Daniel Biemesderfer

Role of NHE3 in Mediating Renal Brush Border Na+-H+ Exchange ADAPTATION TO METABOLIC ACIDOSIS

The aims of the present study were to estimate the fraction of renal brush border membrane Na+-H+ exchange activity mediated by the isoform NHE3 and to evaluate whether the increased brush border Na+-H+ exchange observed in metabolic acidosis is due to increased expression of NHE3 protein. Compared with other isoforms, NHE3 is known to have a unique profile of sensitivity to pharmacologic inhibitors, including relative resistance to amiloride analogs and HOE694. We therefore assessed the inhibitor sensitivity of pH gradient-stimulated 22Na uptake in renal brush border vesicles isolated from normal rats. The I50 values for amiloride (30 μM), dimethylamiloride (10 μM), ethylisopropylamiloride (6 μM), and HOE694 (>100 μM) were markedly dissimilar from those reported for NHE1 and NHE2 but were nearly identical to reported values for NHE3. Na+-H+ exchange activity in renal brush border vesicles isolated from rats with 5 days of NH4Cl-induced metabolic acidosis was increased 1.5-fold compared with control rats, with no change in inhibitor sensitivity. Western blot analysis indicated that NHE3 protein expression was greater in brush border membranes from acidotic compared with control rats. We conclude that virtually all measured Na+-H+ exchange activity in brush border membranes from control and acidotic rats is mediated by NHE3 and that metabolic acidosis causes increased expression of renal brush border NHE3 protein.

Immunolocalization of the electrogenic Na+-HCO3/- cotransporter in mammalian and amphibian kidney

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.

Specific Association of Megalin and the Na+/H+ Exchanger Isoform NHE3 in the Proximal Tubule

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.

Immunolocalization of AE2 anion exchanger in rat kidney

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 AE2cl transcripts in rat kidney mRNA. The results suggest a widespread occurrence of the AE2 protein in several renal epithelial cell types.

Immunochemical characterization of Na+/H+exchanger isoform NHE4

Mammalian Na+/H+exchangers (NHEs) are a family of transport proteins (NHE1-NHE5). To date, the cellular and subcellular localization of NHE4 has not been characterized using immunochemical techniques. We purified a fusion protein containing a portion of rat NHE4 (amino acids 565-675) to use as immunogen. A monoclonal antibody (11H11) was selected by ELISA. It reacted specifically with both the fusion protein and to a 60- to 65-kDa polypeptide expressed in NHE4-transfected LAP1 cells. By Western blot analysis, NHE4 was identified as a 65- to 70-kDa protein that was expressed most abundantly in stomach and in multiple additional epithelial and nonepithelial rat tissues including skeletal muscle, heart, kidney, uterus, and liver. Subcellular localization of NHE4 in the kidney was evaluated by Western blot analysis of membrane fractions isolated by Percoll gradient centrifugation. NHE4 was found to cofractionate with the basolateral markers NHE1 and Na+-K+-ATPase rather than the luminal marker γ-glutamyl transferase. In stomach, NHE4 was detected by immunoperoxidase labeling on the basolateral membrane of cells at the base of the gastric gland. We conclude that NHE4 is a 65- to 70-kDa protein with a broad tissue distribution. In two types of epithelial cells, kidney and stomach, NHE4 is localized to the basolateral membrane.

Sodium-hydrogen exchangers and sodium-bicarbonate co-transporters: Ontogeny of protein expression in the rat brain

We used western blotting to examine the developmental profiles (at embryonic day 16 and postnatal days 1, 13, 23, 33 and 105) of protein expression for three sodium-hydrogen exchanger isoforms (1, 2 and 4) and for a sodium-bicarbonate co-transporter in three CNS regions (cortex, cerebellum and brainstem-diencephalon). In microsomal preparations, sodium-hydrogen exchanger isoform 1 and sodium-bicarbonate co-transporter protein expression in the CNS increases gradually from embryonic day 16 (25-40% of the adult level) to postnatal day 105. In contrast, sodium-hydrogen exchanger isoform 2 and 4 expression reaches a maximum (three to 20 times the adult level) at around three to four weeks of age. There is significant regional heterogeneity in the expression of sodium-hydrogen exchanger and sodium-bicarbonate co-transporter proteins in the rat CNS. Sodium-hydrogen exchanger isoform 1 was highly expressed in the brainstem-diencephalon, whereas the sodium-bicarbonate co-transporter was robustly expressed in the cerebellum and brainstem-diencephalon. These data indicate that the expression of sodium-hydrogen exchanger and sodium-bicarbonate co-transporter proteins varies as a function of both development and specific brain region.

