Opossum Kidney Cells Research Articles

Myosin 1b Regulates Amino Acid Transport by Associating Transporters with the Apical Plasma Membrane of Kidney Cells.

Amino acid transporters (AATers) in the brush border of the apical plasma membrane (APM) of renal proximal tubule (PT) cells mediate amino acid transport (AAT). We found that the membrane-associated class I myosin myosin 1b (Myo1b) localized at the apical brush border membrane of PTs. In opossum kidney (OK) 3B/2 epithelial cells, which are derived from PTs, expressed rat Myo1b-GFP colocalized in patched microvilli with expressed mouse V5-tagged SIT1 (SIT1-V5), which mediates neutral amino acid transport in OK cells. Lentivirus-mediated delivery of opossum Myo1b-specific shRNA resulted in knockdown (kd) of Myo1b expression, less SIT1-V5 at the APM as determined by localization studies, and a decrease in neutral AAT as determined by radioactive uptake assays. Myo1b kd had no effect on Pi transport or noticeable change in microvilli structure as determined by rhodamine phalloidin staining. The studies are the first to define a physiological role for Myo1b, that of regulating renal AAT by modulating the association of AATers with the APM.

Role of Na+/H+ exchanger regulatory factor 1 in forward trafficking of the type IIa Na+-Pi cotransporter.

Na+/H+ exchanger regulatory factor (NHERF1) plays a critical role in the renal transport of phosphate by binding to Na+-Pi cotransporter (NpT2a) in the proximal tubule. While the association between NpT2a and NHERF1 in the apical membrane is known, the role of NHERF1 to regulate the trafficking of NpT2a has not been studied. To address this question, we performed cell fractionation by sucrose gradient centrifugation in opossum kidney (OK) cells placed in low-Pi medium to stimulate forward trafficking of NpT2a. Immunoblot analysis demonstrated expression of NpT2a and NHERF1 in the endoplasmic reticulum (ER)/Golgi. Coimmunoprecipitation demonstrated a NpT2a-NHERF1 interaction in the ER/Golgi. Low-Pi medium for 4 and 8 h triggered a decrease in NHERF1 in the plasma membrane with a corresponding increase in the ER/Golgi. Time-lapse total internal reflection fluorescence imaging of OK cells placed in low-Pi medium, paired with particle tracking and mean square displacement analysis, indicated active directed movement of NHERF1 at early and late time points, whereas NpT2a showed active movement only at later times. Silence of NHERF1 in OK cells expressing green fluorescent protein (GFP)-NpT2a resulted in an intracellular accumulation of GFP-NpT2a. Transfection with GFP-labeled COOH-terminal (TRL) PDZ-binding motif deleted or wild-type NpT2a in OK cells followed by cell fractionation and immunoprecipitation confirmed that the interaction between NpT2a and NHERF1 was dependent on the TRL motif of NpT2a. We conclude that appropriate trafficking of NpT2a to the plasma membrane is dependent on the initial association between NpT2a and NHERF1 through the COOH-terminal TRL motif of NpT2a in the ER/Golgi and requires redistribution of NHERF1 to the ER/Golgi.

Autosomal-Recessive Mutations in SLC34A1 Encoding Sodium-Phosphate Cotransporter 2A Cause Idiopathic Infantile Hypercalcemia.

