Opossum Kidney Proximal Tubule Research Articles

Megalin-mediated siRNA uptake in kidney proximal tubule cells

Small interfering RNA (siRNA) is a clinically validated therapeutic modality, which silences gene expression via RNA interference (RNAi). Systemically delivered siRNA molecules are excreted primarily via the kidneys, where they accumulate in the renal cortex. Proximal epithelial cells within the tubule are thus an attractive target cell for utilizing RNAi, where the primary mode of entry is likely endocytic uptake. Here we report using a well-differentiated, opossum kidney (OK) proximal tubule cell (PTEC) line as a model system to mimic proximal tubule biology and investigate siRNA uptake. Fluorescently tagged siRNA were employed to observed uptake, which was observed in both a time and concentration dependent manner occurring predominantly from the apical surface of OK cells. Live cell imaging revealed significant colocalization of siRNAs with LysoTracker, consistent with endocytic uptake and delivery to lysosomes. We investigated the role of key PTEC solute recovery receptors, megalin and cubilin. siRNA uptake was inhibited by the megalin-binding protein RAP; and was independently reduced in Lrp2 knock-out (KO) cell lines. This work suggests that PTECs employ megalin to facilitate uptake of siRNA via endocytosis. Additionally, we observed colocalization of siRNA molecules and megalin in mouse kidneys from in vivo studies. Elucidating the mechanism(s) for siRNA uptake in the proximal tubule aids in the development of RNAi renal therapeutics and, more broadly, the understanding of class effects associated with siRNA nephrotoxicity. Additional work is focused on intracellular traffcking and megalin mediated siRNA uptake in mice. Project funded by Judo Bio. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

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.

Parathyroid hormone inhibition of Na+/H+ exchanger 3 transcription: Intracellular signaling pathways and transcription factor expression

The main transport mechanism of reabsorption of sodium bicarbonate and fluid in the renal proximal tubules involves Na+/H+ exchanger 3 (NHE3), which is acutely and chronically downregulated by parathyroid hormone (PTH). Although PTH is known to exert an inhibitory effect on NHE3 expression and transcription, the molecular mechanisms involved remain unclear. Here, we demonstrated that, in opossum kidney proximal tubule (OKP) cells, PTH-induced inhibition of Nhe3 gene promoter occurs even in the core promoter that controls expression of the reporter gene. We found that inhibition of the protein kinase A (PKA) and Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathways transformed PTH from an inhibitor of promoter activity into an activator of that same activity, as did point mutations in the EGR1, Sp1, and Sp3 binding consensus elements in the promoter. In nuclear extracts of PTH-treated OKP cells, we also observed increased expression of EGR1 mRNA and of some Sp3 isoforms. Electrophoretic mobility shift assay showed a supershift of the −61 to −42-bp probe with an anti-EGR1 antibody in PTH-treated cells, suggesting that EGR1 binding is relevant for the inhibitory activity of PTH. We conclude that PTH-induced inhibition of NHE3 transcription is related to higher EGR1 expression; to EGR1 binding to the proximal and core promoters; and to PKA and JAK/STAT pathway activation. This mechanism might be responsible, at least in part, for lower NHE3 expression and sodium reabsorption in renal proximal tubules in the presence of high PTH levels.

Mo1751 CGMP-Dependent Kinase 2, Na+/H+ Regulatory Factor 2, and Na+/H+ Exchanger Isoform 3 Dynamically Assemble Within Lipid Rafts in Murine Small Intestinal Brush Border Membrane

