Association of Na(+)-H(+) exchanger isoform NHE3 and dipeptidyl peptidase IV in the renal proximal tubule

In an attempt to identify proteins that assemble with the apical membrane Na(+)-H(+) exchanger isoform NHE3, we generated monoclonal antibodies (mAbs) against affinity-purified NHE3 protein complexes isolated from solubilized renal microvillus membrane vesicles. Hybridomas were selected based on their ability to immunoprecipitate NHE3. We have characterized in detail one of the mAbs (1D11) that specifically co-precipitated NHE3 but not villin or NaPi-2. Western blot analyses of microvillus membranes and immunoelectron microscopy of kidney sections showed that mAb 1D11 recognizes a 110-kDa protein highly expressed on the apical membrane of proximal tubule cells. Immunoaffinity chromatography was used to isolate the antigen against which mAb 1D11 is directed. N-terminal sequencing of the purified protein identified it as dipeptidyl peptidase IV (DPPIV) (EC ), which was confirmed by assays of DPPIV enzyme activity. We also evaluated the distribution of the NHE3-DPPIV complex in microdomains of rabbit renal brush border. In contrast to the previously described NHE3-megalin complex, which principally resides in a dense membrane population (coated pits) in which NHE3 is inactive, the NHE3-DPPIV complex was predominantly in the microvillar fraction in which NHE3 is active. Serial precipitation experiments confirmed that anti-megalin and anti-DPPIV antibodies co-precipitate different pools of NHE3. Taken together, these studies revealed an unexpected association of the brush border Na(+)-H(+) exchanger NHE3 with dipeptidyl peptidase IV in the proximal tubule. These findings raise the possibility that association with DPPIV may affect NHE3 surface expression and/or activity.     

Active (9.6 s) and inactive (21 s) oligomers of NHE3 in microdomains of the renal brush border

We have previously shown that Na(+)-H(+) exchanger isoform NHE3 exists as both 9.6 and 21 S (megalin-associated) oligomers in the renal brush border. To characterize the oligomeric forms of the renal brush border Na(+)-H(+) exchanger in more detail, we performed membrane fractionation studies. We found that similar amounts of NHE3 were present in microvilli and a nonmicrovillar membrane domain of high density (dense vesicles). Horseradish peroxidase-labeled endosomes were not prevalent in the dense membrane fraction. However, megalin, which localizes primarily to the intermicrovillar microdomain of the brush border, was enriched in the dense vesicles, implicating this microdomain as the likely source of these membranes. Immunolocalization of NHE3 confirmed that a major fraction of the transporter colocalized with megalin in the intermicrovillar region of the brush border. Immunoprecipitation studies demonstrated that in microvilli the majority of NHE3 was not bound to megalin, while in the dense vesicles most of the NHE3 coprecipitated with megalin. Moreover, sucrose velocity gradient centrifugation experiments revealed that most NHE3 in microvilli sedimented with an S value of 9.6, while the S value of NHE3 in dense vesicles was 21. Finally, we examined the functional state of NHE3 in both membrane fractions. As assayed by changes in acridine orange fluorescence, imposing an outwardly directed Na(+) gradient caused generation of an inside acid pH gradient in the microvilli, indicating Na(+)-H(+) exchange activity, but not in the dense vesicles. Taken together, these data demonstrate that renal brush border NHE3 exists in two oligomeric states: a 9.6 S active form present in microvilli and a 21 S, megalin-associated, inactive form in the intermicrovillar microdomain of the apical plasma membrane. Thus, regulation of renal brush border Na(+)-H(+) exchange activity may be mediated by shifting the distribution between these forms of NHE3.     

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.     

Effect of angiotensin-II on renal Na+/H+ exchanger-NHE3 and NHE2

The purpose of the present study was to determine the effect of angiotensin II (A-II) on membrane expression of Na+/H+ exchange isoforms NHE3 and NHE2 in the rat renal cortex. A-II (500 ng/kg per min) was chronically infused into the Sprague-Dawley rats by miniosmotic pump for 7 days. Arterial pressure and circulating plasma A-II level were significantly increased in A-II rats as compared to control rats. pH-dependent uptake of 22Na+ study in the presence of 50 microM HOE-694 revealed that Na+ uptake mediated by NHE3 was increased approximately 88% in the brush border membrane from renal cortex of A-II-treated rats. Western blotting showed that A-II increased NHE3 immunoreactive protein levels in the brush border membrane of the proximal tubules by 31%. Northern blotting revealed that A-II increased NHE3 mRNA abundance in the renal cortex by 42%. A-II treatment did not alter brush border NHE2 protein abundance in the renal proximal tubules. In conclusion, chronic A-II treatment increases NHE3-mediated Na+ uptake by stimulating NHE3 mRNA and protein content.     