Idiopathic infantile hypercalcemia (IIH) is characterized by severe hypercalcemia with failure to thrive, vomiting, dehydration, and nephrocalcinosis. Recently, mutations in the vitamin D catabolizing enzyme 25-hydroxyvitamin D3-24-hydroxylase (CYP24A1) were described that lead to increased sensitivity to vitamin D due to accumulation of the active metabolite 1,25-(OH)2D3. In a subgroup of patients who presented in early infancy with renal phosphate wasting and symptomatic hypercalcemia, mutations in CYP24A1 were excluded. Four patients from families with parental consanguinity were subjected to homozygosity mapping that identified a second IIH gene locus on chromosome 5q35 with a maximum logarithm of odds (LOD) score of 6.79. The sequence analysis of the most promising candidate gene, SLC34A1 encoding renal sodium-phosphate cotransporter 2A (NaPi-IIa), revealed autosomal-recessive mutations in the four index cases and in 12 patients with sporadic IIH. Functional studies of mutant NaPi-IIa in Xenopus oocytes and opossum kidney (OK) cells demonstrated disturbed trafficking to the plasma membrane and loss of phosphate transport activity. Analysis of calcium and phosphate metabolism in Slc34a1-knockout mice highlighted the effect of phosphate depletion and fibroblast growth factor-23 suppression on the development of the IIH phenotype. The human and mice data together demonstrate that primary renal phosphate wasting caused by defective NaPi-IIa function induces inappropriate production of 1,25-(OH)2D3 with subsequent symptomatic hypercalcemia. Clinical and laboratory findings persist despite cessation of vitamin D prophylaxis but rapidly respond to phosphate supplementation. Therefore, early differentiation between SLC34A1 (NaPi-IIa) and CYP24A1 (24-hydroxylase) defects appears critical for targeted therapy in patients with IIH.

The Na+/H+ Exchanger-3 (NHE3) Activity Requires Ezrin Binding to Phosphoinositide and Its Phosphorylation.

Na+/H+ exchanger-3 (NHE3) plays an essential role in maintaining sodium and fluid homeostasis in the intestine and kidney epithelium. Thus, NHE3 is highly regulated and its function depends on binding to multiple regulatory proteins. Ezrin complexed with NHE3 affects its activity via not well-defined mechanisms. This study investigates mechanisms by which ezrin regulates NHE3 activity in epithelial Opossum Kidney cells. Ezrin is activated sequentially by phosphatidylinositol-4,5-bisphosphate (PIP2) binding and phosphorylation of threonine 567. Expression of ezrin lacking PIP2 binding sites inhibited NHE3 activity (-40%) indicating that ezrin binding to PIP2 is required for preserving NHE3 activity. Expression of a phosphomimetic ezrin mutated at the PIP2 binding region was sufficient not only to reverse NHE3 activity to control levels but also to increase its activity (+80%) similar to that of the expression of ezrin carrying the phosphomimetic mutation alone. Calcineurin Homologous Protein-1 (CHP1) is part, with ezrin, of the NHE3 regulatory complex. CHP1-mediated activation of NHE3 activity was blocked by expression of an ezrin variant that could not be phosphorylated but not by an ezrin variant unable to bind PIP2. Thus, for NHE3 activity under baseline conditions not only ezrin phosphorylation, but also ezrin spatial-temporal targeting on the plasma membrane via PIP2 binding is required; however, phosphorylation of ezrin appears to overcome the control of NHE3 transport. CHP1 action on NHE3 activity is not contingent on ezrin binding to PIP2 but rather on ezrin phosphorylation. These findings are important in understanding the interrelation and dynamics of a CHP1-ezrin-NHE3 regulatory complex.

Generation of Mice Expressing RFP‐tagged Sodium Phosphate Cotransporter NaPi‐IIa

Homeostasis of inorganic phosphate (Pi) is essential for various physiologic functions. The sodium phosphate cotransporter NaPi‐IIa is located in the brush border membrane (BBM) of the renal proximal tubule and represents the major renal Pi transporter. The plasma concentration of Pi is determined by factors regulating NaPi‐IIa abundance at the BBM. We are developing a novel mouse model expressing NaPi‐IIa tagged with red fluorescent protein (RFP) to analyze the regulation of the cotransporter at the BBM in real time and space and at the molecular level. We have generated RFP‐NaPi‐IIa constructs containing the RFP at three different locations of the cotransporter, the very N‐terminus, within the N‐terminal tail and within the big extracellular loop. Control experiments in opossum kidney cells and in Xenopus laevis oocytes to analyze the cotransporter polarization, PTH sensitivity, Na+‐dependent cotransport activity and kinetic characterization revealed that the RFP within the N‐terminal tail did not impair any of the analyzed parameters. Upon identification of the proper insertion site, we generated three pairs of transcription activator‐like effector nucleases (TALENs) predicted to cut the Slc34a1 (NaPi‐IIa) gene within the desired sequence. The pair with the highest cutting efficiency was chosen for the generation of RFP‐NaPi‐IIa mice. Consequently, we produced RFP donor DNA containing 5' and 3' homologous arms, as well as the RFP coding sequence. TALENs and the RFP donor were injected into one‐cell stage embryos to generate transgenic mice.This work was supported by NCCR Kidney.CH

KSRP and Roquin‐2: Mediators of PTH‐Induced Npt2a mRNA Destabilization?