Background: BB NHE3 (Na+/H+ exchanger 3) plus at least 7 other proteins are assembled into 600 kDa -1000 kDa macro-complexes, which serve as the signaling platforms required for the regulation of NHE3. NHERF proteins are a family of multi-PDZ-scaffolding proteins. NHERF1, NHERF2 and NHERF3 are localized to the areas of and below the BB and are involved in NHE3 regulation. Of these, both NHERF2 and NHERF3 but not NHERF1 were required for the carbachol inhibition of NHE3 activity, suggesting possible roles of NHERF dimerization in NHE3 regulation. The purpose of this study was 1) to compare the dimerization strength of all possible NHERF-NHERF interactions and 2) to test the hypothesis that NHERF2-NHERF3 hetero-dimerization contributes to NHE3 macro-complexes and is required for the acute carbachol inhibition of NHE3. Methods: FLAG-tagged NHERF proteins were used for Co-Immunoprecipitation (CO-IP) with anti-FLAG magnetic beads. Bacterially produced recombinant GST-fusion and MBP-fusion NHERF proteins were purified for pulldown (PD) assays to study direct protein-protein interactions in vitro. NHERF2 PDZ (GYGF to GAGA) mutants and NHERF3-4Ala (last 4 aa were Ala) mutant were used to define their interaction domains. Inhibition of NHE3 activity by carbachol was determined with BCECF/ fluorometry in Caco-2 cells expressing HA-NHE3. shRNAwas used to knock-down NHERF3. Adenovirus was used to express the FLAG-tagged rat wild type and mutant NHERF3. Results: NHERF1-NHERF3 and NHERF2-NHERF3 hetero-dimers were detected by both CO-IP and PD assays. NHERF1 and NHERF2 homo-dimers and NHERF1-NHERF2 hetero-dimers were detected minimally by PD and not by CO-IP. There was no significant NHERF3 homodimerization. Hetero-dimerization of NHERF2-NHERF3 was stronger than that of NHERF1NHERF3. The single-PDZ domain defective mutants of NHERF2 had reduced binding to NHERF3, whereas the double-PDZ domain mutant did not bind NHERF3 at all. NHERF34A did not bind NHERF2. This lack of interactions was confirmed by FRET in live OK (opossum kidney proximal tubule) cells. Macro-complexes could be assembled in vitro from NHERF2, NHERF3 and NHE3. NHE3 activity was inhibited by carbachol in wild-type Caco2 cells, but not in NHERF3 KD cells. This could be rescued by overexpression of shRNAresistant rat NHERF3 but not the rat NHERF3-4Amutant. Conclusions: 1) NHERF2-NHERF3 hetero-dimerization is strongest among all possible NHERF dimerizations. 2) NHERF2 and NHERF3 interact via the PDZ domains of NHERF2 and C-terminal PDZ recognition motif of NHERF3. 3) NHERF3 prefers binding to PDZ1 domain of NHERF2 while NHE3 prefers PDZ2 domain. 4) The NHERF2-NHERF3 hetero-dimerization mediated NHE3 macro-complex is necessary for the inhibition of NHE3 activity by carbachol. 5) Hetero-dimerization of NHER2-NHERF3 explains why both NHERF2 and NHERF3 are required for carbachol inhibition of NHE3.

Mo1747 NHERF2/NHERF3 Hetero-Dimerization Is Required for Carbachol Inhibition of NHE3 Activity That Involves Formation of NHE3 Macro-Complexes

