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Background and objectives Metabolic acidosis is common in patients with CKD and has significant adverse effects on kidney, muscle, and bone. We tested the efficacy and safety of TRC101, a novel, sodium-free, nonabsorbed hydrochloric acid binder, to increase serum bicarbonate in patients with CKD and metabolic acidosis. Design, setting, participants, & measurements One hundred thirty-five patients were enrolled in this randomized, double-blind, placebo-controlled, multicenter, in-unit study (designated the TRCA-101 Study). Patients had a mean baseline eGFR of 35 ml/min per 1.73 m 2 , a mean baseline serum bicarbonate of 17.7 mEq/L, and comorbidities, including hypertension (93%), diabetes (70%), and heart failure (21%). Patients ate a controlled diet and were treated for 14 days with placebo or one of four TRC101 dosing regimens (1.5, 3, or 4.5 g twice daily or 6 g once daily). After treatment, patients were discharged and followed for 7–14 days. Results All TRC101 treatment groups had a mean within-group increase in serum bicarbonate of ≥1.3 mEq/L ( P P Conclusions TRC101 safely and significantly increased the level of serum bicarbonate in patients with metabolic acidosis and CKD.

[][1] On April 25, 2018, Dr. Donald Seldin passed away at the age of 97, and the world lost a great treasure. Born in Coney Island, Brooklyn, Seldin attended James Madison High School and established his intellectual roots early on. He then attended New York University, where he was

Background Urine citrate is reabsorbed exclusively along the renal proximal tubule via the apical Na+-dicarboxylate cotransporter NaDC-1. We previously showed that an acid load in vivo and media acidification in vitro increase NaDC-1 activity through endothelin-1 (ET-1)/endothelin B (ETB) signaling. Here, we further examined the signaling pathway mediating acid-induced NaDC-1 activity.Methods We transiently transfected cultured opossum kidney cells, a model of the proximal tubule, with NaDC-1 and ETB and measured [14C]-citrate uptake after media acidification under various experimental conditions, including inactivation of Pyk2 and c-Src, which were previously shown to be activated by media acidification. Wild-type (Pyk2+/+) and Pyk2-null (Pyk2-/-) mice were exposed to NH4Cl loading and euthanized after various end points, at which time we harvested the kidneys for immunoblotting and brush border membrane NaDC-1 activity studies.Results Inhibition of Pyk2 or c-Src prevented acid stimulation but not ET-1 stimulation of NaDC-1 in vitro Consistent with these results, NH4Cl loading stimulated NaDC-1 activity in kidneys of wild-type but not Pyk2-/- mice. In cultured cells and in mice, ERK1/2 was rapidly phosphorylated by acid loading, even after Pyk2 knockdown, and it was required for acid but not ET-1/ETB stimulation of NaDC-1 in vitro Media acidification also induced the phosphorylation of Raf1 and p90RSK, components of the ERK1/2 pathway, and inhibition of these proteins blocked acid stimulation of NaDC-1 activity.Conclusions Acid stimulation of NaDC-1 activity involves Pyk2/c-Src and Raf1-ERK1/2-p90RSK signaling pathways, but these pathways are not downstream of ET-1/ETB in this process. Background Urine citrate is reabsorbed exclusively along the renal proximal tubule via the apical Na+-dicarboxylate cotransporter NaDC-1. We previously showed that an acid load in vivo and media acidification in vitro increase NaDC-1 activity through endothelin-1 (ET-1)/endothelin B (ETB) signaling. Here, we further examined the signaling pathway mediating acid-induced NaDC-1 activity. Methods We transiently transfected cultured opossum kidney cells, a model of the proximal tubule, with NaDC-1 and ETB and measured [14C]-citrate uptake after media acidification under various experimental conditions, including inactivation of Pyk2 and c-Src, which were previously shown to be activated by media acidification. Wild-type (Pyk2+/+) and Pyk2-null (Pyk22/2) mice were exposed to NH4Cl loading and euthanized after various end points, at which time we harvested the kidneys for immunoblotting and brush border membrane NaDC-1 activity studies. Results Inhibition of Pyk2 or c-Src prevented acid stimulation but not ET-1 stimulation of NaDC-1 in vitro. Consistent with these results, NH4Cl loading stimulated NaDC-1 activity in kidneys of wild-type but not Pyk22/2 mice. In cultured cells and in mice, ERK1/2 was rapidly phosphorylated by acid loading, even after Pyk2 knockdown, and it was required for acid but not ET-1/ETB stimulation of NaDC-1 in vitro. Media acidification also induced the phosphorylation of Raf1 and p90RSK, components of the ERK1/2 pathway, and inhibition of these proteins blocked acid stimulation of NaDC-1 activity. Conclusions Acid stimulation of NaDC-1 activity involves Pyk2/c-Src and Raf1-ERK1/2-p90RSK signaling pathways, but these pathways are not downstream of ET-1/ETB in this process.