Parathyroid hormone (PTH) potently induces phosphaturia through the downregulation of the type IIa sodium-phosphate cotransporter (Npt2a), at the protein and the mRNA level. We previously identified 68 RNA-binding proteins (RBPs) whose phosphorylation status significantly changed in response to 30m or 2h PTH. Preliminary in silico analysis predicts that two of those RBPs – Roquin-2 and KSRP – are capable of interacting with the 3'UTR of Npt2a mRNA. We hypothesize that PTH stimulates Npt2a mRNA degradation through alterations in Roquin-2 and KSRP activity and/or expression. To address this hypothesis, we examined Roquin-2 and KSRP expression in opossum kidney (OK) cells, a proximal tubule cell culture model, treated with 100nM PTH for 15m to 2h. Western blot demonstrated a time-dependent increase in expression of both proteins in response to PTH and a decrease in expression in response to low phosphate media. Nuclear expression of KHSRP increased 10-fold following 2h PTH. Nuclear expression of Roquin-2 increased 207% after 2h PTH, and cytosolic expression increased 342%. Immunoprecipitation of either Roquin-2 or KSRP from control and PTH-treated cells was followed by RNA isolation. RT-qPCR analysis identified Npt2a mRNA from both Roquin-2 and KSRP pulldowns. We conclude that KSRP and Roquin-2 bind Npt2a mRNA and their expression is modulated by known regulators of Npt2a expression. These findings suggest that KSRP and Roquin-2 potentially mediate Npt2a mRNA destabilization in response to PTH. Funding for this research provided by VA to EDL.

Chloride channel ClC-5 binds to aspartyl aminopeptidase to regulate renal albumin endocytosis

ClC-5 is a chloride/proton exchanger that plays an obligate role in albumin uptake by the renal proximal tubule. ClC-5 forms an endocytic complex with the albumin receptor megalin/cubilin. We have identified a novel ClC-5 binding partner, cytosolic aspartyl aminopeptidase (DNPEP; EC 3.4.11.21), that catalyzes the release of N-terminal aspartate/glutamate residues. The physiological role of DNPEP remains largely unresolved. Mass spectrometric analysis of proteins binding to the glutathione-S-transferase (GST)-ClC-5 C terminus identified DNPEP as an interacting partner. Coimmunoprecipitation confirmed that DNPEP and ClC-5 also associated in cells. Further experiments using purified GST-ClC-5 and His-DNPEP proteins demonstrated that the two proteins bound directly to each other. In opossum kidney (OK) cells, confocal immunofluorescence studies revealed that DNPEP colocalized with albumin-containing endocytic vesicles. Overexpression of wild-type DNPEP increased cell-surface levels of ClC-5 and albumin uptake. Analysis of DNPEP-immunoprecipitated products from rat kidney lysate identified β-actin and tubulin, suggesting a role for DNPEP in cytoskeletal maintenance. A DNase I inhibition assay showed a significant decrease in the amount of G actin when DNPEP was overexpressed in OK cells, suggesting a role for DNPEP in stabilizing the cytoskeleton. DNPEP was not present in the urine of healthy rats; however, it was readily detected in the urine in rat models of mild and heavy proteinuria (diabetic nephropathy and anti-glomerular basement membrane disease, respectively). Urinary levels of DNPEP were found to correlate with the severity of proteinuria. Therefore, we have identified another key molecular component of the albumin endocytic machinery in the renal proximal tubule and describe a new role for DNPEP in stabilizing the actin cytoskeleton.