Background: BB NHE3 (Na+/H+ exchanger 3) plus at least 7 other proteins are assembled into 600 kDa -1000 kDa macro-complexes, which serve as the signaling platforms required for the regulation of NHE3. NHERF proteins are a family of multi-PDZ-scaffolding proteins. NHERF1, NHERF2 and NHERF3 are localized to the areas of and below the BB and are involved in NHE3 regulation. Of these, both NHERF2 and NHERF3 but not NHERF1 were required for the carbachol inhibition of NHE3 activity, suggesting possible roles of NHERF dimerization in NHE3 regulation. The purpose of this study was 1) to compare the dimerization strength of all possible NHERF-NHERF interactions and 2) to test the hypothesis that NHERF2-NHERF3 hetero-dimerization contributes to NHE3 macro-complexes and is required for the acute carbachol inhibition of NHE3. Methods: FLAG-tagged NHERF proteins were used for Co-Immunoprecipitation (CO-IP) with anti-FLAG magnetic beads. Bacterially produced recombinant GST-fusion and MBP-fusion NHERF proteins were purified for pulldown (PD) assays to study direct protein-protein interactions in vitro. NHERF2 PDZ (GYGF to GAGA) mutants and NHERF3-4Ala (last 4 aa were Ala) mutant were used to define their interaction domains. Inhibition of NHE3 activity by carbachol was determined with BCECF/ fluorometry in Caco-2 cells expressing HA-NHE3. shRNAwas used to knock-down NHERF3. Adenovirus was used to express the FLAG-tagged rat wild type and mutant NHERF3. Results: NHERF1-NHERF3 and NHERF2-NHERF3 hetero-dimers were detected by both CO-IP and PD assays. NHERF1 and NHERF2 homo-dimers and NHERF1-NHERF2 hetero-dimers were detected minimally by PD and not by CO-IP. There was no significant NHERF3 homodimerization. Hetero-dimerization of NHERF2-NHERF3 was stronger than that of NHERF1NHERF3. The single-PDZ domain defective mutants of NHERF2 had reduced binding to NHERF3, whereas the double-PDZ domain mutant did not bind NHERF3 at all. NHERF34A did not bind NHERF2. This lack of interactions was confirmed by FRET in live OK (opossum kidney proximal tubule) cells. Macro-complexes could be assembled in vitro from NHERF2, NHERF3 and NHE3. NHE3 activity was inhibited by carbachol in wild-type Caco2 cells, but not in NHERF3 KD cells. This could be rescued by overexpression of shRNAresistant rat NHERF3 but not the rat NHERF3-4Amutant. Conclusions: 1) NHERF2-NHERF3 hetero-dimerization is strongest among all possible NHERF dimerizations. 2) NHERF2 and NHERF3 interact via the PDZ domains of NHERF2 and C-terminal PDZ recognition motif of NHERF3. 3) NHERF3 prefers binding to PDZ1 domain of NHERF2 while NHE3 prefers PDZ2 domain. 4) The NHERF2-NHERF3 hetero-dimerization mediated NHE3 macro-complex is necessary for the inhibition of NHE3 activity by carbachol. 5) Hetero-dimerization of NHER2-NHERF3 explains why both NHERF2 and NHERF3 are required for carbachol inhibition of NHE3.

Mo1748 Absorptive Enterocytes Are Poorly Differentiated in the CFTR Knockout Mouse Small Intestine

Background: BB NHE3 (Na+/H+ exchanger 3) plus at least 7 other proteins are assembled into 600 kDa -1000 kDa macro-complexes, which serve as the signaling platforms required for the regulation of NHE3. NHERF proteins are a family of multi-PDZ-scaffolding proteins. NHERF1, NHERF2 and NHERF3 are localized to the areas of and below the BB and are involved in NHE3 regulation. Of these, both NHERF2 and NHERF3 but not NHERF1 were required for the carbachol inhibition of NHE3 activity, suggesting possible roles of NHERF dimerization in NHE3 regulation. The purpose of this study was 1) to compare the dimerization strength of all possible NHERF-NHERF interactions and 2) to test the hypothesis that NHERF2-NHERF3 hetero-dimerization contributes to NHE3 macro-complexes and is required for the acute carbachol inhibition of NHE3. Methods: FLAG-tagged NHERF proteins were used for Co-Immunoprecipitation (CO-IP) with anti-FLAG magnetic beads. Bacterially produced recombinant GST-fusion and MBP-fusion NHERF proteins were purified for pulldown (PD) assays to study direct protein-protein interactions in vitro. NHERF2 PDZ (GYGF to GAGA) mutants and NHERF3-4Ala (last 4 aa were Ala) mutant were used to define their interaction domains. Inhibition of NHE3 activity by carbachol was determined with BCECF/ fluorometry in Caco-2 cells expressing HA-NHE3. shRNAwas used to knock-down NHERF3. Adenovirus was used to express the FLAG-tagged rat wild type and mutant NHERF3. Results: NHERF1-NHERF3 and NHERF2-NHERF3 hetero-dimers were detected by both CO-IP and PD assays. NHERF1 and NHERF2 homo-dimers and NHERF1-NHERF2 hetero-dimers were detected minimally by PD and not by CO-IP. There was no significant NHERF3 homodimerization. Hetero-dimerization of NHERF2-NHERF3 was stronger than that of NHERF1NHERF3. The single-PDZ domain defective mutants of NHERF2 had reduced binding to NHERF3, whereas the double-PDZ domain mutant did not bind NHERF3 at all. NHERF34A did not bind NHERF2. This lack of interactions was confirmed by FRET in live OK (opossum kidney proximal tubule) cells. Macro-complexes could be assembled in vitro from NHERF2, NHERF3 and NHE3. NHE3 activity was inhibited by carbachol in wild-type Caco2 cells, but not in NHERF3 KD cells. This could be rescued by overexpression of shRNAresistant rat NHERF3 but not the rat NHERF3-4Amutant. Conclusions: 1) NHERF2-NHERF3 hetero-dimerization is strongest among all possible NHERF dimerizations. 2) NHERF2 and NHERF3 interact via the PDZ domains of NHERF2 and C-terminal PDZ recognition motif of NHERF3. 3) NHERF3 prefers binding to PDZ1 domain of NHERF2 while NHE3 prefers PDZ2 domain. 4) The NHERF2-NHERF3 hetero-dimerization mediated NHE3 macro-complex is necessary for the inhibition of NHE3 activity by carbachol. 5) Hetero-dimerization of NHER2-NHERF3 explains why both NHERF2 and NHERF3 are required for carbachol inhibition of NHE3.

