Membrane Of Renal Cells Research Articles

Shank2 redistributes with NaPilla during regulated endocytosis.

Serum phosphate levels are acutely impacted by the abundance of sodium-phosphate cotransporter IIa (NaPiIIa) in the apical membrane of renal proximal tubule cells. PSD-95/Disks Large/Zonula Occludens (PDZ) domain-containing proteins bind NaPiIIa and likely contribute to the delivery, retention, recovery, and trafficking of NaPiIIa. Shank2 is a distinctive PDZ domain protein that binds NaPiIIa. Its role in regulating NaPiIIa activity, distribution, and abundance is unknown. In the present in vivo study, rats were maintained on a low-phosphate diet, and then plasma phosphate levels were acutely elevated by high-phosphate feeding to induce the recovery, endocytosis, and degradation of NaPiIIa. Western blot analysis of renal cortical tissue from rats given high-phosphate feed showed NaPiIIa and Shank2 underwent degradation. Quantitative immunofluorescence analyses, including microvillar versus intracellular intensity ratios and intensity correlation quotients, showed that Shank2 redistributed with NaPiIIa during the time course of NaPiIIa endocytosis. Furthermore, NaPiIIa and Shank2 trafficked through distinct endosomal compartments (clathrin, early endosomes, lysosomes) with the same temporal pattern. These in vivo findings indicate that Shank2 is positioned to coordinate the regulated endocytic retrieval and downregulation of NaPiIIa in rat renal proximal tubule cells.

Intrarenal suppression of angiotensin II type 1 receptor binding molecule in angiotensin II-infused mice

ATRAP [ANG II type 1 receptor (AT1R)-associated protein] is a molecule which directly interacts with AT1R and inhibits AT1R signaling. The aim of this study was to examine the effects of continuous ANG II infusion on the intrarenal expression and distribution of ATRAP and to determine the role of AT1R signaling in mediating these effects. C57BL/6 male mice were subjected to vehicle or ANG II infusions at doses of 200, 1,000, or 2,500 ng·kg(-1)·min(-1) for 14 days. ANG II infusion caused significant suppression of ATRAP expression in the kidney but did not affect ATRAP expression in the testis or liver. Although only the highest ANG II dose (2,500 ng·kg(-1)·min(-1)) provoked renal pathological responses, such as an increase in the mRNA expression of angiotensinogen and the α-subunit of the epithelial sodium channel, ANG II-induced decreases in ATRAP were observed even at the lowest dose (200 ng·kg(-1)·min(-1)), particularly in the outer medulla of the kidney, based on immunohistochemical staining and Western blot analysis. The decrease in renal ATRAP expression by ANG II infusion was prevented by treatment with the AT1R-specific blocker olmesartan. In addition, the ANG II-mediated decrease in renal ATRAP expression through AT1R signaling occurred without an ANG II-induced decrease in plasma membrane AT1R expression in the kidney. On the other hand, a transgenic model increase in renal ATRAP expression beyond baseline was accompanied by a constitutive reduction of renal plasma membrane AT1R expression and by the promotion of renal AT1R internalization as well as the decreased induction of angiotensinogen gene expression in response to ANG II. These results suggest that the plasma membrane AT1R level in the kidney is modulated by intrarenal ATRAP expression under physiological and pathophysiological conditions in vivo.

Renal tubular cell membranes inhibit growth but promote aggregation of calcium oxalate monohydrate crystals

Cell membranes have been proposed to serve as promoters for calcium oxalate monohydrate (COM) kidney stone formation. However, direct evidence to demonstrate the modulatory effects of renal tubular cell membranes on COM crystals does not currently exist. We thus examined the effects of intact MDCK cells and their fragmented membranes on COM crystal growth, aggregation and transformation. COM crystals were generated in the absence (control) or presence of intact MDCK cells or their membrane fragments. Intact MDCK cells and their membrane fragments significantly inhibited COM crystal growth (22.6% and 25.2% decreases in size, respectively) and significantly reduced COM total crystal mass (23.1% and 25.6% decreases, respectively). In contrast, both of them markedly promoted crystal aggregation (1.9-fold and 3.2-fold increases, respectively). Moreover, both intact cells and membrane fragments could transform COM to calcium oxalate dihydrate (COD) crystals. Finally, COM crystal growth inhibitory activities of both membrane forms were successfully confirmed by a spectrophotometric oxalate-depletion assay. Our data provide the first direct evidence to demonstrate the dual modulatory effects of MDCK membranes on COM crystals. Although growth of individual COM crystals was inhibited, their aggregation was promoted. These findings provide additional insights into the mechanisms of COM kidney stone formation.

