Functional expression of a pseudohypoaldosteronism type I mutated epithelial Na+ channel lacking the pore-forming region of its alpha subunit.

The autosomal recessive form of type I pseudohypoaldosteronism (PHA-I) is an inherited salt-losing syndrome resulting from diminution-of-function mutations in the 3 subunits of the epithelial Na+ channel (ENaC). A PHA-I stop mutation (alpha(R508stop)) of the ENaC alpha subunit is predicted to lack the second transmembrane domain and the intracellular COOH-terminus, regions of the protein involved in pore function. Nonetheless, we observed a measurable Na+ current in Xenopus laevis oocytes that coexpress the beta and gamma subunits with the truncated alpha subunit. The mutant alpha was coassembled with beta and gamma subunits and was present at the cell surface at a lower density, consistent with the lower Na+ current seen in oocytes with the truncated alpha subunit. The single-channel Na+ conductance for the mutant channel was only slightly decreased, and the appearance of the macroscopic currents was delayed by 48 hours with respect to wild-type. Our data suggest novel roles for the alpha subunit in the assembly and targeting of an active channel to the cell surface, and suggest that channel pores consisting of only the beta and gamma subunits can provide significant residual activity. This activity may be sufficient to explain the absence of a severe pulmonary phenotype in patients with PHA-I.

Defective regulation of the epithelial Na+ channel by Nedd4 in Liddle's syndrome.

Liddle's syndrome is an inherited form of hypertension linked to mutations in the epithelial Na+ channel (ENaC). ENaC is composed of three subunits (alpha, beta, gamma), each containing a COOH-terminal PY motif (xPPxY). Mutations causing Liddle's syndrome alter or delete the PY motifs of beta- or gamma-ENaC. We recently demonstrated that the ubiquitin-protein ligase Nedd4 binds these PY motifs and that ENaC is regulated by ubiquitination. Here, we investigate, using the Xenopus oocyte system, whether Nedd4 affects ENaC function. Overexpression of wild-type Nedd4, together with ENaC, inhibited channel activity, whereas a catalytically inactive Nedd4 stimulated it, likely by acting as a competitive antagonist to endogenous Nedd4. These effects were dependant on the PY motifs, because no Nedd4-mediated changes in channel activity were observed in ENaC lacking them. The effect of Nedd4 on ENaC missing only one PY motif (of beta-ENaC), as originally described in patients with Liddle's syndrome, was intermediate. Changes were due entirely to alterations in ENaC numbers at the plasma membrane, as determined by surface binding and immunofluorescence. Our results demonstrate that Nedd4 is a negative regulator of ENaC and suggest that the loss of Nedd4 binding sites in ENaC observed in Liddle's syndrome may explain the increase in channel number at the cell surface, increased Na+ reabsorption by the distal nephron, and hence the hypertension.

Ras pathway activates epithelial Na+ channel and decreases its surface expression in Xenopus oocytes.

The small G protein K-Ras2A is rapidly induced by aldosterone in A6 epithelia. In these Xenopus sodium reabsorbing cells, aldosterone rapidly activates preexisting epithelial Na+ channels (XENaC) via a transcriptionally mediated mechanism. In the Xenopus oocytes expression system, we tested whether the K-Ras2A pathway impacts on XENaC activity by expressing XENaC alone or together with XK-Ras2A rendered constitutively active (XK-Ras2AG12V). As a second control, XENaC-expressing oocytes were treated with progesterone, a sex steroid that induces maturation of the oocytes similarly to activated Ras. Progesterone or XK-Ras2AG12V led to oocyte maturation characterized by a decrease in surface area and endogenous Na+ pump function. In both conditions, the surface expression of exogenous XENaC's was also decreased; however, in comparison with progesterone-treated oocytes, XK-ras2AG12V-coinjected oocytes expressed a fivefold higher XENaC-mediated macroscopic Na+ current that was as high as that of control oocytes. Thus, the Na+ current per surface-expressed XENaC was increased by XK-Ras2AG12V. The chemical driving force for Na+ influx was not changed, suggesting that XK-Ras2AG12V increased the mean activity of XENaCs at the oocyte surface. These observations raise the possibility that XK-Ras2A, which is the first regulatory protein known to be transcriptionally induced by aldosterone, could play a role in the control of XENaC function in aldosterone target cells.

