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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.

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) γ-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 γ-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, β-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 γ-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.

Myosin VI is a reverse-direction molecular motor implicated in membrane transport events. Because myosin VI is most highly expressed in the kidney, we investigated its renal localization by using high-resolution immunocytochemical and biochemical methods. Indirect immunofluorescence microscopy revealed myosin VI at the base of the brush border in proximal tubule cells. Horseradish peroxidase uptake studies, which labeled endosomes, and double staining for clathrin adapter protein-2 showed that myosin VI was closely associated with the intermicrovillar (IMV) coated-pit region of the brush border. Localization of myosin VI to the IMV region was confirmed at the electron microscopic level by colloidal gold labeling of ultrathin cryosections. In addition, antigen retrieval demonstrated a small but significant pool of myosin VI on the microvilli. To confirm the association of myosin VI with the IMV compartment, these membranes were separated from other membrane compartments by using 15–25% OptiPrep density gradients. Immunoblotting of the gradient fractions confirmed that myosin VI was enriched with markers for the IMV microdomain of the brush border, suggesting that myosin VI associates with proteins in this compartment. Finally, we examined the expression of myosin VI during nephron development. We found myosin VI present in a diffuse cytoplasmic pattern at stage II (S-shaped body phase) and that it was only redistributed fully to the brush border in the stage IV nephron. These studies support a model for myosin VI function in the endocytic process of the proximal tubule.

An acidic luminal pH in the epididymis and vas deferens (VD) helps maintain mature sperm in an immotile state during storage. We have previously shown that the majority of proton secretion in the VD is due to the activity of the vacuolar H+-ATPase. Acidification is dependent on luminal sodium in more proximal regions of the epididymis, and we examined the distribution of the Na+/H+exchanger, NHE3, by immunofluorescence and measured Na+/H+exchange (NHE) activity in isolated epididymal tubules. NHE3 was detected in the apical pole of nonciliated cells of the efferent ducts and principal cells (PC) of the epididymis. No staining was seen in the distal cauda epididymidis and the VD. Isolated tubules from the distal initial segment (DIS) and proximal cauda epididymidis were perfused in vitro and loaded with the pH-sensitive dye 2′,7′-bis(carboxyethyl)-5(6′)-carboxyfluorescein. Ethylisopropyl amiloride (EIPA) (50 μM) reduced the initial rate of intracellular pH recovery (dpHi/d t), in response to an acute acid load, by 51% and 45% in the DIS and cauda epididymidis, respectively. In the DIS, removal of luminal sodium reduced dpHi/d t by 52%. HOE694 (50 μM) inhibited all EIPA-sensitive dpHi/d t in the DIS, despite the previously reported absence of NHE2 in this region (Cheng Chew SB, Leung GPH, Leung PY, Tse CM, and Wong PYD, Biol Reprod 62: 755–758, 2000). These data indicate that HOE694- and EIPA-sensitive Na+/H+exchange may participate, together with the H+-ATPase, in luminal acidification in the male excurrent duct.

Functional and immunohistochemical studies were performed to localize and identify Na+/H+exchanger (NHE) isoforms in macula densa cells. By using the isolated perfused thick ascending limb with attached glomerulus preparation dissected from rabbit kidney, intracellular pH (pHi) was measured with fluorescence microscopy by using 2′,7′-bis-(2-carboxyethyl)-5-(and -6) carboxyfluorescein. NHE activity was assayed by measuring the initial rate of Na+-dependent pHirecovery from an acid load imposed by prior lumen and bath Na+removal. Removal of Na+from the bath resulted in a significant, DIDS-insensitive, ethylisopropyl amiloride (EIPA)-inhibitable decrease in pHi. This basolateral transporter showed very low affinity for EIPA and Hoechst 694 (IC50= 9.0 and 247 μM, respectively, consistent with NHE4). The recently reported apical NHE was more sensitive to inhibition by these drugs (IC50= 0.86 and 7.6 μM, respectively, consistent with NHE2). Increasing osmolality, a known activator of NHE4, greatly stimulated basolateral NHE. Immunohistochemical studies using antibodies against NHE1–4 peptides demonstrated expression of NHE2 along the apical and NHE4 along the basolateral, membrane, whereas NHE1 and NHE3 were not detected. These results suggest that macula densa cells functionally and immunologically express NHE2 at the apical membrane and NHE4 at the basolateral membrane. These two isoforms likely participate in Na+transport, pHi, and cell volume regulation and may be involved in tubuloglomerular feedback signaling by these cells.