NHERF-1 uniquely transduces the cAMP signals that inhibit sodium-hydrogen exchange in mouse renal apical membranes

Sodium-hydrogen exchanger regulatory factor isoform-1 (NHERF-1) and NHERF-2 are two structurally related PDZ-domain-containing protein adapters that effectively transduce cyclic AMP (cAMP) signals that inhibit NHE3, the sodium-hydrogen exchanger isoform present at the apical surface of kidney and gut epithelia. The mouse renal proximal tubule expresses both NHERF isoforms, suggesting their redundant functions as regulators of renal electrolyte metabolism. To define the role of NHERF-1 in the physiological control of NHE3, we analyzed NHE3 activity in isolated brush border membrane (BBM) preparations from renal proximal tubules of wild-type (WT) and NHERF-1 (-/-) mice. Basal Na(+)-H(+) exchange was indistinguishable in BBMs from WT and NHERF-1 (-/-) mice (0.96+/-0.08 and 0.95+/-0.10 nmol/mg protein/10 s, respectively). Activation of membrane bound cAMP-dependent protein kinase (PKA) by cAMP inhibited NHE3 activity in WT BBMs (0.55+/-0.07 nmol/mg protein/10 s or 40+/-9%, P<0.01) but had no discernible effect on Na(+)-H(+) exchange in the NHERF-1 (-/-) BBM (0.97+/-0.07 nmol/mg protein/10 s; P=not significant). This was associated with a significant decrease in cAMP-stimulated phosphorylation of NHE3 immunoprecipitated from solubilized NHERF-1 (-/-) BBMs. As the protein levels for NHE3, NHERF-2, PKA and ezrin were not changed in the NHERF-1 (-/-) BBMs, the data suggest a unique role for NHERF-1 in cAMP-mediated inhibition of NHE3 activity in the renal proximal tubule of the mouse.     

Identification and partial purification of a band 3-like protein from rabbit renal brush border membranes

A monoclonal antibody against the membrane domain of human erythrocyte band 3 was tested for its ability to bind to rabbit renal brush border membranes. A single brush border protein with a molecular mass of 43 kDa was recognized by the band 3 antibody. Using DNase I coupled to an agarose-bead support this 43-kDa protein was partially purified by removing actin and a number of actin-bound proteins from the brush border membranes. The partially purified 43 kDa-band was eluted from sodium dodecyl sulfate-polyacrylamide gels and used to make a highly sensitive and specific guinea pig antiserum. This antiserum, but not serum from control guinea pigs, cross-reacts with purified band 3 from human, rabbit, and bovine erythrocytes confirming the immunologic similarity among these proteins. The 43-kDa protein can be stained by the periodic acid-Schiff base method and binds wheat germ agglutinin and concanavalin A, demonstrating that it is a glycoprotein. Furthermore, in the absence of dithiothreitol, the immunoreactive brush border protein migrates with a molecular mass of 86 kDa on an sodium dodecyl sulfate-polyacrylamide gel suggesting that under nonreducing conditions it exists as a dimer. The 43-kDa protein could be solubilized in octyl glucoside and was further purified using gel filtration chromatography. The amino acid composition of the 43-kDa brush border protein was obtained, and its similarity with erythrocyte band 3 is discussed.     

Effect of angiotensin-II on renal Na/H exchanger-NHE3 and NHE2

The purpose of the present study was to determine the effect of angiotensin II (A-II) on membrane expression of Na⁺/H⁺ exchange isoforms NHE3 and NHE2 in the rat renal cortex. A-II (500 ng/kg per min) was chronically infused into the Sprague-Dawley rats by miniosmotic pump for 7 days. Arterial pressure and circulating plasma A-II level were significantly increased in A-II rats as compared to control rats. pH-dependent uptake of ²²Na⁺ study in the presence of 50 μM HOE-694 revealed that Na⁺ uptake mediated by NHE3 was increased ∼88% in the brush border membrane from renal cortex of A-II-treated rats. Western blotting showed that A-II increased NHE3 immunoreactive protein levels in the brush border membrane of the proximal tubules by 31%. Northern blotting revealed that A-II increased NHE3 mRNA abundance in the renal cortex by 42%. A-II treatment did not alter brush border NHE2 protein abundance in the renal proximal tubules. In conclusion, chronic A-II treatment increases NHE3-mediated Na⁺ uptake by stimulating NHE3 mRNA and protein content.     

