by use of the fluorescent dye fura 2. The following results were obtained: (i) In cell-attached patches, the channel activity was inhibited by a decrease in [Ca]i induced by perfusion with low Ca2+ (10(-8) M), La3+ (100 microM), or EGTA/AM (100 microM) contained in the bath solution. The application of KN-62 (10 microM) or KN-93 (5 microM), inhibitors of CaMK II, also inhibited the channel activity. (ii) The membrane potential measured with nystatin-perforated patches was significantly decreased by the fall in [Ca]i induced by the perfusion with EGTA- or La(3+)-containing solution. Also, the application of KN-62 (10 microM) or KN-93 (5 microM) to the bath significantly decreased the membrane potential. (iii) In inside-out patches, the channel activity was significantly stimulated by the application of CaMK II (300 pM) at 10(-7) M Ca2+ in the bath. Furthermore, the application of KN-62 (10 microM) to the bath significantly decreased the channel activity. Our findings show that the constitutive activity of inwardly rectifying K+ channel at physiological [Ca]i is mediated by the Ca2+/CaMK II pathway in OKPCs.

Ouabain stimulates protein kinase B (Akt) phosphorylation in opossum kidney proximal tubule cells through an ERK-dependent pathway

Endogenous cardiotonic glycosides bind to the inhibitory binding site of the plasma membrane sodium pump (Na(+)/K(+)-ATPase). Plasma levels of endogenous cardiotonic glycosides increase in several disease states, such as essential hypertension and uremia. Low concentrations of ouabain, which do not inhibit Na(+)/K(+)-ATPase, induce cell proliferation. The mechanisms of ouabain-mediated response remain unclear. Recently, we demonstrated that in opossum kidney (OK) proximal tubular cells, low concentrations of ouabain induce cell proliferation through phosphorylation of protein kinase B (Akt) in a calcium-dependent manner. In the present study, we identified ERK as an upstream kinase regulating Akt activation in ouabain-stimulated cells. Furthermore, we provide evidence that low concentrations of ouabain stimulate Na(+)/K(+)-ATPase-mediated (86)Rb uptake in an Akt-, ERK-, and Src kinase-dependent manner. Ouabain-mediated ERK phosphorylation was inhibited by blockade of intracellular calcium release, calcium entry, tyrosine kinases, and phospholipase C. Pharmacological inhibition of phosphoinositide-3 kinase and Akt failed to inhibit ouabain-stimulated ERK phosphorylation. Ouabain-mediated Akt phosphorylation was inhibited by U0126, a MEK/ERK inhibitor, suggesting that ouabain-mediated Akt phosphorylation is dependent on ERK. In an in vitro kinase assay, active recombinant ERK phosphorylated recombinant Akt on Ser(473). Moreover, transient transfection with constitutively active MEK1, an upstream regulator of ERK, increased Akt phosphorylation and activation, whereas overexpression of constitutively active Akt failed to stimulate ERK phosphorylation. Ouabain at low concentrations also promoted cell proliferation in an ERK-dependent manner. These findings suggest that ouabain-stimulated ERK phosphorylation is required for Akt phosphorylation on Ser(473), cell proliferation, and stimulation of Na(+)/K(+)-ATPase-mediated (86)Rb uptake in OK cells.

Ouabain induces cell proliferation through calcium-dependent phosphorylation of Akt (protein kinase B) in opossum kidney proximal tubule cells

