Search

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.

NHE3 and NHERF are targeted to the basolateral membrane in proximal tubules of colchicine-treated rats.BackgroundDepolymerization of microtubules in proximal tubule (PT) cells of colchicine-treated rats causes disruption of vesicle recycling and redistribution of some brush-border membrane (BBM) transporters into cytoplasmic vesicles. NHE3, an isoform of the Na+/H+ exchanger in the PT cell BBM, is acutely regulated by a variety of mechanisms, including protein trafficking and interaction with the PDZ protein, NHERF. The effects of microtubule disruption by colchicine on NHE3 trafficking in PT and the potential role of NHERF in this process have not been studied.MethodsImmunofluorescence and immunogold cytochemistry were performed on cryosections of kidney tissue, and immunoblotting of BBM isolated from the renal cortex and outer stripe of control and colchicine-treated (3.2 mg/kg, IP, a single dose 12 hours before sacrifice) rats.ResultsIn cells of the convoluted PT (S1/S2 segments) of control rats, NHE3 was located mainly in the BBM; subapical endosomes were weakly stained. In cells of the straight PT (S3 segment), NHE3 was present in the BBM and in lysosomes. In colchicine-treated rats, there was a marked redistribution of NHE3 from the BBM into intracellular vesicles and the basolateral plasma membrane in the S1/S2 segments. In the S3 segment, the abundance of BBM NHE3 was not visibly changed, but NHE3-positive intracellular organelles largely disappeared, and the antigen was detectable in the basolateral plasma membrane. The PDZ protein NHERF followed a similar pattern: in control animals, it was strong in the BBM and negative in the basolateral membrane in cells along the PT. After colchicine treatment, expression of NHERF in the basolateral membrane strongly increased in all PT segments, where it colocalized with NHE3.ConclusionsThe data indicate that: (a) microtubules are involved in the apical targeting of NHE3 and NHERF in renal PT cells, and (b) the parallel basolateral insertion of NHE3 and NHERF may represent an indirect targeting pathway that involves transient, microtubule-independent basolateral insertion of these proteins, followed by microtubule-dependent, vesicle-mediated transcytosis to the BBM.

We investigated whether the renal brush border Na+/H+ exchanger NHE3 exists in assemblies with other proteins in native kidney membranes. To this end we generated monoclonal antibodies (mAbs) against affinity purified NHE3 protein complexes. Hybridomas were selected based on ability to immunoprecipitate NHE3. One of the resulting mAbs (10A3) labeled a high molecular mass (>200 kDa) protein and stained primarily the coated pit region of the proximal tubule in a manner similar to that described for megalin (gp330). We then confirmed that both mAb 10A3 and a known anti-megalin mAb immunoprecipitated and immunoblotted the same protein, namely megalin. mAb 10A3 specifically co-precipitated NHE3 but not villin or NaPi-2 from solubilized renal membranes, indicating specificity of the NHE3-megalin interaction. When immunoprecipitations were performed using either 10A3 or anti-NHE3 mAb 2B9 after separation of solubilized renal proteins by sucrose velocity gradient centrifugation, we found that NHE3 exists in two states with distinct sedimentation coefficients, a 9.6 S megalin-free form and a 21 S megalin-bound form, and that when NHE3 assembles with megalin, epitopes within the carboxyl-terminal 131 amino acids of NHE3 are blocked. Taken together, these findings indicate that a significant pool of NHE3 exists as a multimeric complex with megalin in the brush border of the proximal tubule.

