Neuronally differentiated macula densa cells regulate tissue remodeling and regeneration in the kidney.

Tissue regeneration is limited in several organs, including the kidney, contributing to the high prevalence of kidney disease globally. However, evolutionary and physiological adaptive responses and the presence of renal progenitor cells suggest an existing remodeling capacity. This study uncovered endogenous tissue remodeling mechanisms in the kidney that were activated by the loss of body fluid and salt and regulated by a unique niche of a minority renal cell type called the macula densa (MD).

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.

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.

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 Snell's 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(+).

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.

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.

A role of the Lowe syndrome protein OCRL in early steps of the endocytic pathway.

Mutations in the inositol 5-phosphatase OCRL are responsible for Lowe syndrome, whose manifestations include mental retardation and renal Fanconi syndrome. OCRL has been implicated in membrane trafficking, but disease mechanisms remain unclear. We show that OCRL visits late-stage, endocytic clathrin-coated pits and binds the Rab5 effector APPL1 on peripheral early endosomes.

Regulated intramembrane proteolysis of megalin: linking urinary protein and gene regulation in proximal tubule?

Regulated intramembrane proteolysis (RIP) represents an evolutionarily conserved process linking receptor function with transcriptional regulation. Best characterized by the Notch signaling pathway, RIP involves regulated ectodomain shedding followed by gamma-secretase-mediated release of the C-terminal, cytosolic domain. The C-terminus in turn translocates to the nucleus where it interacts with other proteins to regulate expression of specific genes.

Immunolocalization of anion transporter Slc26a7 in mouse kidney.

Previous studies have indicated that a major fraction of the filtered Cl(-) is reabsorbed via apical membrane Cl(-)/base exchange in the proximal tubule. Recent studies in Slc26a6 null mice have suggested that this transporter mediates only a portion of proximal tubule Cl(-)/base exchange, raising the possibility that one or more unidentified apical membrane transporters may additionally contribute. Recent studies have identified Slc26a7 as another Cl(-)/base exchanger expressed in the kidney.

Use of phospho-specific antibodies to determine the phosphorylation of endogenous Na+/H+ exchanger NHE3 at PKA consensus sites.

Transfection studies using mutant constructs have implicated one or both protein kinase A (PKA) consensus phosphorylation sites [serines 552 and 605 in rat Na(+)/H(+) exchanger type 3 (NHE3)] as critical for mediating inhibition of NHE3 in response to several stimuli including dopamine. However, whether one or both of these sites is actually phosphorylated in endogenous NHE3 in proximal tubule cells is unknown. The purpose of this study was to generate phosphospecific antibodies so that the state of phosphorylation of these serine residues in endogenous NHE3 could be assessed in vitro and in vivo.

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.

Does membrane trafficking play a role in regulating the sodium/hydrogen exchanger isoform 3 in the proximal tubule?

Purpose Of Review: The proximal tubule sodium/hydrogen exchanger continuously reabsorbs the bulk of the filtered sodium, controlling salt delivery to the distal nephron which is critical for tubuloglomerular feedback autoregulation and for fine control of salt excretion in the distal nephron. This review focuses on recent studies of the mechanisms of regulation of sodium transport in the proximal tubule, and addresses whether results from studies in proximal tubule cell lines are applicable to the proximal tubule in situ.

Recent Findings: Recent in-vivo studies provided evidence that sodium/hydrogen exchanger isoform 3 can move into and out of the apical microvilli accompanied by parallel changes in renal sodium transport: the exchanger is retracted from the microvilli in response to hypertension, parathyroid hormone or dopamine treatment and moved into the microvilli in response to sympathetic nervous system stimulation, puromycin aminonucleoside induced nephritic syndrome, and insulin treatment.

Expression of KCNA10, a voltage-gated K channel, in glomerular endothelium and at the apical membrane of the renal proximal tubule.

Potassium (K) channels regulate cell membrane potential and modulate a number of important cellular functions. KCNA10 is a cyclic nucleotide-gated, voltage-activated K channel that is detected in kidney, heart, and aorta by Northern blot and postulated to participate in renal K metabolism and to regulate vascular tone. The aim of this study was to establish the cellular and subcellular localization of KCNA10 in kidney and vascular tissues.

Expression of myosin VI within the early endocytic pathway in adult and developing proximal tubules.

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.

NHE3 and NHERF are targeted to the basolateral membrane in proximal tubules of colchicine-treated rats.

Background: Depolymerization 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.

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.

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.

Role of sodium/hydrogen exchanger isoform NHE3 in fluid secretion and absorption in mouse and rat cholangiocytes.

Na+/H+ exchanger (NHE) isoforms play important roles in intracellular pH regulation and in fluid absorption. The isoform NHE3 has been localized to apical surfaces of epithelia and in some tissues may facilitate the absorption of NaCl. To determine whether the apical isoform NHE3 is present in cholangiocytes and to examine whether it has a functional role in cholangiocyte fluid secretion and absorption, immunocytochemical studies were performed in rat liver with NHE3 antibodies and functional studies were obtained in isolated bile duct units from wild-type and NHE3-/- mice after stimulation with forskolin, using videomicroscopic techniques.

Na+/H+-exchange activity and immunolocalization of NHE3 in rat epididymis.

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.

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.

Macula densa Na(+)/H(+) exchange activities mediated by apical NHE2 and basolateral NHE4 isoforms.

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 (pH(i)) 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 pH(i) recovery from an acid load imposed by prior lumen and bath Na(+) removal.

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.

Immunolocalization of the electrogenic Na+-HCO-3 cotransporter in mammalian and amphibian kidney.

Electrogenic cotransport of Na+ and HCO-3 is a crucial element of HCO-3 reabsorption in the renal proximal tubule (PT). An electrogenic Na+-HCO-3 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.

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.

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.