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.

A disintegrin and metalloprotease 10 activity sheds the ectodomain of the amyloid precursor-like protein 2 and regulates protein expression in proximal tubule cells.

A disintegrin and metalloprotease 10 (ADAM10) is a zinc protease that mediates ectodomain shedding of numerous receptors including Notch and members of the amyloid precursor protein family (APP, APLP1, and APLP2). Ectodomain shedding frequently activates a process called regulated intramembrane proteolysis (RIP) that links cellular events with gene regulation. To characterize ADAM10 in kidney and in opossum kidney proximal tubule (OKP) cells, we performed indirect immunofluorescence microscopy and immunoblotting of renal membrane fractions using specific antibodies. These studies show that ADAM10 and APLP2 are coexpressed in the proximal tubule and in OKP cells. To study the role of ADAM10 activity in the proximal tubule, we stably overexpressed wild-type ADAM10 or an inactive mutant ADAM10 in OKP cells. We found a direct correlation between the amount of active ADAM10 expressed and 1) the amount of APLP2 ectodomain shed into the culture supernatant and 2) the amount of Na(+)/H(+) exchanger 3 (NHE3) and megalin mRNA and protein expressed compared with control proteins. To establish a link between ADAM10-mediated shedding of APLP2 and the effect on NHE3 and megalin mRNA expression we performed RNA interference experiments using APLP2-specific short hairpin RNA (shRNA) in OKP cells. Cells expressing the APLP2 shRNA showed >80% knock down of APLP2 protein and mRNA as well as 60-70% reduction in NHE3 protein and mRNA. Levels of megalin and Na-K-ATPase protein and mRNA were not changed. These studies show 1) ADAM10 and APLP2 are expressed in proximal tubule cells and, 2) ADAM10 activity has a pronounced effect on expression of specific brush-border proteins. We postulate that ADAM10 and APLP2 may represent elements of a here-to-fore unknown signaling pathway in proximal tubule that link events at the brush border with control of gene expression.

Regulation of pendrin by pH: dependence on glycosylation

Mutations in the anion exchanger pendrin are responsible for Pendred syndrome, an autosomal recessive disease characterized by deafness and goitre. Pendrin is highly expressed in kidney collecting ducts, where it acts as a chloride/bicarbonate exchanger and thereby contributes to the regulation of acid-base homoeostasis and blood pressure. The present study aimed to characterize the intrinsic properties of pendrin. Mouse pendrin was transfected in HEK (human embryonic kidney) 293 and OKP (opossum kidney proximal tubule) cells and its activity was determined by monitoring changes in the intracellular pH induced by variations of transmembrane anion gradients. Combining measurements of pendrin activity with mathematical modelling we found that its affinity for Cl-, HCO3- and OH- varies with intracellular pH, with increased activity at low intracellular pH. Maximal pendrin activity was also stimulated at low extracellular pH, suggesting the presence of both intracellular and extracellular proton regulatory sites. We identified five putative pendrin glycosylation sites, only two of which are used. Mutagenesis-induced disruption of pendrin glycosylation did not alter its cell-surface expression or polarized targeting to the apical membrane and basal activity, but fully abrogated its sensitivity to extracellular pH. The hither to unknown regulation of pendrin by external pH may constitute a key mechanism in controlling ionic exchanges across the collecting duct and inner ear.