Interactions of cubilin with megalin and the product of the amnionless gene (AMN): effect on its stability

Cubilin, a 456 kDa multipurpose receptor lacking in both transmembrane and cytoplasmic domains is expressed in the apical BBMs (brush border membranes) of polarized epithelia. Cubilin interacts with two transmembrane proteins, AMN, a 45-50 kDa protein product of the amnionless gene, and megalin, a 600 kDa giant endocytic receptor. In vitro, three fragments of cubilin, the 113-residue N-terminus and CUB domains 12-17 and 22-27, demonstrated Ca2+-dependent binding to megalin. Immunoprecipitation and immunoblotting studies using detergent extracts of rat kidney BBMs revealed that cubilin interacts with both megalin and AMN. Ligand (intrinsic factor-cobalamin)-affinity chromatography showed that in renal BBMs, functional cubilin exists as a complex with both AMN and megalin. Cubilin and AMN levels were reduced by 80% and 55-60% respectively in total membranes and BBMs obtained from kidney of megalin antibody-producing rabbits. Immunohistochemical analysis and turnover studies for cubilin in megalin or AMN gene-silenced opossum kidney cells showed a significant reduction (85-90%) in cubilin staining and a 2-fold decrease in its half-life. Taken together, these results indicate that three distinct regions of cubilin bind to megalin and its interactions with both megalin and AMN are essential for its intracellular stability.     

Binding of nicotinamide adenine dinucleotide by the renal brush border membrane from rat kidney cortex

The characteristics of nicotinamide adenine dinucleotide (NAD) binding on brush border membranes prepared from rat renal cortex were investigated with the use of radioactively labelled NAD, [adenine-2,8-3H]NAD+, as a ligand. (1) We found that NAD binds on brush border membrane and that the extent of NAD binding is linearly proportional to the brush border membrane protein, and progressively increases with concentration of NAD in the medium. (2) The rate of NAD binding was dependent on temperature. At 20 degrees C, the equilibrium binding was obtained at 15 min, while NAD binding at 0 degree C was slower, but the final level of binding reached at 120 min was similar to that plateau of binding observed at 20 degrees C. Brush border membrane inactivated by heating at 95 degrees C for 3 min did not bind NAD. Binding of NAD on brush border membranes was reversed by simple dilution or by the addition of unlabelled NAD. Both alpha-NAD and beta-NAD stereoisomers displaced bound [3H]NAD. Reduced NAD (NADH) caused less displacement of bound NAD than oxidized NAD+. Adenine, nicotinamide, pyrophosphate, of 5'-AMP did not displace bound NAD. (3) The NAD binding to brush border membranes was nearly saturable, approximating saturation at 10(-4) M NAD. Kinetic analysis by Scatchard plot indicates two sets of NAD binding sites in brush border membranes: a high-affinity binding site (Kd = 1.9 . 10(-5) M) and a low-affinity binding site (Kd = 2.2 . 10(-3) M). (4) Unlike concentrative uptake of D-[14C]glucose by brush border membrane vesicles, binding of NAD was not dependent on the presence of an outside-in sodium gradient [Na+0 greater than Na+i], nor was it abolished by repeated freezing and thawing of brush border membranes. Unlike D-[14C]glucose uptake, NAD binding by brush border membranes did not change upon decrease of intravesicular volume in hypertonic media. These observations indicate that NAD association with brush border membranes is true binding rather than intravesicular uptake of this compound. (5) The presence of specific binding sites in renal brush border membrane capable of binding of NAD with a high degree of affinity suggests that such sites may be involved in previously observed (Kempson, S.A., Colon-Otero, G., Ou, S.L., Turner, S.T. and Dousa, T.P. (1981) J. Clin. Invest. 67, 1347) modulatory effect of NAD on sodium-gradient-dependent uptake of phosphate across luminal brush border membrane of proximal tubules.     