Electrogenic cotransport of Na+and[Formula: see text] is a crucial element of[Formula: see text] reabsorption in the renal proximal tubule (PT). An electrogenic Na+-[Formula: see text]cotransporter (NBC) has recently been cloned from salamander and rat kidney. In the present study, we generated polyclonal antibodies (pAbs) to NBC and used them to characterize NBC on the protein level by immunochemical methods. We generated pAbs in guinea pigs and rabbits by immunizing with a fusion protein containing the carboxy-terminal 108 amino acids (amino acids 928–1035) of rat kidney NBC (rkNBC). By indirect immunofluorescence microscopy, the pAbs strongly labeled HEK-293 cells transiently expressing NBC, but not in untransfected cells. By immunoblotting, the pAbs recognized a ∼130-kDa band in Xenopus laevis oocytes expressing rkNBC, but not in control oocytes injected with water or cRNA for the Cl−/[Formula: see text]exchanger AE2. In immunoblotting experiments on renal microsomes, the pAbs specifically labeled a major band at ∼130 kDa in both rat and rabbit, as well as a single ∼160-kDa band in salamander kidney. By indirect immunofluorescence microscopy on 0.5-μm cryosections of rat and rabbit kidneys fixed in paraformaldehyde-lysine-periodate (PLP), the pAbs produced a strong and exclusively basolateral staining of the PT. In the salamander kidney, the pAbs labeled only weakly the basolateral membrane of the PT. In contrast, we observed strong basolateral labeling in the late distal tubule, but not in the early distal tubule. The specificity of the pAbs for both immunoblotting and immunohistochemistry was confirmed in antibody preabsorption experiments using either the fusion protein used for immunization or similarly prepared control fusion proteins. In summary, we have developed antibodies specific for NBC, determined the apparent molecular weights of rat, rabbit, and salamander kidney NBC proteins, and described the localization of NBC within the kidney of these mammalian and amphibian species.

The cellular and subcellular localizations of the AE2 anion exchanger in rat kidney have remained elusive despite detection of moderately abundant AE2 mRNA and AE2 polypeptide in all kidney regions. In this report a simple epitope unmasking technique has allowed the immunolocalization of AE2 antigenic sites in basolateral membranes of several rat kidney tubular epithelial cells. AE2 immunostaining was faint or absent in the glomerulus and proximal tubule, present in descending and ascending thin limbs, and stronger in the medullary thick ascending limb (MTAL). A lower staining intensity was found in cortical thick ascending limbs and even less in the distal convoluted tubule. In contrast, there was an enhanced staining in the macula densa. In principal cells (PC) of the connecting segment, AE2 was undetectable but gradually increased in intensity along the collecting duct, with strongest staining in inner medullary collecting duct (IMCD) PC. A sodium dodecyl sulfate-sensitive AE2-related Golgi epitope was also detected in some interstitial and endothelial cells of the inner medulla and in epithelial cells of IMCD and MTAL. Colchicine treatment of the intact animal altered the distribution of this Golgi-associated epitope but left plasmalemmal AE2 undisturbed. Reverse transcription-polymerase chain reaction detected AE2a, AE2b, and AE2c2 but not AE2c1 transcripts in rat kidney mRNA. The results suggest a widespread occurrence of the AE2 protein in several renal epithelial cell types.