Cardiotonic glycosides, like ouabain, inhibit Na(+)-K(+)-ATPase. Recent evidence suggests that low molar concentrations of ouabain alter cell growth. Studies were conducted to examine the effect of ouabain on Akt phosphorylation and rate of cell proliferation in opossum kidney (OK) proximal tubule cells. Cells exposed to 10 nM ouabain displayed increased Akt Ser(473) phosphorylation, as evidenced by an increase in phospho-Akt Ser(473) band density. Ouabain-stimulated Akt Ser(473) phosphorylation was inhibited by pretreatment with phosphatidylinositol 3-kinase (PI3K) inhibitors (LY294002 and wortmannin), a PLC inhibitor (edelfosine), and an Akt inhibitor. Moreover, ouabain-mediated Akt Ser(473) phosphorylation was suppressed by reduction of extracellular calcium (EGTA) or when intracellular calcium was buffered by BAPTA-AM. An inhibitor of calcium store release (TMB-8) and an inhibitor of calcium entry via store-operated calcium channels (SKF96365) also suppressed ouabain-mediated Akt Ser(473) phosphorylation. In fura-2 AM-loaded cells, 10 nM ouabain increased capacitative calcium entry (CCE). Ouabain at 10 nM did not significantly alter baseline cytoplasmic calcium concentration in control cells. However, treatment with 10 nM ouabain caused a significantly higher ATP-mediated calcium store release. After 24 h, 10 nM ouabain increased the rate of cell proliferation. The Akt inhibitor, BAPTA-AM, SKF96365, and cyclopiazonic acid suppressed the increase in the rate of cell proliferation caused by 10 nM ouabain. Ouabain at 10 nM caused a detectable increase in (86)Rb uptake but did not significantly alter Na(+)-K(+)-ATPase (ouabain-sensitive pNPPase) activity in crude membranes or cell sodium content. Taken together, the results point to a role for CCE and Akt phosphorylation, in response to low concentrations of ouabain, that increase the rate of cell proliferation without inhibiting Na(+)-K(+)-ATPase-mediated ion transport.

Role of dipeptidyl peptidase IV in regulating activity of Na+/H+ exchanger isoform NHE3 in proximal tubule cells.

We recently reported that NHE3 exists in multimeric complexes with dipeptidyl peptidase IV (DPPIV) in renal brush-border membranes. To examine the possible role of DPPIV in modulating NHE3 activity, we evaluated whether specific competitive inhibitors that bind to the active site of DPPIV affect NHE3 activity in the OKP line of opossum kidney proximal tubule cells. The DPPIV inhibitors diprotin A and P32/98 significantly reduced NHE3 activity, whereas the inactive isomer P34/98 had no effect. DPPIV inhibitors did not reduce the activity of another brush-border transport process, Na-phosphate cotransport. Effects of DPPIV inhibitors on NHE3 activity were not associated with detectable changes in amount or apparent molecular weight of NHE3 or in NHE3 surface expression. To investigate the signaling mechanisms involved in modulation of NHE3 activity by DPPIV, we used inhibitors of protein kinase pathways known to regulate NHE3. Whereas the PKA inhibitor H-89 failed to block the effect of DPPIV inhibitors, the tyrosine kinase inhibitor genistein alone caused a decrement in NHE3 activity very similar in magnitude to that caused by P32/98. We also found that the effects of genistein and P32/98 on NHE3 activity were not additive. In contrast, forskolin/IBMX and P32/98 had additive inhibitory effects on NHE3 activity. These findings suggested that the effect of DPPIV inhibitors to reduce NHE3 activity results from inhibition of a tyrosine kinase signaling pathway rather than by activation of PKA. We conclude that DPPIV plays an unexpected role in modulating Na+/H+ exchange mediated by NHE3 in proximal tubule cells.

Does membrane trafficking play a role in regulating the sodium/hydrogen exchanger isoform 3 in the proximal tubule?

The proximal tubule sodium/hydrogen exchanger continuously reabsorbs the bulk of the filtered sodium, controlling salt delivery to the distal nephron which is critical for tubuloglomerular feedback autoregulation and for fine control of salt excretion in the distal nephron. This review focuses on recent studies of the mechanisms of regulation of sodium transport in the proximal tubule, and addresses whether results from studies in proximal tubule cell lines are applicable to the proximal tubule in situ. Recent in-vivo studies provided evidence that sodium/hydrogen exchanger isoform 3 can move into and out of the apical microvilli accompanied by parallel changes in renal sodium transport: the exchanger is retracted from the microvilli in response to hypertension, parathyroid hormone or dopamine treatment and moved into the microvilli in response to sympathetic nervous system stimulation, puromycin aminonucleoside induced nephritic syndrome, and insulin treatment. Studies in cultured opossum kidney proximal tubule cells provided evidence for clathrin coated vesicle mediated, dynamin dependent, cytoskeleton dependent internalization of sodium/hydrogen exchanger isoform 3 from the surface to an endosomal pool in response to dopamine or parathyroid hormone. In the intact proximal tubule there is evidence for a two-step internalization process: (1) from villi to the intermicrovillar cleft region and (2) to a higher density membrane pool that may be either below the microvilli or deep in intermicrovillar clefts. Recent studies have described a significant inactive pool of the exchanger in the intermicrovillar region in vivo that may serve as a storage and recruitable pool. The molecular mechanisms responsible for increasing or decreasing sodium transport in the proximal tubule appear to include redistribution of sodium/hydrogen exchanger isoform 3 to or from the microvillar region. Detailed studies in cultured proximal tubule cell lines provide evidence for endocytosis and exocytosis of the exchanger dependent on cytoskeleton and clathrin coated vesicles. In vivo, the apical membrane is differentiated into discrete villar and intermicrovillar membrane domains and the intermicrovillar domain, not observed in cultured cells, may serve as a recruitable storage pool for sodium/hydrogen exchanger isoform 3.