Relationship between sodium-dependent phosphate transporter (NaPi-IIc) function and cellular vacuole formation in opossum kidney cells.

NaPi-IIc/SLC34A3 is a sodium-dependent inorganic phosphate (Pi) transporter in the renal proximal tubules and its mutations cause hereditary hypophosphatemic rickets with hypercalciuria (HHRH). In the present study, we created a specific antibody for opossum SLC34A3, NaPi-IIc (oNaPi-IIc), and analyzed its localization and regulation in opossum kidney cells (a tissue culture model of proximal tubular cells). Immunoreactive oNaPi-IIc protein levels increased during the proliferative phase and decreased during differentiation. Moreover, stimulating cell growth upregulated oNaPi-IIc protein levels, whereas suppressing cell proliferation downregulated oNaPi-IIc protein levels. Immunocytochemistry revealed that endogenous and exogenous oNaPi-IIc proteins localized at the protrusion of the plasma membrane, which is a phosphatidylinositol 4,5-bisphosphate (PIP2) rich-membrane, and at the intracellular vacuolar membrane. Exogenous NaPi-IIc also induced cellular vacuoles and localized in the plasma membrane. The ability to form vacuoles is specific to electroneutral NaPi-IIc, and not electrogenic NaPi-IIa or NaPi-IIb. In addition, mutations of NaPi-IIc (S138F and R468W) in HHRH did not cause cellular PIP2-rich vacuoles. In conclusion, our data anticipate that NaPi-IIc may regulate PIP2 production at the plasma membrane and cellular vesicle formation.

Selection of a novel FGF23-binding peptide antagonizing the inhibitory effect of FGF23 on phosphate uptake.

Fibroblast growth factor 23 (FGF23) is a bone-derived endocrine regulator of phosphate homeostasis and has been considered as a potential therapeutic target for hypophosphatemic disorders. Herein, we isolated a novel FGF23-binding peptide by screening a phage display library with FGF23180-205, the minimal epitope of FGF23 binding to the binary fibroblast growth factor receptor (FGFR)-Klotho complex. The corresponding peptide (referred to as 23-b6) showed high homology to the immunoglobulin-like (Ig-like) domain III (D3) of FGFR1c, the predominant receptor mediating the phosphaturic activity of FGF23. The 23-b6 peptide and panning target FGF23180-205 carried opposite charges and shared similar hydrophilic profiles. Functional analysis indicated that synthetic 23-b6 peptide exhibited antagonistic effect on the inhibition of phosphate uptake by FGF23 in opossum kidney cells (OK cells). The mechanisms of 23-b6 peptide impairing the bioactivity of FGF23 involved blockade of the activation of Erk cascade and up-regulation of NaPi-2a and NaPi-2c expression in OK cells. Our results demonstrate that the 23-b6 peptide is a potent FGF23 antagonist with increased effect on phosphate uptake in kidney cells and might have therapeutic potentials in hypophosphatemic disorders characterized by an abnormally high level of FGF23.

Acute leptin exposure reduces megalin expression and upregulates TGFβ1 in cultured renal proximal tubule cells

Increased leptin concentrations observed in obesity can lead to proteinuria, suggesting that leptin may play a role in obesity-related kidney disease. Obesity reduces activation of AMP-activated protein kinase (AMPK) and increases transforming growth factor-β1 (TGF-β1) expression in the kidney, leading to albuminuria. Thus we investigated if elevated leptin altered AMPK and TGF-β1 signaling in proximal tubule cells (PTCs). In opossum kidney (OK) PTCs Western blot analysis demonstrated that leptin upregulates TGF-β1 secretion (0.50 µg/ml) and phosphorylated AMPKα (at 0.25, and 0.50 µg/ml), and downregulates megalin expression at all concentrations (0.05–0.50 µg/ml). Using the AMPK inhibitor, Compound C, leptin exposure regulated TGF-β1 expression and secretion in PTCs via an AMPK mediated pathway. In addition, elevated leptin exposure (0.50 µg/ml) reduced albumin handling in OK cells independently of megalin expression. This study demonstrates that leptin upregulates TGF-β1, reduces megalin, and reduces albumin handling in PTCs by an AMPK mediated pathway.