Posttranslational mechanisms associated with reduced NHE3 activity in adult vs. young prehypertensive SHR

Abnormalities in renal proximal tubular (PT) sodium transport play an important role in the pathophysiology of essential hypertension. The Na(+)/H(+) exchanger isoform 3 (NHE3) represents the major route for sodium entry across the apical membrane of renal PT cells. We therefore aimed to assess in vivo NHE3 transport activity and to define the molecular mechanisms underlying NHE3 regulation before and after development of hypertension in the spontaneously hypertensive rat (SHR). NHE3 function was measured as the rate of bicarbonate reabsorption by means of in vivo stationary microperfusion in PT from young prehypertensive SHR (Y-SHR; 5-wk-old), adult SHR (A-SHR; 14-wk-old), and age-matched Wistar Kyoto (WKY) rats. We found that NHE3-mediated PT bicarbonate reabsorption was reduced with age in the SHR (1.08 ± 0.10 vs. 0.41 ± 0.04 nmol/cm(2)×s), while it was increased in the transition from youth to adulthood in the WKY rat (0.59 ± 0.05 vs. 1.26 ± 0.11 nmol/cm(2)×s). Higher NHE3 activity in the Y-SHR compared with A-SHR was associated with a predominant microvilli confinement and a lower ratio of phosphorylated NHE3 at serine-552 to total NHE3 (P-NHE3/total). After development of hypertension, P-NHE3/total increased and NHE3 was retracted out of the microvillar microdomain along with the regulator dipeptidyl peptidase IV (DPPIV). Collectively, our data suggest that the PT is playing a role in adapting to the hypertension in the SHR. The molecular mechanisms of this adaptation possibly include an increase of P-NHE3/total and a redistribution of the NHE3-DPPIV complex from the body to the base of the PT microvilli, both predicted to decrease sodium reabsorption.

Epithelial Na+ Channel (ENaC), Hormones, and Hypertension

This minireview examines both the basic science and clinical observations over the past 20 years to show how and why overstimulation of the amiloride-sensitive epithelial Na(+) channel (ENaC) expressed by epithelial principal cells of the renal collecting duct may be responsible for a large portion of hypertension in modern society. This idea is based on the finding that, in Liddle syndrome, a mutation of the beta- and/or gamma-subunits of ENaC produces an activated ion channel, in turn resulting in severe hypertension that is resistant to most forms of conventional antihypertensive therapy. ENaC can also be stimulated to conduct sodium by two hormones: aldosterone and insulin. These hormones are both often elevated in obese individuals with therapy-resistant hypertension. Thus, overstimulation of ENaC by metabolic abnormalities in obese individuals may be a likely cause of the hypertension that accompanies obesity. The molecular mechanisms underlying both Liddle syndrome and obesity-related hypertension are different (i.e. genetic and hormonal, respectively), but both have the same end result, namely increased ENaC activity.

An apical expression signal of the renal type IIc Na+-dependent phosphate cotransporter in renal epithelial cells

The type IIc Na(+)-dependent phosphate cotransporter (NaPi-IIc) is specifically targeted to, and expressed on, the apical membrane of renal proximal tubular cells and mediates phosphate transport. In the present study, we investigated the signals that determine apical expression of NaPi-IIc with a focus on the role of the N- and the C-terminal tails of mouse NaPi-IIc in renal epithelial cells [opossum kidney (OK) and Madin-Darby canine kidney cells]. Wild-type NaPi-IIc, the cotransporter NaPi-IIa, as well as several IIa-IIc chimeras and deletion mutants, were fused to enhanced green fluorescent protein (EGFP), and their cellular localization was analyzed in polarized renal epithelial cells by confocal microscopy and by cell-surface biotinylation. Fluorescent EGFP-fused NaPi-IIc transporter proteins are correctly expressed in the apical membrane of OK cells. The apical expression of N-terminal deletion mutants (deletion of N-terminal 25, 50, or 69 amino acids) was not affected by truncation. In contrast, C-terminal deletion mutants (deletion of C-terminal 45, 50, or 62 amino acids) did not have correct apical expression. A more detailed mutational analysis indicated that a domain (amino acids WLHSL) in the cytoplasmic C terminus is required for apical expression of NaPi-IIc in renal epithelial cells. We conclude that targeting of NaPi-IIc to the apical cell surface is regulated by a unique amino acid motif in the cytoplasmic C-terminal domain.