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.     

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.     

Transcobalamin II Receptor Interacts with Megalin in the Renal Apical Brush Border Membrane

Purified human transcobalamin II receptor (TC II-R) binds to megalin, a 600 kDa endocytic receptor with an association constant, K(a), of 66 n M and bound(max) of 1.1 mole of TC II-R/mole of megalin both in the presence and absence of its ligand, transcobalamin II (TC II). Immunoprecipitation followed by immunoblotting of Triton X-100 extracts of the apical brush border membrane (BBM) from rabbit renal cortex revealed association of these two proteins. (35)[S]-TC II complexed with cobalamin (Cbl; Vitamin B(12)) bound to Sepharose-megalin affinity matrix and the binding was enhanced 5-fold when TC II-R was prebound to megalin. Megalin antiserum inhibited both the TC II-R-dependent and -independent binding of (35)[S]-TC II-Cbl to megalin, while TC II-R antiserum inhibited only the TC II-R-dependent binding. In rabbits with circulating antiserum to megalin, renal apical BBM megalin was present as an immune complex, but its levels were not altered. However, the protein levels of both TC II-R and the cation-independent mannose 6-phosphate receptor (CIMPR) were drastically reduced and the urinary excretion of TC II, albumin, and other low-molecular weight proteins was significantly increased. These results suggest that megalin contains a distinct single high-affinity binding site for TC II-R and their association in the native renal BBM is important for tubular reabsorption of many proteins, including TC II.     

Identification and purification of a renal amiloride-binding protein with properties of the Na+-H+ exchanger

The aim of this study was to identify and purify the Na+-H+ exchanger from rabbit renal brush border membranes by use of affinity chromatography. Triton-solubilized membranes were equilibrated with an affinity matrix consisting of the amiloride analogue A35 (5-N-(3-aminophenyl)amiloride) covalently coupled to Sepharose CL-4B beads through a triglycine spacer arm. The matrix was then washed extensively with buffer and sequentially eluted with buffer, buffer containing 5 mM amiloride, and 1% sodium dodecyl sulfate (SDS). Eluates were concentrated and subjected to SDS-polyacrylamide gel electrophoresis. The silver-stained gel revealed a 25-kDa protein that was not visible in the initial solubilized brush border membrane extract, was not eluted from the affinity matrix by buffer alone, but was eluted with 5 mM amiloride. A subsequent elution with 1% SDS did not release any more of the 25-kDa protein, indicating that it had been completely eluted from the affinity matrix by amiloride. The presence of 5 mM amiloride during equilibration of the solubilized brush border extract with the affinity matrix completely blocked adsorption of the 25-kDa protein. The relative abundance of this protein correlated closely with Na+-H+ exchange activity when preparations of cortical brush border membrane vesicles, outer medullary brush border membrane vesicles, and cortical basolateral membrane vesicles were compared. Moreover, binding of the protein to the affinity matrix was inhibited by amiloride and amiloride analogues with a rank order identical to that for inhibition of Na+-H+ exchange activity. These findings strongly suggest that the 25-kDa protein is a structural component of the Na+-H+ exchanger.     

Apolipoprotein A-II is catabolized in the kidney as a function of its plasma concentration

We investigated in vivo catabolism of apolipoprotein A-II (apo A-II), a major determinant of plasma HDL levels. Like apoA-I, murine apoA-II (mapoA-II) and human apoA-II (hapoA-II) were reabsorbed in the first segment of kidney proximal tubules of control and hapoA-II-transgenic mice, respectively. ApoA-II colocalized in brush border membranes with cubilin and megalin (the apoA-I receptor and coreceptor, respectively), with mapoA-I in intracellular vesicles of tubular epithelial cells, and was targeted to lysosomes, suggestive of degradation. By use of three transgenic lines with plasma hapoA-II concentrations ranging from normal to three times higher, we established an association between plasma concentration and renal catabolism of hapoA-II. HapoA-II was rapidly internalized in yolk sac epithelial cells expressing high levels of cubilin and megalin, colocalized with cubilin and megalin on the cell surface, and effectively competed with apoA-I for uptake, which was inhibitable by anti-cubilin antibodies. Kidney cortical cells that only express megalin internalized LDL but not apoA-II, apoA-I, or HDL, suggesting that megalin is not an apoA-II receptor. We show that apoA-II is efficiently reabsorbed in kidney proximal tubules in relation to its plasma concentration.     