Luminal heterodimeric amino acid transporter defective in cystinuria.

Mutations of the glycoprotein rBAT cause cystinuria type I, an autosomal recessive failure of dibasic amino acid transport (b(0,+) type) across luminal membranes of intestine and kidney cells. Here we identify the permease-like protein b(0,+)AT as the catalytic subunit that associates by a disulfide bond with rBAT to form a hetero-oligomeric b(0,+) amino acid transporter complex. We demonstrate its b(0,+)-type amino acid transport kinetics using a heterodimeric fusion construct and show its luminal brush border localization in kidney proximal tubule. These biochemical, transport, and localization characteristics as well as the chromosomal localization on 19q support the notion that the b(0,+)AT protein is the product of the gene defective in non-type I cystinuria.

Functional heterodimeric amino acid transporters lacking cysteine residues involved in disulfide bond.

The protein mediating system L amino acid transport, AmAT-L, is a disulfide-linked heterodimer of a permease-related light chain (AmAT-L-lc) and the type II glycoprotein 4F2hc/ CD98. The Schistosoma mansoni protein SPRM1 also heterodimerizes with h4F2hc, inducing amino acid transport with different specificity. In this study, we show that the disulfide bond is formed by heavy chain C109 with a Cys residue located in the second putative extracellular loop of the multi-transmembrane domain light chain (C164 and C137 for XAmAT-L-lc and SPRM1, respectively). The non-covalent interaction of Cys-mutant subunits is not sufficient to allow coimmunoprecipitation, but cell surface expression of the light chains is maintained to a large extent. The non-covalently linked transporters display the same transport characteristics as disulfide bound heterodimers, but the maximal transport rates are reduced by 30-80%.

Enhanced osteopontin expression and macrophage infiltration in MRL-Fas(lpr) mice with lupus nephritis.

MRL-Fas(lpr) mice spontaneously develop a chronic lupus-like renal disease, characterized by immune complex-mediated glomerulonephritis and abundant mononuclear cell infiltration in the interstitium. In the present study we have examined whether the macrophage chemoattractant osteopontin (Opn) could be important in the recruitment of macrophages in this murine model of autoimmune renal injury. We have examined the expression of Opn in the kidney of MRL-Fas(lpr) mice and have correlated Opn synthesis with the degree of macrophage infiltration. Immunofluorescence staining revealed prominent expression of Opn by proximal tubules in MRL-Fas(lpr) mice but not in MRL-++ control mice. Northern blot analysis demonstrated that steady-state transcript levels for Opn mRNA were also significantly increased in MRL-Fas(lpr) kidneys compared with control kidneys. Furthermore, in situ hybridization showed massive Opn mRNA transcripts in proximal tubules in MRL-Fas(lpr) mice but not in controls. The diffuse macrophage infiltration in the kidney of MRL-Fas(lpr) correlated with the enhanced Opn expression. Opn secretion in vitro by cultured renal tubular epithelial cells was upregulated by TNF-alpha and 1,25(OH)2-vitamin D3, whereas no regulation was observed in a control macrophage cell line. We conclude that the enhanced expression of the chemotactic molecule Opn by tubular cells is a prominent feature of murine lupus nephritis and might be promoted by the proinflammatory cytokine environment in MRL-Fas(lpr). The chronic upregulation of Opn could participate in the recruitment of monocytes in the kidney of MRL-Fas(lpr) mice, thereby contributing to the pathogenesis of autoimmune renal disease.

Morphology of interstitial cells in the healthy kidney.