Membrane Topology of NHE3 EPITOPES WITHIN THE CARBOXYL-TERMINAL HYDROPHILIC DOMAIN ARE EXOPLASMIC

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.

Increased Tubular Proliferation as an Adaptive Response to Glomerular Albuminuria

Renal tubular atrophy accompanies many proteinuric renal diseases, suggesting that glomerular proteinuria injures the tubules. However, local or systemic inflammation and filtration of abnormal proteins known to directly injure tubules are also present in many of these diseases and animal models; therefore, whether glomerular proteinuria directly causes tubular injury is unknown. Here, we examined the renal response to proteinuria induced by selective podocyte loss. We generated mice that express the diphtheria toxin receptor exclusively in podocytes, allowing reproducible dose-dependent, specific ablation of podocytes by administering diphtheria toxin. Ablation of <20% of podocytes resulted in profound albuminuria that resolved over 1-2 weeks after the re-establishment of normal podocyte morphology. Immediately after the onset of albuminuria, proximal tubule cells underwent a transient burst of proliferation without evidence of tubular damage or increased apoptosis, resulting in an increase in total tubular cell numbers. The proliferative response coincided with detection of the growth factor Gas6 in the urine and phosphorylation of the Gas6 receptor Axl in the apical membrane of renal tubular cells. In contrast, ablation of >40% of podocytes led to progressive glomerulosclerosis, profound tubular injury, and renal failure. These data suggest that glomerular proteinuria in the absence of severe structural glomerular injury activates tubular proliferation, potentially as an adaptive response to minimize the loss of filtered proteins.

Altered renal proximal tubular endocytosis and histology in mice lacking myosin-VI†

Myosin VI (Myo6) is an actin‐based molecular motor involved in clathrin‐mediated endocytosis that is highly expressed in the renal proximal tubule brush border. We investigated the renal physiological consequences of loss of Myo6 function by performing renal clearance and physiological measurements on Myo6 functional null Snells waltzer (sv/sv) and control heterozygous (+/sv) mice. Sv/sv mice showed reduced body weight and elevated blood pressure compared with controls; no differences were observed for glomerular flow rate, urine volume, blood acid‐base parameters, and plasma concentrations and urinary excretions of Na+ and K+. To assess the integrity of endocytosis‐mediated protein absorption by the kidney, urinary albumin excretion was measured, and the proximal tubular uptake of intravenously injected endocytic marker horseradish peroxidase (HRP) was examined. Albumin excretion was increased nearly 4‐fold in sv/sv mice relative to controls. Conversely, HRP uptake was reduced and delayed in proximal tubule cells of the sv/sv kidney observed by electron microscopy at 5 and 30 min after injection. Consistent with impaired endocytosis, we also observed defects indicating alterations along the endocytic pathway in sv/sv proximal tubule cells: (1) decreased membrane association of the clathrin adaptor subunit, adaptin beta, and Disabled‐2 (Dab2) after sedimentation of renal homogenates and (2) reduced apical vacuole number. In addition, proximal tubular dilation and fibrosis, likely secondary effects of the loss of Myo6, were observed in sv/sv kidneys. These results indicate that Myo6 plays a key role in endocytosis‐mediated protein absorption in the mouse kidney proximal tubule.

MiRP3 acts as an accessory subunit with the BK potassium channel

MinK-related peptides (MiRPs) are single-span membrane proteins that assemble with specific voltage-gated K+ (Kv) channel alpha-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 alpha-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 ( approximately 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.