Transcriptional regulation of human organic anion transporter 1 by B-cell CLL/lymphoma 6

The human organic anion transporter 1 (OAT1) is crucial for the excretion of organic anions in renal proximal tubular cells and has been classified as a clinically relevant transporter in the kidneys. Our previous study indicated that renal male-predominant expression of rat Oat1 and Oat3 appears to be regulated by transcription factor B-cell CLL/lymphoma 6 (BCL6). The aim of this study was to characterize the effect of BCL6 on human OAT1 promoter and on the transcription of OAT1 mediated by hepatocyte nuclear factor-1α (HNF-1α). Luciferase assays were carried out in opossum kidney (OK) cells transiently transfected with promoter constructs of OAT1, expression vectors for BCL6 and HNF-1α, and the empty control vectors. BCL6 and HNF-1α binding on OAT1 promoter was analyzed using electrophoretic mobility shift assay (EMSA). Protein expression of HNF-1α was investigated by Western blot analysis. Site-directed mutagenesis was used to introduce mutations into BCL6 and HNF-1α binding sites within the OAT1 promoter. BCL6 enhanced the promoter activity of OAT1 independently of predicted BCL6 binding sites but was dependent on HNF-1α response element and HNF-1α protein. Coexpression of BCL6 and HNF-1α induced an additive effect on OAT1 promoter activation compared with BCL6 or HNF-1α alone. BCL6 does not bind directly or indirectly to OAT1 promoter but increases the protein expression of HNF-1α and thereby indirectly enhances OAT1 gene transcription. BCL6 constitutes a promising candidate gene for the regulation of human OAT1 transcription and other renal and/or hepatic drug transporters that have been already shown to be activated by HNF-1α.

Kidney tubular-cell secretion of osteoblast growth factor is increased by kaempferol: a scientific basis for "the kidney controlling the bone" theory of Chinese medicine.

To study, at the cytological level, the basic concept of Chinese medicine that "the Kidney (Shen) controls the bone". Kaempferol was isolated form Rhizoma Drynariae (Gu Sui Bu, GSB) and at several concentrations was incubated with opossum kidney (OK) cells, osteoblasts (MC3T3 E1) and human fibroblasts (HF) at cell concentrations of 2×10(4)/mL. Opossum kidney cell-conditioned culture media with kaempferol at 70 nmol/L (70kaeOKM) and without kaempferol (0OKM) were used to stimulate MC3T3 E1 and HF proliferation. The bone morphological protein receptors I and II (BMPR I and II) in OK cells were identified by immune-fluorescence staining and Western blot analysis. Kaempferol was found to increase OK cell growth (P<0.05), but alone did not promote MC3T3 E1 or HF cell proliferation. However, although OKM by itself increased MC3T3 E1 growth by 198% (P<0.01), the 70kaeOKM further increased the growth of these cells by an additional 127% (P<0.01). It indicates that the kidney cell generates a previously unknown osteoblast growth factor (OGF) and kaempferol increases kidney cell secretion of OGF. Neither of these media had any significant effect on HF growth. Kaempferol also was found to increase the level of the BMPR II in OK cells. This lends strong support to the original idea that the Kidney has a significant influence over bone-formation, as suggested by some long-standing Chinese medical beliefs, kaempferol may also serve to stimulate kidney repair and indirectly stimulate bone formation.

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.

Lysophosphatidic acid stimulation of NHE3 exocytosis in polarized epithelial cells occurs with release from NHERF2 via ERK-PLC-PKCδ signaling.