Renal interstitial cells play an important role in renal function and renal diseases. We describe the morphology of renal interstitial cells in the healthy kidney. We distinguish within the renal interstitium (1) renal fibroblasts and (2) cells of the immune system. Fibroblasts are in the majority and constitute the scaffold of the kidney; they are interconnected by junctions, and are attached to tubules and vessels. Although the phenotype of fibroblasts shows some variation depending on their location in the kidney and on their functional stage, their recognition as fibroblasts is possible on account of structural features. Among the cell types of the second group, antigen-presenting dendritic cells are the most abundant in in the peritubular interstitial spaces of healthy kidneys. Their incidence is highest in the inner stripe of the outer medulla. They share some morphological features with fibroblasts but lack others--junctional complexes, morphologically defined connections with tubules and vessels, and the prominent layer of actin filaments under the plasma membrane--that are characteristic for fibroblasts. Dendritic cells in healthy kidneys are morphologically different from macrophages, which are characterized by abundant primary and secondary lysosomes. In healthy kidneys macrophages are restricted to the connective tissue of the renal capsule and the pelvic wall, and to the periarterial connective tissue. Lymphocytes are rare in healthy kidneys. The distinction of cell types by morphology is supported by differences of membrane proteins. Among all interstitial cells in the renal cortex, fibroblasts alone exhibit ecto-5'-nucleotidase. Dendritic cells constitutively have a high abundance of MHC class II protein. Both proteins are mutually exclusive. Rat macrophages display the membrane antigen ED 2 and lymphocytes exhibit specific surface antigens, depending on their type and functional stage, e.g., CD4 or CD8.

Postnatal maturation of renal cortical peritubular fibroblasts in the rat.

The stromal cells in the renal cortex and medulla of adult rats reveal different phenotypes. Cortical peritubular fibroblasts are ecto-5'nucleotidase (5'NT)-positive and lack alpha-smooth muscle actin (alphaSMA) and vimentin immunoreactivity, whereas medullary fibroblasts are 5'NT-negative and vimentin-positive. We have studied by immunohistochemistry the postnatal (neonatal up to 8 weeks) development of renal cortical stromal cells with respect to 5'NT and to the cytoskeletal proteins alphaSMA and vimentin. Both alphaSMA and vimentin are characteristic for the renal myofibroblasts that replace stromal fibroblasts in interstitial nephritis. In new-born and 1-week-old rats, stromal cells in the cortex and medulla display alphaSMA and vimentin, but lack 5'NT. During the second postnatal week, alphaSMA and vimentin immunoreactivity in cortical interstitial cells gradually declines, whereas 5'NT reactivity becomes progressively apparent between the convoluted tubules in the juxtamedullary labyrinth. For a short time, all three proteins are found to be coexpressed in the same cells. At the end of the third week, interstitial 5'NT-immunoreactivity becomes evident also in the superficial cortical labyrinth, and alphaSMA and vimentin are no longer detectable in cortical peritubular cells. From the fourth week on, the distribution pattern and phenotype of 5'NT-positive cortical fibroblasts correspond to that in adult rats. The temporal pattern of maturation of cortical peritubular fibroblasts seems to parallel the functional maturation of cortical tubules. It is suggested that the local phenotype of peritubular fibroblasts in healthy and possibly also in injured kidneys may be controlled, at least in part, by the local tubular environment, conditioned by tubular metabolism and function.

OS058. Aldosterone deficiency adversely affects pregnancy outcome in mice.

INTRODUCTION: In pregnancy, plasma volume is expanded due to high aldosterone levels to support placental perfusion and fetal nutrition. Inadequately low aldosterone levels as present in preeclampsia, a life-threatening disease for both mother and child, are discussed to be involved in its pathogenesis or severity. OBJECTIVES: We used aldosterone synthase deficient (AS(-/-)) mice to test whether the absence of aldosterone is sufficient to impair pregnancy or even to cause preeclampsia. METHODS: AS(-/-) and AS(+/+) females were mated with AS(+/+) and AS(-/-) males, respectively, always generating AS(+/-) offspring. Blood pressure was measured by tail cuff, fetal and placental number and size as well as placental histology were assessed. Placental expression of HIF-1αand angiogenic factors was assessed by semiquantitative RT-PCR. RESULTS: With maternal aldosterone deficiency in AS(-/-) mice, systolic blood pressure was low before and further reduced during pregnancy and with no increase in proteinuria. Yet, AS(-/-) had smaller litters due to loss of fetuses as indicated by a high number of necrotic placentas with massive lymphocyte infiltrations at gestational day 18. Surviving fetuses and their placentas from AS(-/-) females were smaller. High salt diet before and during pregnancy increased systolic blood pressure only before pregnancy in both genotypes and reduced blood pressure during late pregnancy as compared to normal salt controls. Litter size from AS(-/-) was slightly improved and the differences in placental and fetal weights between AS(+/+) and AS(-/-) mothers disappeared. Overall an increased placental efficiency was observed in both groups. CONCLUSION: Our results demonstrate that aldosterone deficiency has profound adverse effects on placental function. High dietary salt intake improved placental function and lowered blood pressure in wild-type mice. In this animal model, aldosterone deficiency did not cause preeclampsia.

