The vacuolar-ATPase B1 subunit in distal tubular acidosis: novel mutations and mechanisms for dysfunction.

Mutations in the B1 subunit of the multisubunit vacuolar ATPase cause autosomal-recessive distal renal tubular acidosis and sensorineural deafness. Here, we report a novel frameshift mutation that truncates the C-terminus of the human B1 subunit. This mutant protein failed to assemble with other subunits in the cytosol to form the complex that can be targeted to vesicular structures in mammalian cells. Loss of proton pump activity was demonstrated in a functional complementation assay in B-subunit null yeast. The mutation caused loss of a discreet C-terminal region critical for subunit interaction not related to the C-terminal PDZ motif. Co-expression studies failed to demonstrate dominant negative effects of this truncated mutant over wild-type B1. Analysis of 12 reported B1 subunit missense mutations showed one polymorphic allele had intact pump function, two point mutants had intact assembly but defective proton pumping, and the remaining nine had disrupted assembly with no pump function. One presumed polymorphic allele was actually an inactivating mutation. Our study shows that multiple mechanisms of pump dysfunction result from B1 subunit mutations with a common outcome being defective assembly. Polymorphisms of the B1 subunit in the general population may affect renal acidification and urinary chemistry.

Activation of protein kinase A acutely inhibits and phosphorylates Na/H exchanger NHE-3.

In the mammalian renal proximal tubule, protein kinase A (PKA) plays an important role in mediating hormonal regulation of apical membrane Na/H exchanger activity. This exchanger is likely encoded by NHE-3. The present studies examined regulation of NHE-3 by PKA. NHE-3 was stably expressed in Na/H exchanger-deficient fibroblasts (AP-1/NHE-3 cells). PKA activation (0.1 mM 8-BrcAMP x 20 min) inhibited NHE-3 activity by 39% (P < 0.01) with no change in NHE-3 protein abundance in the plasma membrane. To define the structural requirements for PKA-mediated inhibition, full-length NHE-3 and a cytoplasmic domain-truncated mutant (NHE-3 delta cyto) were expressed in Xenopus laevis oocytes. 8-BrcAMP inhibited NHE-3 activity by 27% (P < 0.05), an effect that was blocked by 10(-7) M PKA inhibitor peptide. NHE-3 delta cyto had baseline activity similar to that of full-length NHE-3 but its activity was not regulated by 8-BrcAMP. The purified recombinant cytoplasmic domain of NHE-3 was phosphorylated in vitro by the catalytic subunit of PKA on serine residues. In AP-1/NHE-3 cells, NHE-3 was immunoprecipitated as a approximately 87-kD phosphoprotein. Addition of 0.1 mM 8-BrcAMP increased the phosphocontent of NHE-3 by threefold. In summary, acute activation of PKA inhibits NHE-3 activity, an effect that is likely mediated by phosphorylation of its cytoplasmic domain.

Acid activation of immediate early genes in renal epithelial cells.

These studies examined the effect of acidosis on immediate early (IE) gene expression in renal tubule cells. In MCT cells, an SV40 transformed mouse proximal tubule cell line, incubation in acid media led to transient increases in c-fos, c-jun, junB, and egr-1 mRNA abundance, peaking at 30 min to 1 h. In vivo metabolic acidosis caused more prolonged increases in these mRNA species in renal cortex. Nuclear runon studies demonstrated increased rates of transcription for these IE genes. In addition, pretreatment of cells with cycloheximide caused superinduction of these mRNA by acid incubation. These responses are similar to those elicited by growth factors. Inhibition of tyrosine kinase pathways prevented IE gene activation by acid, while inhibition of protein kinase C and/or increases in cell calcium had no effect. In 3T3 cells, acid activated IE genes by a different mechanism in that the increase in mRNA did not include c-jun, was more prolonged, and was blocked by cycloheximide. In summary, incubation of renal cells in acid media leads to activation of IE genes that is similar to growth factor-induced IE gene activation, and is likely mediated by tyrosine kinase pathways.

Chronic hyperosmolality increases NHE3 activity in OKP cells.