Microdomains of distinctive glycoprotein composition in the kidney proximal tubule brush border

Two membrane proteins, maltase and gp330 (the pathogenic antigen of Heymann nephritis), present in the proximal tubule brush border have recently been independently purified and found to be large glycoproteins of similar molecular weight (Mr = approximately 300,000) by SDS PAGE. To determine the relationship between the two, monoclonal antibodies raised against the purified proteins were used for comparative immunochemical analyses and immunocytochemical localization. When a detergent extract of [35S]methionine-labeled rat renal cortex was used for immunoprecipitation with monoclonal antimaltase IgG, a single band of approximately 300 kdaltons was precipitated, whereas a single 330-kdalton band was precipitated with monoclonal anti-gp330 IgG. Monoclonal antimaltase (gp300) IgG also immunoprecipitated maltase activity from solubilized renal maltase preparations, whereas monoclonal anti-gp330 IgG failed to do so. When cyanogen bromide-generated peptide maps of the two proteins were compared, there were many similar peptides, but some differences. When maltase and gp330 were localized by indirect immunofluorescence and by indirect immunoperoxidase and immunogold techniques at the electron microscope level, they were found to be differently distributed in the brush border of the initial (S1 and S2) segments of the proximal tubule: maltase was concentrated (approximately 90%) on the microvilli, and gp330 was concentrated (approximately 90%) in the clathrin-coated apical invaginations located at the base of the microvilli. We conclude that maltase (gp300) and the Heymann nephritis antigen (gp330) are structurally related membrane glycoproteins with a distinctive distribution in the proximal tubule brush border which may serve as markers for the microvillar and coated microdomains, respectively, of the apical plasmalemma.     

Immunodetection and localization of protein(s) related to retinal S-antigen (arrestin) in kidney.

S-antigen (arrestin) is a cytosolic protein which regulates phototransduction in retinal rods. A protein immunologically related to S-antigen was identified in fractions from soluble extract of bovine kidney enriched by gel filtration or by immunoaffinity chromatography using a polyclonal antibody to retinal S-antigen. On immunoblots, this protein was recognized by a panel of monoclonal antibodies (mAbs S2D2, S1A3 and S9E2) directed against different S-antigen epitopes and displayed the same apparent molecular mass (48 kDa) as retinal S-antigen. All three mAbs revealed a specific immunoreactivity by indirect immunocytochemical technique on rat kidney sections. The three mAbs recognized some but not all glomerular cells, identified as epithelial cells by immunoelectron microscopy using the mAb S9E2. Both mAbs S2D2 and S1A3 gave a diffuse cytoplasmic staining in all tubule cells. Proximal tubule cells exhibited a weak immunoreactivity, whereas distal and collecting tubule cells were strongly labeled. In contrast, the mAb S9E2 immunoreaction was restricted to a cell subpopulation from distal and collecting tubules corresponding to intercalated cells identified by immunoelectron microscopy. With the mAb S9E2, the labeling of proximal tubule cells was localized in the apical region of the cytoplasm. These results suggest that two or more 48-kDa proteins immunologically cross-reactive with retinal S-antigen are present in kidney. The observed pattern of distribution is in keeping with the hypothesis that such proteins could play a role in the regulation of G-protein-related receptors present in renal glomerulus and tubule epithelial cells.     

Developmentally regulated 75-kilodalton protein expressed in LLC-PK1 cultures is a component of the renal Na+/glucose cotransport system

Na+/D-glucose symport is a secondary active glucose transport mechanism expressed only in kidney proximal tubule and in small intestine. A monoclonal antibody that recognized the Na+/glucose symporter of pig renal brush border membranes also recognized a 75-kD protein in apical membranes isolated from highly differentiated LLC-PK1 cultures, an epithelial cell line of pig renal proximal tubule origin. The 75-kD antigen was enriched from solubilized LLC-PK1 apical membranes by means of high-pressure liquid chromatography. The symporter antigen became apparent on the apical membrane surface after the development of a confluent monolayer in correlation with the expression of transport activity. Long-term treatment of cultures with the differentiation inducer hexamethylene bisacetamide was accompanied by a dramatically increased expression of the symporter antigen as detected quantitatively by Western blot analysis and qualitatively by immunofluorescence staining. The number of symporter-positive cells was dramatically increased after inducer treatment as predicted for differentiation-regulated expression. These results identify a 75-kD protein as a component of a developmentally regulated renal Na+/glucose symporter expressed in cell culture.     