Long-term effects of vasopressin on the subcellular localization of ENaC in the renal collecting system.

Previous studies revealed that chronic (days) vasopressin treatment stimulates amiloride-sensitive sodium transport in isolated renal cortical collecting ducts and increases the abundance of beta- and gamma-subunits of the epithelial sodium channel (ENaC) in the kidney. The aim of the present work was to investigate in vivo the cellular basis of these effects. The long-term effect of V2 vasopressin agonist (1-deamino-8-D-arginine vasopressin (dDAVP)) on the abundance and subcellular localization of ENaC along the rat renal collecting system was determined by immunohistochemistry and laser confocal microscopy. Moreover, we studied by real-time reverse transcriptase-polymerase chain reaction the effect of vasopressin on proteins implicated in the regulation of ENaC (Nedd4-2, prostasin, Sgk1). After 5 days of administration, dDAVP markedly increased the intracellular pool of the beta- and gamma-ENaC subunits in the principal cells, with an increasing gradient from connecting tubule to the outer medullary collecting duct, but did not increase any subunit at the cell surface. The apical immunostaining of ENaC increased in response to sodium restriction, as expected, but dDAVP did not further enhance this apical labelling. dDAVP increased the gene expression of prostasin in the cortex but not that of Nedd4-2 and Sgk1. These findings suggest that the previously reported increase in sodium transport induced by sustained stimulation of vasopressin V2 receptor is probably mediated by other mechanism than an increase in the apical density of ENaC.

The connecting tubule is the main site of the furosemide-induced urinary acidification by the vacuolar H+-ATPase.

Final urinary acidification is achieved by electrogenic vacuolar H(+)-ATPases expressed in acid-secretory intercalated cells (ICs) in the connecting tubule (CNT) and the cortical (CCD) and initial medullary collecting duct (MCD), respectively. Electrogenic Na(+) reabsorption via epithelial Na(+) channels (ENaCs) in the apical membrane of the segment-specific CNT and collecting duct cells may promote H(+)-ATPases-mediated proton secretion by creating a more lumen-negative voltage. The exact localization where this supposed functional interaction takes place is unknown. We used several mouse models performing renal clearance experiments and assessed the furosemide-induced urinary acidification. Increasing Na(+) delivery to the CNT and CCD by blocking Na(+) reabsorption in the thick ascending limb with furosemide enhanced urinary acidification and net acid excretion. This effect of furosemide was abolished with amiloride or benzamil blocking ENaC action. In mice deficient for the IC-specific B1 subunit of the vacuolar H(+)-ATPase, furosemide led to only a small urinary acidification. In contrast, in mice with a kidney-specific inactivation of the alpha subunit of ENaC in the CCD and MCD, but not in the CNT, furosemide alone and in combination with hydrochlorothiazide induced normal urinary acidification. These results suggest that the B1 vacuolar H(+)-ATPase subunit is necessary for the furosemide-induced acute urinary acidification. Loss of ENaC channels in the CCD and MCD does not affect this acidification. Thus, functional expression of ENaC channels in the CNT is sufficient for furosemide-stimulated urinary acidification and identifies the CNT as a major segment in electrogenic urinary acidification.

Ecto-5'-nucleotidase is expressed by pericytes and fibroblasts in the rat heart.