This study investigated the effect of chronic hypertonicity on the OKP cell Na/H antiporter, encoded by Na/H exchanger 3 (NHE3). Chronic (48 h) increases in extracellular glucose, mannitol, or raffinose concentration caused a significant increase in Na/H antiporter activity, while increases in urea concentration were without effect. This effect was seen with changes in osmolality of only 20 mOsm/liter, a magnitude that is observed clinically in poorly controlled diabetes mellitus. Increases in mannitol concentration acutely inhibited and chronically stimulated Na/H antiporter activity. The increase in Na/H antiporter activity induced by hypertonic incubation was resistant to 10(-7) and 5 x 10(-6) M but inhibited by 10(-4) M ethylisopropyl amiloride, consistent with regulation of NHE3. In addition, hypertonicity increased total cellular and plasma membrane NHE3 protein abundance twofold, with only a small increase in NHE3 mRNA abundance. We conclude that chronic pathophysiologically relevant increases in tonicity lead to increases in NHE3 protein abundance and activity. This may be responsible for increased proximal tubule apical membrane Na/H antiporter activity in poorly controlled diabetes mellitus, which could then contribute to hypertension, glomerular hyperfiltration and diabetic nephropathy.

Endothelin-1/endothelin-B receptor-mediated increases in NHE3 activity in chronic metabolic acidosis.

Decreases in blood pH activate NHE3, the proximal tubular apical membrane Na/H antiporter. In cultured renal epithelial cells, activation of the endothelin-B (ET(B)) receptor increases NHE3 activity. To examine the role of the ET(B) receptor in the response to acidosis in vivo, the present studies examined ET(B) receptor-deficient mice, rescued from neonatal lethality by expression of a dopamine beta-hydroxylase promoter/ET(B) receptor transgene (Tg/Tg:ET(B)(-/-) mice). In proximal tubule suspensions from Tg/Tg:ET(B)(+/-) mice, 10(-8) M endothelin-1 (ET-1) increased NHE3 activity, but this treatment had no effect on tubules from Tg/Tg:ET(B)(-/-) mice. Acid ingestion for 7 days caused a greater decrease in blood HCO(3)(-) concentration in Tg/Tg:ET(B)(-/-) mice compared with Tg/Tg:ET(B)(+/+) and Tg/Tg:ET(B)(+/-) mice. Whereas acid ingestion increased apical membrane NHE3 by 42-46% in Tg/Tg:ET(B)(+/+) and Tg/Tg:ET(B)(+/-) mice, it had no effect on NHE3 in Tg/Tg:ET(B)(-/-) mice. In C57BL/6 mice, excess acid ingestion increased renal cortical preproET-1 mRNA expression 2.4-fold and decreased preproET-3 mRNA expression by 37%. On a control diet, Tg/Tg:ET(B)(-/-) mice had low rates of ammonium excretion, which could not be attributed to an inability to acidify the urine, as well as hypercitraturia, with increased titratable acid excretion. Acid ingestion increased ammonium excretion, citrate absorption, and titratable acid excretion to the same levels in Tg/Tg:ET(B)(-/-) and Tg/Tg:ET(B)(+/+) mice. In conclusion, metabolic acidosis increases ET-1 expression, which increases NHE3 activity via the ET(B) receptor.

Renin expression in renal proximal tubule.

Angiotensinogen, angiotensin-converting enzyme, and renin constitute the components of the renin-angiotensin system. The mammalian renal proximal tubule contains angiotensinogen, angiotensin-converting enzyme, and angiotensin receptors. Previous immunohistochemical studies describing the presence of renin in the proximal tubule could not distinguish synthesized renin from renin trapped from the glomerular filtrate. In the present study, we examined the presence of renin activity and mRNA in rabbit proximal tubule cells in primary culture and renin mRNA in microdissected proximal tubules. Renin activity was present in lysates of proximal tubule cells in primary culture. Cellular renin content in cultured proximal tubule cells was increased by incubation with 10(-5) M isoproterenol and 10(-5) M forskolin by 150 and 110%, respectively. In addition, renin transcripts were detected in poly(A)+ RNA from cultured proximal tubule cells by RNA blots under high stringency conditions. In microdissected tubules from normal rats, renin mRNA was not detectable with reverse transcription and polymerase chain reaction. However, in tubules from rats administered the angiotensinogen-converting-enzyme inhibitor, enalapril, renin was easily detected in the S2 segment of the proximal tubule. We postulate the existence of a local renin-angiotensin system that enables the proximal tubule to generate angiotensin II, thereby providing an autocrine system that could locally modulate NaHCO3 and NaCl absorption.