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.

Nedd4-2 Functionally Interacts with ClC-5: INVOLVEMENT IN CONSTITUTIVE ALBUMIN ENDOCYTOSIS IN PROXIMAL TUBULE CELLS

Constitutive albumin uptake by the proximal tubule is achieved by a receptor-mediated process in which the Cl(-) channel, ClC-5, plays an obligate role. Here we investigated the functional interaction between ClC-5 and ubiquitin ligases Nedd4 and Nedd4-2 and their role in albumin uptake in opossum kidney proximal tubule (OK) cells. In vivo immunoprecipitation using an anti-HECT antibody demonstrated that ClC-5 bound to ubiquitin ligases, whereas glutathione S-transferase pull-downs confirmed that the C terminus of ClC-5 bound both Nedd4 and Nedd4-2. Nedd4-2 alone was able to alter ClC-5 currents in Xenopus oocytes by decreasing cell surface expression of ClC-5. In OK cells, a physiological concentration of albumin (10 mug/ml) rapidly increased cell surface expression of ClC-5, which was also accompanied by the ubiquitination of ClC-5. Albumin uptake was reduced by inhibiting either the lysosome or proteasome. Total levels of Nedd4-2 and proteasome activity also increased rapidly in response to albumin. Overexpression of ligase defective Nedd4-2 or knockdown of endogenous Nedd4-2 with small interfering RNA resulted in significant decreases in albumin uptake. In contrast, pathophysiological concentrations of albumin (100 and 1000 mug/ml) reduced the levels of ClC-5 and Nedd4-2 and the activity of the proteasome to the levels seen in the absence of albumin. These data demonstrate that normal constitutive uptake of albumin by the proximal tubule requires Nedd4-2, which may act via ubiquitination to shunt ClC-5 into the endocytic pathway.

Linking Receptor-mediated Endocytosis and Cell Signaling: EVIDENCE FOR REGULATED INTRAMEMBRANE PROTEOLYSIS OF MEGALIN IN PROXIMAL TUBULE

Megalin, a member of the low density lipoprotein receptor gene family, is required for efficient protein absorption in the proximal tubule. Recent studies have shown that the low density lipoprotein receptor-related protein, another member of this gene family, is proteolytically processed by gamma-secretase implying a role for low density lipoprotein receptor-related protein in a Notchlike signaling pathway. This pathway has been shown to involve: 1) metalloprotease-mediated ectodomain shedding and gamma-secretase-mediated intramembrane proteolysis of some receptors. Experiments were performed to determine whether megalin undergoes similar processing. By immunocytochemistry, immunoblotting, and a fluorogenic enzyme assay presenilin-1 (required for gamma-secretase activity) and gamma-secretase activity were found in the brush border of proximal kidney tubules where megalin is localized. Using a fluorogenic peptide containing an amyloid precursor protein gamma-secretase cleavage site and Compound E, a specific gamma-secretase inhibitor, we found high levels of gamma-secretase activity in renal brush border membrane vesicles. Immunoblotting analysis of renal microsomes and opossum kidney proximal tubule (OKP) cells using antibodies directed to the cytosolic domain of megalin showed a 35-40-kDa, membrane-associated, carboxyl-terminal fragment of megalin (MCTF). When cells were incubated with 200 nm phorbol 12-myristate 13-acetate, the appearance of the MCTF increased 2.5-fold and was blocked by metalloprotease inhibitors. When the cells were incubated with gamma-secretase inhibitor Compound E, it caused a 2-fold increase in MCTF. Finally, incubating the cells with 1 microm vitamin D-binding protein resulted in a 25% increase in the appearance of the MCTF. In summary, the MCTF is produced by protein kinase C regulated, metalloprotease-mediated ectodomain shedding and is the substrate for gamma-secretase. We postulate that the enzymatic processing of megalin represents part of a novel ligand-dependent signaling pathway in the proximal tubule that links receptor-mediated endocytosis with cell signaling.