Experimental data indicate that the relatively hydrophilic carboxyl-terminal domains of Na+-H+ exchangers mediate the regulation of transporter activity through interactions with cytoskeletal effectors. It has therefore been assumed that this entire domain lies on the cytoplasmic surface of the plasma membrane. The purpose of the present study was to determine the membrane orientation of the COOH-terminal 131 amino acids of Na+-H+exchanger isoform NHE3 by use of three monoclonal antibodies that recognize at least two distinct epitopes within this region. Enzyme-linked immunosorbent assay studies demonstrated binding of these monoclonal antibodies (mAbs) to intact right-side-out renal brush border membrane vesicles in the absence of detergent. Moreover, when coupled to an affinity matrix to isolate membrane vesicles, the anti-NHE3 mAbs bound structures that were morphologically identical to intact microvilli. To confirm the identity of the exoplasmic antigen bound by the antibodies, immunoprecipitation studies were performed. Intact right-side-out brush border membrane vesicles were incubated with the mAbs in the absence of detergent. The membranes were pelleted, supernatant with unbound antibody was removed, the pellet was solubilized, and then immunoprecipitation with secondary antibody was performed. Immunoblot analysis indicated that NHE3 was precipitated after binding of the mAbs to intact membranes. Finally, the localization of the mAb epitopes was determined using high resolution immunocytochemistry. Ultrathin cryosections of rat kidney were labeled with the mAbs and bound antibody detected with the colloidal gold technique. Labeling was restricted to the exoplasmic surface of microvilli of the proximal tubule. Taken together, these findings indicate that epitopes within the carboxyl terminus of the Na+-H+ exchanger isoform NHE3 are exposed to the outside of the plasma membrane.

The “housekeeping” sodium/hydrogen exchanger, NHE1, mediates the electroneutral 1:1 exchange of Na+ and H+ across the plasma membrane. NHE1 is ubiquitous and is studied extensively for regulation of pH i, cell volume, and response to growth factors. We describe a spontaneous mouse mutant, s low-w ave e pilepsy, (swe), with a neurological syndrome including ataxia and a unique epilepsy phenotype consisting of 3/sec absence and tonic-clonic seizures. swe was fine-mapped on Chromosome 4 and identified as a null allele of Nhe1. Mutants show selective neuronal death in the cerebellum and brainstem but otherwise are healthy. This first example of a disease-causing mutation in an Nhe gene provides a new tool for studying the delicate balance of neuroexcitability and cell survival within the CNS.

Cadherins are recognized as the principal mediators of homotypic cellular recognition and play a demonstrated role in the morphogenic direction of tissue development. We report here the identification of a structurally unique, kidney-specific member of the cadherin multigene family (Ksp-cadherin). cDNA cloning and molecular analysis of the 130-kDa protein confirmed that it was novel and indicated that it most closely resembled members of the LI-cadherin/HPT-1 cadherin subgroup. The predicted protein possesses the definitive cadherin-specific sequence motifs LDRE, DXND, and DXD in well conserved sequential arrangement, and the characteristic cysteine residues found in the last ectodomains of almost all known cadherins. Like LI-cadherin and HPT-1, Ksp-cadherin lacks the prosequence and HAV adhesion recognition sequence typical of most classical cadherins, and possesses a truncated cytoplasmic domain (18-22 amino acids). When expressed in a transient Vaccinia/T7 expression system, Ksp-cadherin displayed the classic calcium sensitivity to trypsin proteolysis that is observed in all cadherins. Immunolocalization studies and Northern analysis indicated that expression of Ksp-cadherin was kidney-specific and limited to the basolateral membranes of renal tubular epithelial cells. In summary, we have identified and cloned a novel, kidney-specific member of the cadherin multigene family that we propose be designated Ksp-cadherin.