Adenosine triphosphate citrate lyase mediates hypocitraturia in rats.

Chronic metabolic acidosis increases proximal tubular citrate uptake and metabolism. The present study addressed the effect of chronic metabolic acidosis on a cytosolic enzyme of citrate metabolism, ATP citrate lyase. Chronic metabolic acidosis caused hypocitraturia in rats and increased renal cortical ATP citrate lyase activity by 67% after 7 d. Renal cortical ATP citrate lyase protein abundance increased by 29% after 3 d and by 141% after 7 d of acid diet. No significant change in mRNA abundance could be detected. Hypokalemia, which causes only intracellular acidosis, caused hypocitraturia and increased renal cortical ATP citrate lyase activity by 28%. Conversely, the hypercitraturia of chronic alkali feeding was associated with no change in ATP citrate lyase activity. Inhibition of ATP citrate lyase with the competitive inhibitor, 4S-hydroxycitrate, significantly abated hypocitraturia and increased urinary citrate excretion fourfold in chronic metabolic acidosis and threefold in K+-depletion. In summary, the hypocitraturia of chronic metabolic acidosis is associated with an increase in ATP citrate lyase activity and protein abundance, and is partly reversed by inhibition of this enzyme. These results suggest an important role for ATP citrate lyase in proximal tubular citrate metabolism.

Chronic metabolic acidosis enhances NHE-3 protein abundance and transport activity in the rat thick ascending limb by increasing NHE-3 mRNA.

Chronic metabolic acidosis (CMA) is associated with an adaptive increase in the bicarbonate absorptive capacity of the rat medullary thick ascending limb (MTAL). To specify whether NHE-3, the apical MTAL Na/H exchanger, is involved in this adaptation, NHE-3 mRNA was quantified by a competitive RT-PCR using an internal standard which differed from the wild-type NHE-3 mRNA by an 80-bp deletion. CMA increased NHE-3 mRNA from 0.025+/-0.003 to 0.042+/-0.009 amol/ng total RNA (P < 0.005). NHE-3 transport activity was measured as the initial proton flux rate calculated from the Na-dependent cell pH recovery of Na-depleted acidified MTAL cells in the presence of 50 microM HOE694 which specifically blocks NHE-1, the basolateral MTAL NHE isoform. CMA caused a 68% increase in NHE-3 transport activity (P < 0.001). In addition, CMA was associated with a 71% increase in NHE-3 protein abundance (P < 0.05) as determined by Western blot analysis on MTAL membranes using a polyclonal antiserum directed against a cytoplasmic epitope of rat NHE-3. Thus, NHE-3 adapts to CMA in the rat MTAL via an increase in the mRNA transcript that enhances NHE-3 protein abundance and transport activity.

Glucocorticoids enhance acid activation of the Na+/H+ exchanger 3 (NHE3).

In the absence of exogenous glucocorticoids, decreasing media pH (from 7.4 to 6.8) for 24 hours increased the Na+/H+ exchanger 3 (NHE3) activity in opossum kidney (OKP) cells. 10(-7) M and 10(-8) M hydrocortisone increased NHE3 activity, and in their presence, acid incubation further increased NHE3 activity. Hydrocortisone (10(-9) M) had no effect on NHE3 activity, but in its presence, the effect of acid incubation on NHE3 activity increased twofold. Aldosterone (10(-8) M) had no effect. In the absence of hydrocortisone, acid incubation increased NHE3 protein abundance by 47%; in the presence of 10(-9) M hydrocortisone, acid incubation increased NHE3 protein abundance by 132%. The increase in NHE3 protein abundance was dependent on protein synthesis. However, 10(-9) M hydrocortisone did not modify the effect of acid incubation to cause a twofold increase in NHE3 mRNA abundance. In the absence of protein synthesis, 10(-9) M hydrocortisone did potentiate an effect of acid on NHE3 activity, which was due to trafficking of NHE3 to the apical membrane. These results suggest that glucocorticoids and acid interact synergistically at the level of NHE3 translation and trafficking.