Heterodimer requirement for gene regulation by Vitamin D in variant OK cells

OK cells are a transformed cell line derived from opossum kidney proximal tubule cells. Prior studies have utilized this cell line to study both positive and negative transcriptional responses to Vitamin D. However, there was a noticeable decrease in sensitivity on the part of these cells to respond to Vitamin D treatment in transfection assays, particularly when assessing transcriptional activity from a heterologous promoter construct that used the chicken parathyroid hormone (cPTH) repressor Vitamin D response element (VDRE). Western blotting revealed the apparently diminished expression of both the Vitamin D receptor (VDR) together with its heterodimeric DNA-binding partner, the retinoid X receptor (RXR), in these cellular extracts. Co-transfection of either a VDR or RXR expression vector alone had little effect on hormone-dependent enhancer transcriptional activity from the human osteocalcin (hOC) reporter construct, or the degree of repression from the cPTH construct. Indeed, significant effects on repressor or enhancer activity were only observed in these cells when expression vectors for both the VDR and RXR were simultaneously introduced into the cells via transfection experiments. Analogous results were obtained irrespective of the identity of RXR isoform; co-transfection of either RXRα or RXRβ expression vectors together with the VDR-produced similar improvements in repressor activity. Titration of Vitamin D hormone under conditions of co-expression of the two receptors indicated that half-maximal responses were comparable for both VDREs and occurred at <1 nM concentration. In summary, these results are consistent with prior in vitro studies indicating interaction of the VDR with these VDREs occurs as a heterodimer complex with RXR. The decreased expression of both heterodimer partners observed in these cells could explain the requirement for additional VDR/RXR expression, in particular in order to compensate for the reportedly lower binding affinity of the heterodimer with the repressor cPTH VDRE. The extent of expression of both heterodimer partners, therefore, may act to modulate the available responses to Vitamin D in target cells.

Endothelin-1 Chronically Inhibits Na/H Exchanger-3 in ETB-Overexpressing OKP Cells

Endothelin-1 (ET-1) acutely increases Na/H antiporter activity in OKPETB6 cells, an opossum kidney proximal tubule cell line transfected with ETB receptor cDNA. The purpose of the present study was to examine the chronic effect of ET-1 on Na/H antiporter activity in OKP cells and to examine whether Na/H exchanger (NHE)-3 mRNA and protein abundance are regulated by ET-1. Quiescent OKPETB6 cells were treated with 10 nM ET-1 for 3, 6 or 24 h and Na/H antiporter activity was assayed. The Na/H antiporter activity in 3-h ET-1-treated cells was not different from controls. However, Na/H antiporter activity was significantly decreased by 29% at 6 h and 72% at 24 h. The effect of ET-1 on Na/H antiporter activity was blocked by BQ788, an ETB receptor antagonist, but not BQ123, an ETA receptor antagonist. The NHE-3 mRNA abundance in ET-1-treated cells was not different from controls at 3 h. However, there was a significant decrease in NHE-3 mRNA abundance at 6 and 24 h. There was also a significant decrease in NHE-3 protein abundance at 6 and 24 h. In summary, ET-1 chronically inhibits NHE-3 in OKPETB6 cells.