Disposal of ferritin in the glomerular mesangium of rats. Mechanisms involved in the mesangial disposal of exogenous macromolecules were investigated in rats using native horse-spleen ferritin as a probe. After intravenous injection of ferritin into rats, renal tissue was examined at intervals from 1min to 28 days. Immunofluorescence microscopy studies showed that the biodegradable protein component, apoferritin, was detectable in glomeruli within 10min, was most prominent by 6 hr, and became undetectable at 14 to 28 days. By contrast, glomerular staining for the iron core of ferritin was strongest at 24hr and did not noticeably change during the remaining study period. Apoferritin and iron were localized in a mesangial pattern including the stalk and lacis area. Transmission electron microscopy showed entry of ferritin into the mesangial matrix within 10min and incorporation into cells in the mesangium by 30min. After day 3, all ferritin was found intracellularly. No shift of ferritin from the glomerular tuft to the vascular pole was observed and ferritin was not found in adjacent interstitial areas or macula densa cells. Counts of mesangial Ia-antigen bearing cells, demonstrated in cryostat sections by immunofluorescence, increased from a mean of 2.1 per glomerular tuft section (range, 0 to 6), in noninjected rats, to 2.8 (range, 0 to 8) 14 days after ferritin injection. However, simultaneous phase contrast microscopy revealed that only a proportion of Ia-positive cells was associated with mesangial ferritin granules and that the bulk of ferritin was located to Ia-negative cells. Counts of infiltrating monocytes, monitored by histochemical staining for nonspecific esterase, rose from 0.1 stained cells per tuft section in controls to a maximum of 1.3 (range, 0 to 3) on day 3, at a time when most cells in the mesangium showed ferritin uptake by electron microscopy. The results demonstrate that the glomerulus disposes of ferritin, upon entry into the mesangial space, by endocytosis by mesangial cells involving degradation of apoferritin and storage of iron cores in phagolysosomes. The great majority of these cells do not bear Ia-antigens. The contribution of infiltrating monocytes to this process appears small. While the fraction of ferritin which is removed from the mesangium by an extracellular route remains undetermined, there is no appreciable egress of ferritin from the glomerulus via the vascular pole to the adjacent interstitium or tubules. Elimination de la ferritine dans le mesangium glomerulaire de rats. Les mecanismes impliques dans l'elimination mesangiale de macromolecules exogenes ont ete etudies chez des rats en utilisant de la ferritine native de rate de cheval. Apres injection intraveineuse de ferritine aux rats, le tissu renal etait examine a des intervalles compris entre 1min et 28 jours. Les etudes en microscopie avec immunofluorescence ont montre que le composant proteique biodegradable, l'apoferritine, etait detectable dans les glomerules en 10min, qu'il etait predominant en 6hr et devenait indetectable de 14 a 28 jours. Par contraste, la coloration glomerulaire pour le noyau ferrique de la ferritine etait tres forte a 24hr et ne changeait pas de facon notable pendant le reste de la periode d'etude. L'apoferritine et le fer etaient localises dans un site mesangial comprenant la tige et l'aire du lacis. La microscopie electronique par transmission a montre l'entree de ferritine dans la matrice mesangiale en 10min et l'incorporation dans les cellules du mesangium en 30min. Apres le jour 3, toute la ferritine etait trouvee intracellulaire. Aucun deplacement de la ferritine de la touffe glomerulaire au pole vasculaire n'a ete observe, et la ferritine n'etait pas trouvee dans les aires interstitielles adjacentes, ni dans les cellules de la macula densa. Le compte des cellules mesangiales comportant de l'antigene Ia, mises en evidence dans des sections au cryostat par immunofluorescence s'elevaient d'une moyenne de 2,1 par section de touffe glomerulaire (intervalles zero a 6) chez les rats non injectes, a 2,8 (intervalle zero a 8) 14 jours apres l'injection de ferritine. Cependant, la microscopie en contraste de phase simultanee a revele que seulement une proportion de cellules Ia positives etait associee avec des granules mesangiaux de ferritine et que la majorite de la ferritine etait localisee dans des cellules Ia negatives. Le compte des monocytes infiltrants, reperes par coloration histochimique pour l'esterase non specifique, se sont eleves de 0,1 cellules colorees par section de touffe dans les controles, un maximum de 1,3 (intervalle de zero a 3) au jour 3, a un moment ou la plupart des cellules du mesangium montraient une captation de la ferritine par microscopie electronique. Ces resultats demontrent que le glomerule se debarrasse de la ferritine lors de son entree dans l'espace mesangial, par endocytose par les cellules mesangiales mettant en jeu une degradation de l'apo-ferritine et un stockage des noyaux ferriques dans des phagolysosomes. La grande majorite de ces cellules n'a pas d'antigene Ia. La contribution des monocytes infiltrants a ce processus apparait faible. Bien que la fraction de ferritine enlevee a partir du mesangium par une voie extracellulaire reste indeterminee, il n'y a pas de sortie appreciable de ferritine du glomerule par le pole vasculaire vers l'interstitium ou les tubules adjacents.