Differential regulation of Na/H antiporter by acid in renal epithelial cells and fibroblasts.

Increased Na/H antiporter activity has been demonstrated after in vivo chronic metabolic acidosis as well as in vitro acid preincubation of cultured rabbit renal tubule cells. To study the underlying molecular mechanisms of this adaptive increase in Na/H antiporter activity, the present studies examined the effect of low pH media on Na/H antiporter activity and mRNA abundance in cultured renal tubule cells. Na/H antiporter activity was increased by 60% in a mouse renal cortical tubule cell line (MCT), and by 90% in an opossum kidney cell line (OKP) after 24 h of preincubation in acid (low [HCO3]) media. The ethylisopropylamiloride sensitivity of the Na/H antiporters were different in these two cell lines (MCT IC50 = 65 nM; OKP IC50 = 4.5 microM). In MCT cells, Na/H antiporter mRNA abundance measured by RNA blots increased by two- to fivefold after 24 h in low [HCO3] media. Na/H antiporter mRNA abundance was also increased in MCT cells with high CO2 preincubation as well as in rat renal cortex with in vivo chronic acid feeding. In contrast to renal epithelia, acid preincubation of NIH 3T3 fibroblasts led to suppression of Na/H antiporter activity. RNA blots of 3T3 fibroblasts revealed the same size Na/H antiporter transcript as in MCT cells. However, Na/H antiporter mRNA levels were suppressed by acid preincubation. These studies demonstrate differential regulation of Na/H antiporter activity and mRNA abundance in renal epithelial cells and fibroblasts in response to an acidotic environment.

Hypercalciuria from acid load: renal mechanisms.

Excessive acid load is considered a risk factor for both Nephrolithiasis and osteoporosis. The mechanisms by which acid confer the risk involves multiple organs, and is highly complex and incompletely understood. At least one component of the acid effect is due to renal leak of calcium. This article summarizes the effect of acid on renal handling of calcium in the proximal tubule, thick ascending limb, and distal convoluted tubule.

Renin regulation in cultured proximal tubular cells.

Recent studies have documented the presence of a complete renin-angiotensin system in the proximal tubule of the kidney: however, little is known about the regulation of renin in this proximal tubular system. Therefore, we performed the present studies to learn whether the behavior of the renin system in cultured proximal tubule is similar to that of the juxtaglomerular renin system. Basal renin secretion from rabbit proximal tubular cells in primary culture was low and not affected by isoproterenol (10(-5) mol/L), diltiazem (10(-5) mol/L), or a zero-calcium bath (O nmol/L). Only the calcium ionophore A23187 (10(-4) mol/L) significantly reduced renin secretion in these cells (from 2.44 +/- 0.37 to 1.14 +/- O.08 ng angiotensin I/mg protein per hour, P<.05). When the proximal tubular cells were lysed so the effects of the test agents on intracellular renin content could be assessed, isoproterenol caused a significant twofold (107 percent) increase (from 2.02 +/- 0.56 to 4.18 +/- 0.81 ng angiotensin I/mg protein per hour, P<.05), whereas diltiazem, A23187, and zero- and high-calcium baths did not produce a significant change. The effects of these agents on renin mRNA were examined in rabbit and rat proximal tubular cells in primary culture with the use of an S1 nuclease protection assay. Densitometry analysis of renin mRNA and either GAPDH mRNA (rat) or alpha-actin (rabbit) showed no significant alterations in renin mRNA abundance. In summary, these results confirm the presence of renin mRNA in cultured proximal tubular cells and suggest that a low-level, constitutive secretion of renin occurs in this system that is decreased by A23187. Moreover, the results also suggest that proximal tubular renin is regulated, albeit differently from the juxtaglomerular renin system. Finally, short-term increments in proximal tubular renin occur without a change in renin mRNA.

Factitious hyperkalemia.