Nitric oxide reduces the molecular activity of Na+,K+-ATPase in opossum kidney cells

Nitric oxide (NO) directly inhibits fluid and solute reabsorption in the proximal tubule. In the present study, we investigated the effect of NO on the Na+, K+-ATPase of opossum kidney (OK) cells, a proximal tubule cell line, and its mechanisms. Na+,K+-ATPase activity in the membrane fraction of OK cells was measured as the ouabain-sensitive ATP hydrolytic activity. The enzyme unit number on intact cells was measured by ouabain-binding assay. Incubation with 0.5 mM sodium nitroprusside (SNP), a NO donor, for two hours inhibited the catalytic activity of the membrane-associated Na+,K+-ATPase in OK cells to 65.5 +/- 9.7% of control (N = 6, P < 0.05 vs. control). This effect of SNP was concentration- and time-dependent. The NO scavenger hemoglobin blunted, while another NO donor spermine NONOate (5 microM) mimicked this effect of SNP. At all concentrations and time points tested, SNP did not alter the molecular number of Na+,K+-ATPase on intact OK cells, indicating that NO inhibited the molecular activity of Na+,K+-ATPase. The soluble guanylate cyclase inhibitor, 1H-[1,2,4]oxadiazolo-[4, 3-a]quinoxalin-1-one (ODQ), blunted the inhibitory effect of SNP on the Na+,K+-ATPase activity. An exogenous cGMP analog similarly inhibited the Na+,K+-ATPase activity. Neither lipid soluble antioxidants vitamin E/probucol or thiol group compound DL-dithiothreitol (DTT) altered the inhibitory effect of SNP on the Na+,K+-ATPase activity. NO inhibited the molecular activity of the Na+,K+-ATPase of the OK proximal tubule cell line probably via cGMP-dependent mechanisms.

Lysophosphatidic acid-induced calcium mobilization and proliferation in kidney proximal tubular cells.

Patients with proteinuria tend to develop progressive renal disease with proximal tubular cell atrophy and interstitial scarring. It has been suggested that the nephrotoxicity of albuminuric states may be due to the protein molecule itself or by lipids, such as lysophosphatidic acid (LPA), that albumin carries. LPA was found to cause a transient increase in intracytoplasmic free Ca2+ ([Ca2+]i) in opossum kidney proximal tubule cells (OK) that was maximal at 100 microM LPA and was dose dependent with an EC50 of 2.6 x 10(-6) M. This Ca2+ mobilization was from both internal stores and across the plasma membrane and was pertussis toxin (PTX) insensitive. Treatment of OK cells with 100 microM LPA for 5 min was found to cause a twofold increase in [3H]thymidine incorporation and a three- to fivefold increase over control after 24 h. This was highly PTX sensitive and insensitive to pretreatment with the tyrosine kinase inhibitors genistein and herbimycin A. These findings may be of significance in the progression of renal disease and indicate the potential importance of lipids in modulating proximal tubule cell function and growth.

A nucleoside-sensitive organic cation transporter in opossum kidney cells.

Renal secretion of organic cations and anions are pleiotropic, active processes in mammals. Some nucleosides such as deoxyadenosine (dAdo), 2-chlorodeoxyadenosine, and azidothymidine are secreted by human and rodent kidneys. Previous work (J. A. Nelson, J. F. Kuttesch, Jr., and B. H. Herbert. Biochemical Pharmacology 32: 2323-2327, 1983) indicated a role for the classic organic cation transporter (OCT) in the secretion of the dAdo analog, 2'-deoxytubercidin, by mouse kidney. Using [14C]tetraethylammonium bromide ([14C]TEA) as a substrate, we tested several renal cell lines for a nucleoside-sensitive OCT. American opossum kidney proximal tubule cells (OK) express a cimetidine-sensitive and metabolic-dependent ability to efflux TEA. Other classic OCT inhibitors and several nucleosides also inhibit TEA efflux by these cells in a manner reflecting structural specificity for the carrier. Inhibition of OCT by nucleosides is not a universal feature of OCTs, since TEA transport mediated by cloned rat kidney OCT2 in the Xenopus laevis oocyte system was not inhibited by the same nucleosides. In conclusion, OK cells appear to possess an OCT that may also transport some nucleosides by a novel carrier.