Ecto-5'-nucleotidase is anchored at the outer surface of cell membranes and thus its reaction product adenosine is released into the extracellular space. Extracellular adenosine displays via specific receptors a wide range of physiological effects in heart. There are discrepancies in the literature concerning the distribution of ecto-5'-nucleotidase in heart. Since we suspected that these may be due to technical problems, in the present study on ecto-5'-nucleotidase in rat heart we attempted to circumvent some technical pitfalls. Good preservation of the tissue with open capillary lumina, providing a clear identification of endothelium, was obtained by perfusion fixation. At the light microscopic level, the distribution of ecto-5'-nucleotidase studied by enzyme histochemistry and immunohistochemistry using a monoclonal and a polyclonal antibody yielded congruent results. The enzyme was rather homogeneously distributed throughout the myocardium, with a slightly higher incidence of stained cells in the outer thirds than in the inner third of the wall. Consistently high levels of ecto-5'-nucleotidase were seen only in interstitial cells. The walls of large vessels and heart muscle cells were constantly negative for ecto-5'-nucleotidase. The endothelia of capillaries were mostly negative but a few profiles occasionally displayed a weak immunoreaction. The interstitial cells staining positive for ecto-5'-nucleotidase could be identified as pericytes and as fibroblasts according to their shapes and localizations. The immunoreactivity of fibroblasts was confirmed by electron microscopy. These data indicate that adenosine may be formed extracellularly in the interstitium of the myocardium, where it would have direct access to important targets such as myocytes, arterioles and nerve endings.

Upregulated renal tubular CD44, hyaluronan, and osteopontin in kdkd mice with interstitial nephritis.

BACKGROUND: The hyaluronan (HA) receptor CD44 is upregulated on parenchymal cells in various inflammatory lesions and could play a role in immune injury. The purpose of the present study was to examine CD44 and its ligands HA and osteopontin (Opn) in a murine model of tubulointerstitial nephritis (TIN). METHODS: The expression of CD44 was investigated by immunofluorescence staining and RNA analysis in kidneys of kdkd mice with autoimmune TIN. The CD44 expression was then correlated with the location of its ligands HA and Opn. RESULTS: CD44 is expressed de novo by tubular epithelial cells (TEC) in areas of tubular injury in kdkd kidneys, but not in normal control kidneys. CD44 positive lymphocytes and macrophages also infiltrate the kidney to kdkd mice. RT-PCR and Southern blot analysis demonstrate that transcripts encoding standard and variant forms of CD44 are increased in kdkd mice with TIN. In parallel the CD44 ligand HA also accumulates in kdkd kidneys in the interstitial space, particularly in cortical areas of tubular injury. Furthermore, the expression of the chemotactic protein Open is enhanced in kdkd kidney, predominantly in areas of tubular injury. Opn mRNA expression also increases markedly in kdkd kidneys compared with normal kidneys, and correlates with disease severity. CONCLUSIONS: Prominent CD44 expression by TEC in areas of tubulointerstitial lesions is a characteristic feature of kdkd mice. The de novo appearance of CD44 on injured TEC might allow interaction with the ligands HA and Opn in vivo. Interaction of CD44 with these ligands could participate in the tubulointerstitial inflammatory response in kdkd mice.

Modulation of salt transport rate affects DNA synthesis in vivo in rat renal tubules.

In adult male Wistar rats we investigated whether cell proliferation contributes to salt load-induced hypertrophy of distal tubules. In one treatment group salt transport in the thick ascending limb (TAL) was inhibited by furosemide (7.5 mg/100 g body wt/24 hr, via osmotic minipump) and stimulated in the successive distal segments by simultaneous high salt intake (F + Salt). Controls without furosemide treatment had a standard salt intake. All animals received the thymidine analog bromodeoxyuridine (BrdU) during 24 and 72 hours, respectively. In cryostat sections of the perfusion-fixed kidneys DNA synthesis was assessed by immunohistochemistry for BrdU, and for endogenous proliferating cellular nuclear antigen (PCNA). Incidence of BrdU- and PCNA-labeled nuclei were quantified in proximal tubules, medullary TAL, and cortical distal segments downstream the TAL. In control animals low labeling indices were found in all investigated segments. After 24 and 72 hours of F + Salt, indices of labeled nuclei were markedly increased in distal segments downstream the TAL, whereas they were significantly reduced in TAL. In proximal tubules increased DNA synthesis rate was apparent only after 72 hours. The data demonstrate that (1.) DNA synthesis rate in nephron segments in vivo varies in parallel with changes of their salt transport activity; (2.) increased DNA synthesis, thus probably cellular proliferation, is a component of the structural response of nephron segments following increased salt transport activity.

Mediators of aldosterone action in the renal tubule.