Abstract 1522: Identification of pifithrin-α as a potent inhibitor of OCT2-mediated transport of cisplatin

Abstract Members of the organic cation transporter (OCT) solute carrier family mediate the cellular uptake of a large number of structurally diverse molecules, including a variety of endogenous compounds and xenobiotics such as metformin, cimetidine, and cisplatin. OCT2 is highly expressed on the basolateral membrane of renal tubular cells, where it facilitates the renal secretion of substrate drugs. We previously found that the urinary excretion of cisplatin was drastically reduced in mice lacking the ortholog transporters Oct1 and Oct2 [Oct1/2(-/-) mice], and that, compared to wildtype mice, these animals were resistant to severe (grade 4) cisplatin-induced renal tubular necrosis. Since kidney damage was not completely abolished in Oct1/2(-/-) mice, we performed microarray analyses (Affymetrix Mouse Genome 430 v2.0) on kidney biopsies following cisplatin (10 mg/kg, i.p.) administration in order to further understand the mechanism of cisplatin-induced nephrotoxicity in these mice. Using a false-discovery rate of 5% and an average fold change of ≥2.0, we identified complex gene expression changes and a drug-response signature comprising 1063 up-regulated and 1072 down-regulated genes in wildtype mice that was strikingly different quantitatively in Oct1/2(-/-) mice. Next, we performed a KEGG pathway analysis to identify processes that are specifically altered in response to cisplatin. Ten out of 193 analyzed pathways showed significant (P<0.001) alteration in wildtype mice and these changes were largely absent in Oct1/2(-/-) mice. The most significantly altered category involved genes associated with the p53 signaling network, both in wildtype mice (P=2.40×10−11) and Oct1/2(-/-) mice (P=1.92×10−8). Several well-characterized transcriptional p53 target genes such as Cdnk1a (p21), Mdm2, Bbc3 (PUMA), and Rmr2 showed strong induction in kidneys of treated wildtype mice, while their alteration was weaker or not detected in treated Oct1/2(-/-) mice. There were no mouse genotype-dependent differences in gene expression at baseline (P>0.13). During the course of our screening work on identifying inhibitors of OCT2-mediated transport of cisplatin, we observed that pifithrin-α, an investigational inhibitor of p53-dependent transcriptional activation and apoptosis, is a highly potent non-competitive inhibitor of OCT2-mediated transport of tetraethylammonium, a prototypical OCT2 substrate, with a Ki of 3.0 μM. Furthermore, pifithrin-α completely blocked OCT2-mediated transport of cisplatin in vitro even at a substrate-to-inhibitor concentration ratio of 5:1. Collectively, this study suggests that (i) the ability to mount an effective p53 response in response to cisplatin is differentially influencing treatment sensitivity in wildtype mice and Oct1/2(-/-) mice, and (ii) pifithrin-α should be explored as a unique dual OCT2/p53-inhibitor for preventing cisplatin nephrotoxicity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1522.