Pseudohyperkalemia, or factitious hyperkalemia, constitutes an artificially high plasma potassium level (P(K)) from a variety of possible causes. Occasionally, the cause cannot be elucidated. Three patients who showed unusually large differences between free-flowing and tourniquet (stasis) potassium levels prompted us to investigate the influence of tourniquets in routine phlebotomy in eight healthy volunteers. P(K) showed a consistent but rather small average increase of 0.2 mEq/L (P < 0.001) during tourniquet use; however, the range was 10-fold, from 0.05 to 0.5 mEq/L in our subjects. We suggest there may be large variability leading to an excessive increase in P(K) in some individuals. In the three patients presented, average excessive increases in P(K) of 1.6, 1.3, and 1.7 mEq/L were seen. Although diagnosing and treating true hyperkalemia remains paramount, recognizing factitious hyperkalemia is important to preclude unnecessary investigations and potentially hazardous intervention.

The renal proximal tubule renin-angiotensin system.

In conclusion, a complete RAS is present in the mammalian proximal tubule that is potentially autocrine and paracrine in nature. Maneuvers that stimulate renin in JG cells and renal vasculature appear to also stimulate renin in the proximal tubule. The subcellular localization of the different components and the regulation of this epithelial RAS still remain to be defined. This RAS may be important in regulation of proximal tubule NaCl and NaHCO3 transport. Finally, one can speculate that activation of this RAS may play a pathogenetic role in some patients with essential hypertension and in the hypertension and cyst growth seen in autosomal dominant polycystic kidney disease.

Coordination of pancreatic HCO3- secretion by protein-protein interaction between membrane transporters.

Increasing evidence suggests that protein-protein interaction is essential in many biological processes including epithelial transport. In this report, we discuss the significance of protein interactions to HCO(3)(-) secretion in pancreatic duct cells. In pancreatic ducts HCO(3)(-) secretion is mediated by cystic fibrosis transmembrane conductance regulator (CFTR) activated luminal Cl(-)/HCO(3)(-) exchange activity and HCO(3)(-) absorption is achieved by Na(+)-dependent mechanisms including Na(+)/H(+) exchanger 3 (NHE3). We found biochemical and functional association between CFTR and NHE3. In addition, protein binding through PDZ modules is needed for this regulatory interaction. CFTR affected NHE3 activities in two ways. Acutely, CFTR augmented the cAMP-dependent inhibition of NHE3. In a chronic mechanism, CFTR increases the luminal expression of Na(+)/H(+) exchange in pancreatic duct cells. These findings reveal that protein complexes in the plasma membrane of pancreatic duct cells are highly organized for efficient HCO(3)(-) secretion.

NaPi-IIa and interacting partners.

Regulation of renal proximal tubular reabsorption of phosphate (Pi) is one of the critical steps in Pi homeostasis. Experimental evidence suggests that this regulation is achieved mainly by controlling the apical expression of the Na+-dependent Pi cotransporter type IIa (NaPi-IIa) in proximal tubules. Only recently have we started to obtain information regarding the molecular mechanisms that control the apical expression of NaPi-IIa. The first critical observation was the finding that truncation of only its last three amino acid residues has a strong effect on apical expression. A second major finding was the observation that the last intracellular loop of NaPi-IIa contains sequence information that confers parathyroid hormone (PTH) sensitivity. The use of the above domains of the cotransporter in yeast two-hybrid (Y2H) screening allowed the identification of proteins interacting with NaPi-IIa. Biochemical and morphological, as well as functional, analyses have allowed us to obtain insights into the physiological roles of such interactions, although our present knowledge is still far from complete.

Sodium-hydrogen exchange in renal epithelia: mechanisms of acute regulation.

Five isoforms of sodium-hydrogen exchangers are present in the kidney. Specific isoforms serve specialized functions and are strategically located in polarized distributions in different nephron segments. This review updates understanding of the renal distribution and highlights some of the recent advances in understanding the mechanisms of acute regulation of sodium-hydrogen exchangers in the kidney.

Abnormal regulation of proximal tubule renin mRNA in the Dahl/Rapp salt-sensitive rat.