The aldosterone-sensitive distal nephron extends from the second part of the distal convoluted tubule to the inner medullary collecting duct. As recently shown, aldosterone increases within two hours the abundance of the alpha-subunit of the epithelial sodium channel along the entire aldosterone-sensitive distal nephron, whereas it induces only in an initial portion of the aldosterone-sensitive distal nephron an apical translocation of all three epithelial sodium channel subunits. This suggests that another factor or factors determines the length of the aldosterone-sensitive distal nephron portion in which aldosterone controls epithelial sodium channel surface expression. Since the glucocorticoid-induced kinase SGK1 was identified as aldosterone-induced protein in 1999, it has been postulated to play a key regulatory role. The in-vivo localization of its induction to segment-specific cells of the aldosterone-sensitive distal nephron, and the in-vitro correlation of the amount of its hyperphosphorylated form with transepithelial sodium transport, support this hypothesis. Other recent studies unravel pathways other than those activated by aldosterone and insulin that impact on SGK1 expression and/or function, and thus shed some light onto the complex network that appears to control sodium transport. In view of the ongoing research, the question of how, and formally also whether, SGK1 acts on the epithelial sodium channel should be resolved in the near future.

Distribution of ecto-5'-nucleotidase in the rat liver: effect of anaemia.

In the kidney a striking parallel exists between the expression of ecto-5'-nucleotidase and of erythropoietin by renal fibroblasts. It was therefore hypothesized that the expression of ecto-5'-nucleotidase in fibroblasts might be controlled by oxygen tension. In order to test this hypothesis, we examined the distribution of the enzyme in a tissue which displays a defined zonation in respect to oxygen tension, namely in the liver; anaemia was used in order to exaggerate this zonation. The distribution of ecto-5'-nucleotidase was investigated by light and electron microscopy using enzyme and immunohistochemical methods in the livers of healthy and of anaemic rats. Anaemia was produced by haemolysis combined with X-ray irradiation. The enzyme was detected in the bile canaliculi, in the connective tissue of the portal triads and of the central veins, and in fat-storing cells probably corresponding to a special form of fibroblasts. In healthy animals the perisinusoidal ecto-5'-nucleotidase activity was slightly higher in the pericentral than in the periportal area of the acinus whereas the inverse was observed for the staining of bile canaliculi. Anaemia provoked an increase of ecto-5'-nucleotidase in fat-storing cells in the pericentral zone of the acinus and in fibroblasts around the central veins, resulting in steepended gradients along the sinusoids. The intralobular gradient of ecto-5'-nucleotidase in perisinusoidal cells and the effect thereon of anaemia suggest that the expression of the ecto-5'-nucleotidase might be directly or indirectly controlled by local oxygen tension.

Exocytosis is not involved in activation of Cl- secretion via CFTR in Calu-3 airway epithelial cells.

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel, which mediates transepithelial Cl- transport in a variety of epithelia, including airway, intestine, pancreas, and sweat duct. In some but not all epithelial cells, cAMP stimulates Cl- secretion in part by increasing the number of CFTR Cl- channels in the apical plasma membrane. Because the mechanism whereby cAMP stimulates CFTR Cl- secretion is cell-type specific, our goal was to determine whether cAMP elevates CFTR-mediated Cl- secretion across serous airway epithelial cells by stimulating the insertion of CFTR Cl- channels from an intracellular pool into the apical plasma membrane. To this end we studied Calu-3 cells, a human airway cell line with a serous cell phenotype. Serous cells in human airways, such as Calu-3 cells, express high levels of CFTR, secrete antibiotic-rich fluid, and play a critical role in airway function. Moreover, dysregulation of CFTR-mediated Cl- secretion in serous cells is thought to contribute to the pathophysiology of cystic fibrosis lung disease. We report that cAMP activation of CFTR-mediated Cl- secretion across human serous cells involves stimulation of CFTR channels present in the apical plasma membrane and does not involve the recruitment of CFTR from an intracellular pool to the apical plasma membrane.

Butyrate increases apical membrane CFTR but reduces chloride secretion in MDCK cells.