Synergistic Interaction between Ciliary Genes Reflects the Importance of Mutational Load in Ciliopathies

The formation of renal cysts occurs in roughly 10 to 15% of the population,1 and although simple cysts are largely asymptomatic, the formation of multiple cysts can be extremely detrimental. The two major multicystic syndromes, autosomal recessive polycystic kidney disease (PKD) and autosomal dominant PKD, are primarily linked to disruptions in the pkhd1 and pkd1 / pkd2 genes, respectively2; however, several additional syndromes are also characterized by kidney cyst development, including nephronophthisis (NPHP), Joubert syndrome, Bardet-Biedl syndrome, Orofaciodigital syndrome 1, and Meckel syndrome (MKS). During the past decade, there has been a great upsurgence in identifying genes affected in these syndromes. One of the most striking discoveries is that protein products of these genes localize to the cilium and/or the associated basal body and affect ciliogenesis and cilia morphology or function when mutated,3 collectively identifying these syndromes as ciliopathies. Why do kidney cysts form? An increasing body of evidence indicates that mutations affecting ciliogenesis or cilia function can give rise to cysts. Primary cilia extend from the apical membrane of renal epithelial cells into the lumen, where they act as mechanosensors. One model suggests that fluid flow within the kidney bends the cilia and triggers intracellular calcium signaling, which, in turn, regulates epithelial tubule morphology.2 Polycystin 1 and polycystin 2, the protein products of the pkd1 and pkd2 genes, respectively, localize to renal cilia4 and may act as the mediators of mechanosensation in these structures.5 Mutations in pkd1 or pkd2 do not result in morphologic cilia defects but rather affect calcium influx, which leads to cyst formation. In addition, mutations that result in aberrant cilia …

Sodium-Hydrogen Exchanger Regulatory Factor 1 (NHERF-1) Transduces Signals That Mediate Dopamine Inhibition of Sodium-Phosphate Co-transport in Mouse Kidney

Dopamine inhibited phosphate transport in isolated renal brush border membrane vesicles and in cultured renal proximal tubule cells from wild-type but not from NHERF-1 null mice. Co-immunoprecipitation experiments established that NHERF-1 associated with D1-like receptors. In wild-type mice, dopamine stimulated cAMP accumulation and protein kinase C (PKC) activity in renal proximal tubule cells, an effect that was abolished by SCH-23390, a D1-like receptor antagonist. In NHERF-1 null kidney tissue; however, dopamine failed to stimulate either cAMP accumulation or PKC activity. Infection of proximal tubule cells from NHERF-1 null mice with adenovirus-green fluorescent protein-NHERF-1 restored the ability of dopamine to stimulate cAMP and PKC. Finally, in (32)P-labeled wild-type proximal tubule cells and in opossum kidney cells, dopamine increased NHERF-1 phosphorylation at serine 77 of the PDZ I domain of NHERF-1, a site previously shown to attenuate binding of cellular targets including the Npt2a (sodium-dependent phosphate transporter 2a). Together, these studies establish that NHERF-1 plays a key role in dopamine signaling and is also a downstream target of D1-like receptors in the mouse kidney. These studies suggest a novel role for the PDZ adapter protein NHERF-1 in coordinating dopamine signals that inhibit renal phosphate transport.

Topological Location and Structural Importance of the NBCe1-A Residues Mutated in Proximal Renal Tubular Acidosis

NBCe1-A electrogenically cotransports Na(+) and HCO(3)(-) across the basolateral membrane of renal proximal tubule cells. Eight missense mutations and 3 nonsense mutations in NBCe1-A cause severe proximal renal tubular acidosis (pRTA). In this study, the topologic properties and structural importance of the 8 endogenous residues mutated in pRTA and the in situ topology of NBCe1-A were examined by the substituted cysteine accessibility method. Of the 55 analyzed individually introduced cysteines, 8 were labeled with both membrane permeant (biotin maleimide (BM)) and impermeant (2-((5(6)-tetramethylrhodamine)carboxylamino)ethyl methanethiosulfonate (MTS-TAMRA)) sulfhydryl reagents, 4 with only BM, and 3 with only MTS-TAMRA. The location of the labeled and unlabeled introduced cysteines clearly indicates that the transmembrane region of NBCe1-A contains 14 transmembrane segments (TMs). In this in situ based NBCe1-A topology, residues mutated in pRTA (pRTA residues) are assigned as: Ser(427), TM1; Thr(485) and Gly(486), TM3; Arg(510) and Leu(522), TM4; Ala(799), TM10; and Arg(881), TM12. Substitution of pRTA residues with cysteines impaired the membrane trafficking of R510C and R881C, the remaining membrane-processed constructs had various impaired transport function. Surprisingly, none of the membrane-processed constructs was accessible to labeling with BM and MTS-TAMRA, nor were they functionally sensitive to the inhibition by (2-aminoethyl)methanethiosulfonate. Functional analysis of Thr(485) with different amino acid substitutions indicated it resides in a unique region important for NBCe1-A function. Our findings demonstrate that the pRTA residues in NBCe1-A are buried in the protein complex/lipid bilayer where they perform important structural roles.