The Na(+)/H(+) exchanger 3 (NHE3) is a brush border (BB) Na(+)/H(+) antiporter that accounts for the majority of physiologic small intestinal and renal Na(+) absorption. It is regulated physiologically and in disease via changes in endocytosis/exocytosis. Paradoxically, NHE3 is fixed to the microvillar (MV) actin cytoskeleton and has little basal mobility. This fixation requires NHE3 binding to the multi-PDZ domain scaffold proteins Na(+)/H(+) exchanger regulatory factor (NHERF)1 and NHERF2 and to ezrin. Coordinated release of NHE3 from the MV cytoskeleton has been demonstrated during both stimulation and inhibition of NHE3. However, the signaling molecules involved in coordinating NHE3 trafficking and cytoskeletal association have not been identified. This question was addressed by studying lysophosphatidic acid (LPA) stimulation of NHE3 in polarized renal proximal tubule opossum kidney (OK) cells that occurs via apical LPA5 receptors and is NHERF2 dependent and mediated by epidermal growth factor receptor (EGFR), Rho/Rho-associated kinase (ROCK), and ERK. NHE3 activity was determined by BCECF/fluorometry and NHE3 microvillar mobility by FRAP/confocal microscopy using NHE3-EGFP. Apical LPA (3 μM)/LPA5R stimulated NHE3 activity, increased NHE3 mobility, and decreased the NHE3/NHERF2 association. The LPA stimulation of NHE3 was also PKCδ dependent. PKCδ was necessary for LPA stimulation of NHE3 mobility and NHE3/NHERF2 association. Moreover, the LPA-induced translocation to the membrane of PKCδ was both ERK and phospholipase C dependent with ERK acting upstream of PLC. We conclude that LPA stimulation of NHE3 exocytosis includes a signaling pathway that regulates fixation of NHE3 to the MV cytoskeleton. This involves a signaling module consisting of ERK-PLC-PKCδ, which dynamically and reversibly releases NHE3 from NHERF2 to contribute to the changes in NHE3 MV mobility.

Analysis of the uptake of native versus modified albumin by renal tubular epithelial cells (LB59)

Albumin is most abundant protein in blood and plays important homeostatic roles in the regulation of oncotic pressure and as a carrier of many small proteins, peptides and hormones. Albumin in the circulation can be covalently modified at surface lysine residues and thus the circulation contains a mixture of native albumin (i.e. not modified) and modified albumin. A significant amount of albumin is lost from the blood each day as a result of the filtration occurring in the capillaries of the renal glomeruli. This filtered albumin in the early filtrate is thought to be mostly taken up by and degraded in the early renal proximal tubular epithelial cells, while a small proportion is excreted in the final urine. This degradation of albumin by proximal tubules operates via the megalin scavenger receptor and an endocytic pathway.A major gap in our knowledge is whether the megalin‐based albumin uptake is a non‐selective process, or whether megalin has a significantly different affinity for modified versus native albumin.Using a well‐established albumin uptake assay in Opossum Kidney (OK) cells, modified albumin probes (Alexa488‐BSA and FITC‐BSA) were shown taken up into OK cells via the endocytic pathway. This uptake was efficiently competed by a molar excess of modified albumin, whereas an excess of rHA had little effect upon the uptake, suggesting a difference in binding affinity. The addition of 14C‐rHA to OK cells resulted in a very low percentage (3%) of the probe being taken up by cells compared to a 30% uptake using Alexa488‐BSA. In addition, the uptake of 14C‐rHA was not saturated in a dose‐response assay suggesting non‐specific binding. This was confirmed when an inhibitor of the endocytic pathway failed to inhibit the uptake of 14C‐rHA by OK cells.The results indicate that the megalin pathway has a much greater affinity for modified compared to native albumin. This may be an important mechanism for the removal of modified albumin from the body as it can exert toxic effects.

Transcriptional regulation of renal organic anion transporter 1 by B‐cell CLL/lymphoma 6 (892.33)