Sodium butyrate and its derivatives are useful therapeutic agents for the treatment of genetic diseases including urea cycle disorders, sickle cell disease, thalassemias, and possibly cystic fibrosis (CF). Butyrate partially restores cAMP-activated Cl(-) secretion in CF epithelial cells by stimulating DeltaF508 cystic fibrosis transmembrane conductance regulator (DeltaF508-CFTR) gene expression and increasing the amount of DeltaF508-CFTR in the plasma membrane. Because the effect of butyrate on Cl(-) secretion by renal epithelial cells has not been reported, we examined the effects of chronic butyrate treatment (15-18 h) on the function, expression, and localization of CFTR fused to the green fluorescent protein (GFP-CFTR) in stably transfected MDCK cells. We report that sodium butyrate reduced Cl(-) secretion across MDCK cells, yet increased apical membrane GFP-CFTR expression 25-fold and increased apical membrane Cl(-) currents 30-fold. Although butyrate also increased Na-K-ATPase protein expression twofold, the drug reduced the activity of the Na-K-ATPase by 55%. Our findings suggest that butyrate inhibits cAMP-stimulated Cl(-) secretion across MDCK cells in part by reducing the activity of the Na-K-ATPase.

Cellular mechanisms of the age-related decrease in renal phosphate reabsorption.

The aging process in humans and in the rat is associated with an impairment in renal tubular reabsorption of Pi and renal tubular adaptation to a low Pi diet. The purposes of the present study were to determine whether changes in the abundance of type II Na-Pi contransporter (NaPi-2) protein and/or mRNA play a role in the age-related decrease in Na-Pi cotransport activity, and to further determine the cellular mechanisms of impaired adaptation to a low Pi diet. In studies performed in 3- to 4-month-old young adult rats and 32-to 16-month-old aged rats we found that there was an age-related twofold decrease in proximal tubular apical brush border membrane (BBM) Na-Pi cotransport activity, which was associated with similar decreases in BBM NaPi-2 protein abundance and renal cortical NaPi-2 mRNA level. Immunohisto-chemistry showed lower NaPi-2 protein expression in the BBM of proximal tubules of superficial, midcortical, and juxtamedullary nephrons. We also found that in response to chronic (7 days) and/or acute (4 hr) feeding of a low Pi diet there were similar adaptive increases in BBM Na-Pi cotransport activity and BBM NaPi-2 protein abundance in both young and aged rats. However, BBM Na-Pi cotransport activity and BBM NaPi-2 protein abundance were still significantly lower in aged rats, in spite of a significantly lower serum Pi concentration in aged rats. The results indicate that impaired expression of the type II renal Na-Pi cotransporter protein at the level of the apical BBM plays an important role in the age-related impairment in renal tubular reabsorption of Pi and renal tubular adaptation to a low Pi diet.

Expression of NHE-3 in the apical membrane of rat renal proximal tubule and thick ascending limb.

Apical membrane Na/H exchange is a principal mechanism of renal proximal tubule Na absorption and H secretion, and thick ascending limb H secretion. Based on current data on Na/H exchanger isoforms (NHE-1 to 5), NHE-3 is the likeliest candidate for the apical membrane isoform. The present study localizes NHE-3 in rat kidney using polyclonal antisera against cytoplasmic epitopes of rat NHE-3. These antisera recognized an approximately 87 kD protein in Na/H exchanger-deficient cells transfected with the rat NHE-3 gene but not in mock-transfected cells. All antisera labeled an approximately 87 kD protein in plasma membranes from cortex and outer medulla. Fractionation of cortical membranes showed labeling in apical but not basolateral membranes. Cross linking studies suggested existence of oligomeric forms of the transporter. Immunohistochemistry showed strong staining of the apical membrane of S1 convoluted, and S2 convoluted tubule with lesser staining of the S2 straight tubule and absent staining of S3. Weak staining was observed in thin descending limbs in the inner stripe and intense staining was seen in the apical membrane of medullary and cortical thick ascending limbs. NHE-3 staining was absent in the remainder of the nephron. In summary, NHE-3 is the isoform responsible for NaCl and NaHCO3 absorption in the proximal convoluted tubule, and NaHCO absorption in the thick ascending limb. In the S3 proximal tubule and the distal convoluted tubule, apical membrane Na/H exchange activity is likely mediated by other isoform(s) of the NHE family.