High‐Throughput Yeast Screen to Identify Vacuolar H+‐ATPase subunit a specific inhibitors

Vacuolar H+‐ATPases (V‐ATPases) are multi‐subunit proteins critical for acidification of intracellular compartments. V‐ATPases are important for biological processes such as bone resorption and disease related processes including metastasis. V‐ATPase localization is determined by the subunit a isoform. There are four a isoforms in humans, a1‐a4. V‐ATPases at the plasma membrane of osteoclasts have a3 and those at the plasma membrane of certain renal cells have a4. Genetic defects in a3 and a4 lead to osteopetrosis and renal tubular acidosis, respectively. We would like to exploit the specificity of a isoform function to develop isoform specific inhibitors that can be used as therapeutics for diseases. A high‐throughput screen will be developed in which each of the human a isoforms will be expressed in a yeast strain lacking the endogenous a subunit genes. These yeast strains will be grown in the presence of compounds from small molecule libraries. Isoform‐specific inhibitors will be identified by their ability to selectively block growth at neutral pH in yeast strains expressing the appropriate a isoforms. Thus far our experiments indicate that expression of the human a subunit alone does not complement the loss of the yeast a subunits. Thus, to develop the inhibitor screen we must determine the minimal number of mammalian V0 subunits necessary to have a functional V‐ATPase containing the human a subunit.

Polycystin-dependent fluid flow sensing targets histone deacetylase 5 to prevent the development of renal cysts

Polycystin 1 and polycystin 2 are large transmembrane proteins, which, when mutated, cause autosomal dominant polycystic kidney disease (ADPKD), a highly prevalent human genetic disease. The polycystins are thought to form a receptor-calcium channel complex in the plasma membrane of renal epithelial cells and elicit a calcium influx in response to mechanical stimulation, such as fluid flow across the apical surface of renal epithelial cells. The functional role of the polycystins in mechanosensation remains largely unknown. Here, we found that myocyte enhancer factor 2C (MEF2C) and histone deacetylase 5 (HDAC5), two key regulators of cardiac hypertrophy, are targets of polycystin-dependent fluid stress sensing in renal epithelial cells in mice. We show that fluid flow stimulation of polarized epithelial monolayers induced phosphorylation and nuclear export of HDAC5, which are crucial events in the activation of MEF2C-based transcription. Kidney-specific knockout of Mef2c, or genetrap-inactivation of a MEF2C transcriptional target, MIM, resulted in extensive renal tubule dilation and cysts, whereas Hdac5 heterozygosity or treatment with TSA, an HDAC inhibitor, reduced cyst formation in Pkd2(-/-) mouse embryos. These findings suggest a common signaling motif between myocardial hypertrophy and maintenance of renal epithelial architecture, and a potential therapeutic approach to treat ADPKD.

Effect of lamivudine on uptake of organic cations by rat renal brush-border and basolateral membrane vesicles.

The effect of lamivudine on uptake of a representative organic cation, tetraethylammonium (TEA), by rat renal brush-border membrane vesicles (BBMV) and basolateral membrane vesicles (BLMV) has been investigated. The pH-driven uptake of TEA by BBMV (pHin = 6.0, pHout = 7.5) was inhibited by lamivudine. The IC50 value (concentration resulting in 50% inhibition) for the concentration-dependent effect of lamivudine on TEA uptake by BBMV after 30 s was 2668 microM whereas IC50 values for cimetidine and trimethoprim were < 2.5 microM and < 25 microM, respectively. The early uptake of TEA by BLMV was also reduced significantly by lamivudine. The IC50 value for the concentration-dependent effect of lamivudine on uptake of TEA by BLMV at 30 s was > 25 mM, whereas the IC50 values for cimetidine and trimethoprim were 2116 microM and 445 microM, respectively. These findings suggest that compared with other cationic drugs, such as trimethoprim and cimetidine, lamivudine is a weak inhibitor of organic cation transport into the tubules by the brush-border and basolateral membranes of renal epithelial cells. It is unlikely lamivudine will have any significant effect on the excretion of co-administered cationic drugs by the renal tubules.