Structural and functional study of the rat distal nephron: Effects of potassium adaptation and depletion. To examine the relationship between tubular transport of potassium and cell structure in segments of the superficial distal nephron, we performed potassium transport and quantitative electron microscopic studies in rats after potassium adaptation and potassium depletion. In distal nephrons continuously microperfused in vivo, potassium adaptation stimulated potassium secretion by 200%. Microperfused distal convoluted tubules (earliest portion of accessible distal nephron) did not, however, secrete potassium in potassium-adapted animals, Morphometric analysis of the distal convoluted tubule also revealed no detectable effect of potassium diet on the structure of the distal cell type. In contrast, examination of the connecting tubule and the initial collecting tubule of the distal nephron demonstrated a striking increase in basolateral membrane in potassium-adapted animals. This change was limited to the connecting tubule cell and the principal cell type. No structural change of the intercalated cell type in either segment was associated with altered potassium transport. We conclude that cells of the distal convoluted tubule do not secrete potassium. Functional and morphologic evidence suggests that potassium is secreted by the connecting tubule cell and the principal cell of the connecting tubule and the initial collecting tubule, respectively. Etude structurale et fonctionnelle du nephron distal du rat: Effets de l'adaptation au potassium et de la depletion en potassium. Afin d'etudier la relation entre le transport tubulaire de potassium et la structure cellulaire dans des segments du nephron distal superficiel, le transport de potassium a ete etudie et la microscopie electronique quantitative realisee chez des rats apres adaptation au potassium et depletion en potassium. Dans les nephrons distaux microperfuses en continu in vivo, l'adaptation a potassium stimule la secretion de potassium par un facteur deux. Chez les animaux adaptes a potassium, cependant, les tubes contournes distaux microperfuses ne secretent pas de potassium. L'analyse morphometrique du tube contourne distal ne montre pas d'effet decelable du contenu en potassium de l'alimentation sur la structure des cellules tubulaires distales. Par contre l'etude du tube connecteur et de la partie initiale du tube collecteur montre une augmentation considerable des membranes baso-laterales chez les animaux adaptes a potassium. Cette modification est limitee aux cellules de type connecteur et de type principal. Aucune modification structurale des cellules intercalaires n'a ete observee dans ces deux segments au cours de modifications du transport de potassium. Nous concluons que les cellules du tube contourne distal ne secretent pas de potassium. Il existe des arguments fonctionnels et morphologiques en faveur d'une secretion de potassium par les cellules du tube connecteur et les cellules principales du tube collecteur initial.

Monoclonal antibody to Na,K-ATPase: Immunocytochemical localization along nephron segments. To obtain a highly specific reagent that could be utilized for ultrastructural localization of Na,K-ATPase, monoclonal antibodies were produced using microsomal preparations of outer renal medulla of dog and rat enriched for Na,K-ATPase. The monoclonal antibody (C62.4) raised against dog antigen, immunoprecipitated a 96,000 Dalton protein from membranes labeled either with 35 S methionine or 3 H NAB ouabain. Na,K-ATPase, Na-ATPase, and KpNPPase activity were 25, 60, and 100% maximal after reaction with C62.4. Na,K-ATPase activated with SDS was inhibited, but Na,K-ATPase in tight right-side-out membrane vesicles was not. C62.4 inhibited ouabain binding in the presence of Na,K, and Mg, but did not inhibit ouabain binding in the presence of Mg and Pi. Labeling of broken membranes was readily seen using C62.4 labeled with colloidal gold. Intact right-side-out vesicles showed no evidence of labeling, demonstrating that the antibody is directed to an epitope of the cytoplasmic domain of the enzyme. Differential localization of C62.4 along the nephron was identified. Glomeruli showed no significant antibody binding except by occasional cells in the mesangial regions. Only basal lateral membranes of cells from all tubule segments labeled with C62.4. There was no evidence of specific apical labeling. The thick ascending limb of Henle's loop demonstrated the greatest concentration of antibody binding. In the cortical and outer medullary collecting duct, only principal cells showed abundant antibody binding. Intercalated cells showed no detectable evidence of antibody binding on any surface. These studies demonstrate that Na,K-ATPase is localized exclusively to the basal lateral membrane of renal tubular epithelial cells and varies in density and distribution in different nephron segments.