The human organic anion transporters 1 (OAT1) is crucial for the excretion of organic anions in renal proximal tubular cells and was classified as clinically relevant transporter in the kidneys. Our previous study indicated that renal male‐predominant expression of rat Oat1 and Oat3 appears to be regulated by transcription factor B‐cell CLL/lymphoma 6 (BCL6). The aim of this study was to characterize the effect of BCL6 on human OAT1 promoter.Luciferase assays were carried out on Opossum kidney (OK) cells transiently transfected with promoter constructs of OAT1 and expression vectors for BCL6. Transcription factor (TF) activation profiling plate array was used for monitoring the activation of multiple transcription factors in nuclear extracts of pcDNA3‐BCL6 and pcDNA3 transfected OK cells. The protein expression of several transcription factors was investigated using fluorescence microscopy and Western blot.BCL6 enhanced the promoter activity of OAT1, independently of predicted BCL6‐binding sites. Promoter sequence ‐63 to +1 bp of OAT1, which contained HNF1a binding sites, is the minimal regions required for BCL6 dependent activation. BCL6 enhanced the protein expression of HNF1α, a known OAT1 activator. HNF1α dependent activation of OAT1 promoter was increased by BCL6.BCL6 constitutes a promising candidate gene for the regulation of human OAT1 transcription, possibly via activation of HNF1α.

Hepatectomy-Related Hypophosphatemia

Marked hypophosphatemia is common after major hepatic resection, but the pathophysiologic mechanism remains unknown. We used a partial hepatectomy (PH) rat model to investigate the molecular basis of hypophosphatemia. PH rats exhibited hypophosphatemia and hyperphosphaturia. In renal and intestinal brush-border membrane vesicles isolated from PH rats, Na(+)-dependent phosphate (Pi) uptake decreased by 50%-60%. PH rats also exhibited significantly decreased levels of renal and intestinal Na(+)-dependent Pi transporter proteins (NaPi-IIa [NaPi-4], NaPi-IIb, and NaPi-IIc). Parathyroid hormone was elevated at 6 hours after PH. Hyperphosphaturia persisted, however, even after thyroparathyroidectomy in PH rats. Moreover, DNA microarray data revealed elevated levels of nicotinamide phosphoribosyltransferase (Nampt) mRNA in the kidney after PH, and Nampt protein levels and total NAD concentration increased significantly in the proximal tubules. PH rats also exhibited markedly increased levels of the Nampt substrate, urinary nicotinamide (NAM), and NAM catabolites. In vitro analyses using opossum kidney cells revealed that NAM alone did not affect endogenous NaPi-4 levels. However, in cells overexpressing Nampt, the addition of NAM led to a marked decrease in cell surface expression of NaPi-4 that was blocked by treatment with FK866, a specific Nampt inhibitor. Furthermore, FK866-treated mice showed elevated renal Pi reabsorption and hypophosphaturia. These findings indicate that hepatectomy-induced hypophosphatemia is due to abnormal NAM metabolism, including Nampt activation in renal proximal tubular cells.

Phosphatidylinositol 3-phosphate 5-kinase (PIKfyve) is an AMPK target participating in contraction-stimulated glucose uptake in skeletal muscle

PIKfyve (FYVE domain-containing phosphatidylinositol 3-phosphate 5-kinase), the lipid kinase that phosphorylates PtdIns3P to PtdIns(3,5)P2, has been implicated in insulin-stimulated glucose uptake. We investigated whether PIKfyve could also be involved in contraction/AMPK (AMP-activated protein kinase)-stimulated glucose uptake in skeletal muscle. Incubation of rat epitrochlearis muscles with YM201636, a selective PIKfyve inhibitor, reduced contraction- and AICAriboside (5-amino-4-imidazolecarboxamide riboside)-stimulated glucose uptake. Consistently, PIKfyve knockdown in C2C12 myotubes reduced AICAriboside-stimulated glucose transport. Furthermore, muscle contraction increased PtdIns(3,5)P2 levels and PIKfyve phosphorylation. AMPK phosphorylated PIKfyve at Ser307 both in vitro and in intact cells. Following subcellular fractionation, PIKfyve recovery in a crude intracellular membrane fraction was increased in contracting versus resting muscles. Also in opossum kidney cells, wild-type, but not S307A mutant, PIKfyve was recruited to endosomal vesicles in response to AMPK activation. We propose that PIKfyve activity is required for the stimulation of skeletal muscle glucose uptake by contraction/AMPK activation. PIKfyve is a new AMPK substrate whose phosphorylation at Ser307 could promote PIKfyve translocation to endosomes for PtdIns(3,5)P2 synthesis to facilitate GLUT4 (glucose transporter 4) translocation.