Expression of Kidney and Liver Bilitranslocase in Response to Acute Biliary Obstruction

Background/Aim: It has been recently demonstrated that acute obstructive jaundice is associated with modifications in the renal expression and function of organic anion transporters such as Oat1, Oat3, Oatp1 and Mrp2. This study examined the expression and function of bilitranslocase in liver and kidney from rats with bile duct ligation (BDL). Methods: Bilitranslocase expression was evaluated in renal homogenates (H), renal basolateral plasma membranes (KBLM) and liver plasma membranes (LPM) by immunoblotting. Bilitranslocase function was studied by measuring the kinetic parameters of electrogenic bromosulfophthalein (BSP) uptake in KBLM and LPM by a spectrophotometric technique. Results: An increased abundance of bilitranslocase in KBLM without modifications in renal H and in LPM from BDL rats was observed compared with Sham rats. BDL rats showed a higher V_max for BSP uptake in KBLM. No differences between groups were observed for Michaelis-Menten parameters in LPM. Conclusion: The higher renal expression and function of bilitranslocase in renal basolateral membranes from rats with obstructive cholestasis might also contribute to the dramatic increase in BSP renal excretion observed in this experimental model. This would be another compensation mechanism to overcome the hepatic dysfunction in the elimination of organic anions.

Rhesus Glycoprotein P2 (Rhp2) Is a Novel Member of the Rh Family of Ammonia Transporters Highly Expressed in Shark Kidney

Rhesus (Rh) glycoproteins are a family of membrane proteins capable of transporting ammonia. We isolated the full-length cDNA of a novel Rh glycoprotein, Rhp2, from a kidney cDNA library from the banded hound shark, Triakis scyllium. Molecular cloning and characterization indicated that Rhp2 consists of 476 amino acid residues and has 12 putative transmembrane spans, consistent with the structure of other family members. The shark Rhp2 gene was found to consist of only one coding exon. Northern blotting and in situ hybridization revealed that Rhp2 mRNA is exclusively expressed in the renal tubules of the sinus zone but not in the bundle zone and renal corpuscles. Immunohistochemical staining with a specific antiserum showed that Rhp2 is localized in the basolateral membranes of renal tubule cells. Double fluorescence labeling with phalloidin or labeling of the Na(+)/K(+)-ATPase further narrowed the location to the second and fourth loops in the sinus zone. Vacuolar type H(+)-ATPase was localized in apical membranes of the Rhp2-expressing tubule cells. Quantitative real-time PCR analysis and Western blotting showed that expression of Rhp2 was increased in response to elevation of environmental salinity. Functional analysis using the Xenopus oocyte expression system showed that Rhp2 has transport activity for methylammonium, an analog of ammonia. This transport activity was inhibited by NH(4)Cl but not trimethylamine-N-oxide and urea. These results suggested that Rhp2 is involved in ammonia reabsorption in the kidney of the elasmobranch group of cartilaginous fish comprising the sharks and rays.

New Approaches to Pathogenesis and Management of Hypertension

New Approaches to Pathogenesis and Management of Hypertension

Renal glutathione transport: Identification of carriers, physiological functions, and controversies

Glutathione (GSH) is an endogenous tripeptide composed of the amino acids L-glutamate, L-cysteine, and glycine. It is found in virtually all aerobic cells and plays critical roles in maintenance of cellular redox homeostasis and drug metabolism. An important component of its regulation is transport across biological membranes. Because GSH is a charged, hydrophilic molecule, transport occurs via catalysis by specific carrier proteins rather than by simple diffusion. Although it has been clearly understood that efflux of GSH across membranes such as the canalicular and sinusoidal plasma membranes in hepatocytes and the brush-border plasma membrane in renal proximal tubules is a key step in GSH turnover and interorgan metabolism, the existence and physiological functions of uptake of GSH across various epithelial plasma membranes has been subject to some debate. Besides transport across plasma membranes, GSH transport across intracellular membranes, most notably the mitochondrial inner membrane, has received some attention in recent years because of the importance of mitochondrial redox status and the mitochondrial GSH pool in cellular physiology and pathology. This commentary will focus on renal transport processes for GSH and will discuss some of the controversies that have existed and still seem to exist in the literature, specifically regarding uptake of intact GSH by basolateral membranes of renal proximal tubular cells and uptake of intact GSH by the mitochondrial inner membrane.