BACKGROUND: The precise pathogenesis of salt-sensitive hypertension in the Dahl rat is unknown. Abnormalities in renal hemodynamics and NaCl handling have been implicated, and may relate to changes in the activity of the intrarenal renin-angiotensin system. METHODS: Circulating, juxtaglomerular and intrarenal (glomerular and proximal tubular) renin were studied in Dahl/Rapp salt-sensitive and salt-resistant rats fed with a normal (0.5%) or high (4%) NaCl diet. Circulating and juxtaglomerular renin were assessed by measurement of plasma renin activity and renin secretory rates. Glomerular and proximal tubular renin mRNA were assessed by microdissection and quantitative competitive RT-PCR. RESULTS: Circulating and juxtaglomerular renin were suppressed by high dietary NaCl in salt-sensitive rats (plasma renin activity, 0.5%, 10.9 +/- 0.7 vs. 4%, 7.9 +/- 0.3 ng/ml/hr, P < 0.05; renin secretory rate, 0.5% 220 +/- 32 vs. 4%, 58 +/- 5 ng/mg/hr, P < 0.05). Glomerular renin mRNA was also suppressed by the higher salt diet in salt-sensitive animals (0.5%, 411 +/- 84 vs. 4%, 67 +/- 22 x 103 copies/glomerulus, P < 0.05). In contrast, proximal tubular renin was not suppressed by a high NaCl diet in salt-sensitive animals (0.5%, 13.9 +/- 2.7 vs. 4%, 12.1 +/- 3.6 x 103 copies/mm tubule, P = NS), but was suppressed in salt-resistant rats (0.5%, 9.5 +/- 2.8 vs. 4%, 3.2 +/- 1.2 x 103 copies/mm, P < 0. 05). CONCLUSIONS: Failure to suppress proximal tubular renin in response to high dietary NaCl may result in increased local generation of angiotensin II and enhanced proximal tubular NaCl absorption, and thereby contribute to the generation of salt sensitive hypertension.

Acute regulation of Na/H exchanger NHE3 activity by protein kinase C: role of NHE3 phosphorylation.

Acute hormonal modulation of NHE3 activity is partly mediated by kinases, including protein kinase C (PKC). We examined the role of NHE3 phosphorylation in regulating its activity in response to PKC activation by phorbol 12-myristate 13-acetate (PMA). In pooled NHE-deficient fibroblasts transfected with NHE3, PMA increased NHE3 activity and phosphorylation. When six potential PKC target serines were mutated, NHE3 phosphorylation was drastically reduced and PMA failed to regulate NHE3 phosphorylation or function. To examine whether NHE3 phosphorylation is sufficient for functional regulation by PKC, we exploited the heterogeneous response of NHE3 activity to PMA in individual clones of transfectants. Clones with stimulatory, inhibitory, or null responses to PMA were observed. Despite the diverse functional response, changes in NHE3 phosphorylation as revealed by tryptic phosphopeptide maps were similar in all clones. We conclude that although phosphorylation appears to be necessary, it is insufficient to mediate PKC regulation of NHE3 function and factors extrinsic to the NHE3 protein must be involved.

Thyroid hormone stimulates the renal Na/H exchanger NHE3 by transcriptional activation.

Thyroid hormone stimulates renal proximal tubule NaCl and NaHCO3 absorption in part by activating the apical membrane Na/H exchanger NHE3. We used a renal epithelial cell line, the opossum kidney (OK) cell, to define the mechanism by which 3,5,3'-triiodothyronine (T3) increases NHE3 activity. T3 stimulated NHE3 activity, an effect that was blocked by inhibition of cellular transcription or translation. The increase in activity was associated with increases in steady-state cell surface and total cellular NHE3 protein and NHE3 transcript abundance. T3 stimulated transcription of the NHE3 gene and had no effect on NHE3 transcript stability. The transcriptional activity of the 5'-flanking region of the rat NHE3 gene was stimulated by T3 when expressed in OK cells. When heterologously expressed rat NHE3 transcript levels were clamped constant with a constitutive promoter in OK cells, T3 has no effect on rat NHE3 protein abundance, suggesting the absence of regulation of NHE3 protein stability or translation. These studies demonstrate that T3 stimulates NHE3 activity by activating NHE3 gene transcription and increasing NHE3 transcript and protein abundance.