Previous immunochemical studies have shown that NHE3 is an apical Na+/H+ exchanger in some renal epithelia. The purpose of the present study was to develop high-affinity, isoform-specific monoclonal antibodies (MAbs) that would be useful for carrying out high-resolution immunocytochemical studies of NHE3 in the adult and neonatal mammalian kidney. Three MAbs were developed to a fusion protein containing amino acids 702-832 of rabbit NHE3. Specificity was established by immunoblotting membranes from NHE-deficient LAP cells that had been transfected with either NHE1,-2, -3, or -4. With the use of high-resolution immunocytochemical techniques, NHE3 was found in vesicles in the apical cytoplasm of proximal tubule cells, as well as in the apical plasma membrane of the proximal tubule, and in both the thin and thick limbs of the loop of Henle. When localized in the 1-day-old rat kidney, NHE3 was first detected in the late stages of the S-shaped body. In later stages of nephron development, the pattern of NHE3 staining was similar to that seen in the adult. This study demonstrates 1) the specificity of three MAbs for Na+/H+ exchanger isoform NHE3; 2) NHE3 is present in an intracellular vesicular compartment in cells of the proximal tubule, consistent with possible regulation by membrane recycling; and 3) NHE3 is expressed on the apical membrane in early stages of the developing nephron.

MinK-related peptides (MiRPs) are single-span membrane proteins that assemble with specific voltage-gated K+(Kv) channel α-subunits to establish gating kinetics, unitary conductance, expression level, and pharmacology of the mixed complex. MiRP3 (encoded by the KCNE4 gene) has been shown to alter the behavior of some Kv α-subunits in vitro but its natural partners and physiologic functions are unknown. Seeking in vivo partners for MiRP3, immunohistochemistry was used to localize its expression to a unique subcellular site, the apical membrane of renal intercalated cells, where one potassium channel type has been recorded, the calcium- and voltage-gated channel BK. Overlapping staining of these two proteins was found in rabbit intercalated cells, and MiRP3 and BK subunits expressed in tissue culture cells were found to form detergent-stable complexes. Electrophysiologic and biochemical evaluation showed MiRP3 to act on BK to reduce current density in two fashions: shifting the current-voltage relationship to more depolarized voltages in a calcium-dependent fashion (∼10 mV at normal intracellular calcium levels) and accelerating degradation of MiRP3-BK complexes. The findings suggest a role for MiRP3 modulation of BK-dependent urinary potassium excretion.

The cellular ultrastructure of the renal distal nephron of the salamander, Amphiuma means, was examined by electron-microscopic and stereological techniques before and after exposure to potassium in the ambient environment. The distal nephron of Amphiuma is composed of three ultrastructurally distinct segments: early distal (or diluting segment), late distal, and collecting tubule. The early distal tubule structurally resembles the mammalian thick ascending limb of Henle's loop. Large renin-like granules are present in the smooth muscle cells of the afferent arteriole in the vicinity of the early distal tubule, suggesting the presence of a rudimentary juxtaglomerular apparatus. Late distal tubules are composed of one large cell type characterized by extensive basal membrane invaginations, often extending to the luminal membrane. Collecting tubules contain principal and intercalated cells that are ultrastructurally similar to cells of the mammalian cortical collecting tubule. Exposure to potassium had no effect on the ultrastructure of early distal cells but led to a sharp increase in the basolateral membrane surface density of principal cells in the collecting tubule (1.17 +/- 0.08-1.63 +/- 0.13 micron2/micron3). Potassium adaptation leads to a similar structural response in the mammalian collecting tubule. Since Amphiuma collecting tubules can be isolated and perfused in vitro and impaled with ion- and voltage-sensitive microelectrodes, the observed structural adaptation suggests that the collecting tubule may be a useful preparation to study the cellular mechanisms of potassium adaptation.