PTH transiently increases the percent mobile fraction of Npt2a in OK cells as determined by FRAP

Renal sodium-dependent phosphate transporter 2a (Npt2a) binds to a number of PDZ adaptor proteins including sodium-hydrogen exchanger regulatory factor-1 (NHERF-1), which regulates its retention in the apical membrane of renal proximal tubule cells and the response to parathyroid hormone (PTH). The present experiments were designed to study the lateral mobility of enhanced green fluorescent protein (EGFP)-Npt2a in proximal tubule-like opossum kidney (OK) cells using fluorescence recovery after photobleaching (FRAP) and to determine the role of PDZ binding proteins in mediating the effects of PTH. The mobile fraction of wild-type Npt2a (EGFP-Npt2a-TRL) under basal conditions was approximately 17%. Treatment of the cells with Bis(sulfosuccinimidyl) suberate, a water-soluble cross-linker, abolished recovery nearly completely, indicating that recovery represented lateral diffusion in the plasma membrane and not the exocytosis or synthesis of unbleached transporter. Substitution of the C-terminal amino acid PDZ binding sequence TRL with AAA (EGFP-Npt2a-AAA) resulted in a nearly twofold increase in percent mobile fraction of Npt2a. Treatment of cells with PTH resulted in a rapid increase in the percent mobile fraction to >30% followed by a time-dependent decrease to baseline or below. PTH had no effect on the mobility of EGFP-Npt2a-AAA expressed in native OK cells or on wild-type EGFP-Npt2a-TRL expressed in OK-H cells deficient in NHERF-1. These findings indicate that the association of Npt2a with PDZ binding proteins limits the lateral mobility of the transporter in the apical membrane of renal proximal tubule cells. Treatment with PTH, presumably by dissociating NHERF-1/Npt2a complexes, transiently increases the mobility of Npt2a, suggesting that freeing of Npt2a from the cytoskeleton precedes PTH-mediated endocytosis.

PTH-induced internalization of apical membrane NaPi2a: role of actin and myosin VI

Parathyroid hormone (PTH) plays a critical role in the regulation of renal phosphorous homeostasis by altering the levels of the sodium-phosphate cotransporter NaPi2a in the brush border membrane (BBM) of renal proximal tubular cells. While details of the molecular events of PTH-induced internalization of NaPi2a are emerging, the precise events governing NaPi2a removal from brush border microvilli in response to PTH remain to be fully determined. Here we use a novel application of total internal reflection fluorescence microscopy to examine how PTH induces movement of NaPi2a out of brush border microvilli in living cells in real time. We show that a dynamic actin cytoskeleton is required for NaPi2a removal from the BBM in response to PTH. In addition, we demonstrate that a myosin motor that has previously been shown to be coregulated with NaPi2a, myosin VI, is necessary for PTH-induced removal of NaPi2a from BBM microvilli.

Kidney Damage in Rats Bearing an Adrenocorticotropin and Prolactin Secreting Tumor

The effect of chronic high plasma corticosteroids' concentration upon renal function was studied in rats bearing a transplantable pituitary mammotropic tumor which produces large quantities of ACTH and prolactin (MtTF4S). Kidney splanchnomegaly and degenerative changes of renal cortex, particularly in proximal tubules, as well as cytolysis and appearance of vacuoles were noticed in tumor bearing rats. (Na+ + K+)-ATPase activity in renal plasma membranes decreased 67% in rats with a tumor secreting ACTH and prolactin, and 64% in rats with a tumor secreting growth hormone and prolactin when compared with controls. After adrenalectomy of MtTF4S rats, kidney weight as well as plasma concentrations of urea, sodium, chloride and phosphate ions were normalized indicating the involvement of adrenal glands in the development of disturbances in renal function.