Effect of limited trypsin digestion on the renal Na+−H+ exchanger and its regulation by cAMP-Dependent protein kinase

Summary The Na⁺–H⁺ exchanger from solubilized rabbit renal brush border membranes is inhibited by cAMP-dependent protein kinase (PKA) mediated protein phosphorylation. To characterize this inhibitory response and its sensitivity to limited proteolysis, the activity of the transporter was assayed after reconstitution of the proteins into artificial lipid vesicles. Limited trypsin digestion increased the basal rate of proton gradient-stimulated, amiloride-inhibitable sodium uptake in reconstituted proteoliposomes and blocked the inhibitory response to PKA-mediated protein phosphorylation. To determine if the inhibitory response to PKA-mediated protein phosphorylation could be restored to the trypsin-treated solubilized proteins, nontrypsinized solubilized brush border membrane proteins were separated by column chromatography. The addition of small molecular weight polypeptides, fractionated on Superose-12 FPLC (V _e=0.7), to trypsinized solubilized brush border membrane proteins restored the inhibitory response to PKA-mediated protein phosphorylation. Similarly, the addition of the 0.1m NaCl fraction from an anion exchange column, Mono Q-FPLC, also restored the inhibitory response to PKA. Both protein fractions contained a common 42–43 kDa protein which was preferentially phosphorylated by PKA.These results indicate that limited trypsin digestion dissociates the activity of the renal Na⁺–H⁺ exchanger from its regulation by PKA. It is suggested that trypsin cleaves an inhibitory component of the transporter and that this component is the site of PKA-mediated regulation. Phosphoprotein analysis of fractions that restored PKA regulation raises the possibility that a polypeptide of 42–43 kDa is involved in the inhibition of the renal Na⁺–H⁺ exchanger by PKA-mediated, protein phosphorylation.     

Monoclonal antibodies that bind the renal Na+/glucose symport system. 1. Identification

Phlorizin is a specific, high-affinity ligand that binds the active site of the Na+/glucose symporter by a Na+-dependent mechanism but is not itself transported across the membrane. We have isolated a panel of monoclonal antibodies that influence high-affinity, Na+-dependent phlorizin binding to pig renal brush border membranes. Antibodies were derived after immunization of mice either with highly purified renal brush border membranes or with apical membranes purified from LLC-PK1, a cell line of pig renal proximal tubule origin. Antibody 11A3D6, an IgG2b, reproducibly stimulated Na+-dependent phlorizin binding whereas antibody 18H10B12, an IgM, strongly inhibited specific binding. These effects were maximal after 30-min incubation and exhibited saturation at increased antibody concentrations. Antibodies did not affect Na+-dependent sugar uptake in vesicles but significantly prevented transport inhibition by bound phlorizin. Antibodies recognized a 75-kDa antigen identified by Western blot analysis of brush border membranes, and a 75-kDa membrane protein could be immunoprecipitated by 18H10B12. These properties, taken together with results in the following paper [Wu, J.-S.R., & Lever, J.E. (1987) Biochemistry (following paper in this issue)], provide compelling evidence that the 75-kDa antigen recognized by these antibodies is a component of the renal Na+/glucose symporter.     

Studies on the brush border membrane of the mouse duodenum

Summary Brush border membranes have been isolated from villus epithelial cells of the adult Swiss mouse duodenum. Preparations of these membranes are not contaminated by other organelles as judged from electron-micrographs of sectioned pellets of brush borders. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins from brush borders solubilized in Tris-sodium dodecyl sulfate buffer reveals a reproducible Coomassie Brilliant Blue pattern of 17 bands. By comparing the brush border protein band positions with those of standard proteins run concurrently on sodium dodecyl sulfate-polyacrylamide gel slabs it is estimated that the 17 brush border proteins and subunits have molecular weights ranging from over 250,000 to around 16,000. Periodate-fuchsin sulfite staining shows that the five more slowly migrating, high molecular weight proteins are glycoproteins. The two proteins of smallest molecular size react positively with Oil Red O but have very small amounts of lipophilic amino acid residues, which indicates that the lipid extractable from the gels in these areas is a contaminant and is not bound to the proteins.