The classic definition of hypercalciuria, an upper normal limit of 200 mg/day, is based on a constant diet restricted in calcium, sodium, and animal protein; however, random diet data challenge this. Here our retrospective study determined the validity of the classic definition of hypercalciuria by comparing data from 39 publications analyzing urinary calcium excretion on a constant restricted diet and testing whether hypercalciuria could be defined when extraneous dietary influences were controlled. These papers encompassed 300 non-stone-forming patients, 208 patients with absorptive hypercalciuria type I (presumed due to high intestinal calcium absorption), and 234 stone formers without absorptive hypercalciuria; all evaluated on a constant restricted diet. In non-stone formers, the mean urinary calcium was well below 200 mg/day, and the mean for all patients was 127±46 mg/day with an upper limit of 219 mg/day. In absorptive hypercalciuria type I, the mean urinary calcium significantly exceeded 200 mg/day in all studies with a combined mean of 259±55 mg/day. Receiver operating characteristic curve analysis showed the optimal cutoff point for urinary calcium excretion was 172 mg/day on a restricted diet, a value that approximates the traditional limit of 200 mg/day. Thus, on a restricted diet, a clear demarcation was seen between urinary calcium excretion of kidney stone formers with absorptive hypercalciuria type I and normal individuals. When dietary variables are controlled, the classic definition of hypercalciuria of nephrolithiasis appears valid.

Exposure to an acid load increases apical membrane Na+/H+ antiporter (NHE3) activity, a process that involves exocytic trafficking of the transporter to the apical membrane. We have previously shown that an intact microfilament structure is required for this exocytic process (Yang X, Amemiya M, Peng Y, Moe OW, Preisig PA, Alpern RJ. Am J Physiol Cell Physiol 279: C410–C419, 2000). The present studies demonstrate that acid-induced stress fiber formation is required for stimulation of NHE3 activity. Formation of stress fibers is associated with acid-induced tyrosine phosphorylation and increases in protein abundance of two focal adhesion proteins, p125FAK and paxillin. The Rho kinase inhibitor Y27632 completely blocks acid-induced stress fiber formation and the increases in apical membrane NHE3 abundance and activity, but it has no effect on acid-induced tyrosine phosphorylation of p125FAK or paxillin. Herbimycin A completely blocks acid-induced tyrosine phosphorylation of p125FAK and paxillin but only partially blocks stress fiber formation and NHE3 activation. These studies demonstrate that Rho kinase mediates acid-induced stress fiber formation, which is required for NHE3 exocytosis, and increases in NHE3 activity. Acid-induced tyrosine phosphorylation of the focal adhesion proteins p125FAK and paxillin is not Rho kinase dependent. Thus these two acid-mediated effects are associated, yet independent processes.

Reductions in federal support and clinical revenue jeopardize biomedical research and, in turn, clinical medicine.

Endothelin-1 (ET-1) increases the activity of Na+/H+exchanger 3 (NHE3), the major proximal tubule apical membrane Na+/H+antiporter. This effect is seen in opossum kidney (OKP) cells expressing the endothelin-B (ETB) and not in cells expressing the endothelin-A (ETA) receptor. However, ET-1 causes similar patterns of protein tyrosine phosphorylation, adenylyl cyclase inhibition, and increases in cell [Ca2+] in ETA- and ETB-expressing OKP cells, implying that an additional mechanism is required for NHE3 stimulation by the ETBreceptor. The present studies used ETAand ETBreceptor chimeras and site-directed mutagenesis to identify the ET receptor domains that mediate ET-1 regulation of NHE3 activity. We found that binding of ET-1 to the ETAreceptor inhibits NHE3 activity, an effect for which the COOH-terminal tail is necessary and sufficient. ET-1 stimulation of NHE3 activity requires the COOH-terminal tail and the second intracellular loop of the ETBreceptor. Within the second intracellular loop, a consensus sequence was identified, KXXXVPKXXXV, that is required for ET-1 stimulation of NHE3 activity. This sequence suggests binding of a homodimeric protein that mediates NHE3 stimulation.

An autocrine role for endothelin-1 in the regulation of proximal tubule NHE3. Background Chronic metabolic acidosis leads to an increase in NHE3 activity that is mediated by endothelin-1 (ET-1) expression and activation of the proximal tubule endothelin B receptor. Chronic metabolic acidosis increases preproET-1 mRNA abundance in kidney cortex, but the cell responsible has not been identified. Methods PreproET-1 mRNA abundance was quantified by competitive reverse transcription-polymerase chain reaction (RT-PCR) on tissue harvested from control rats or rats in which chronic metabolic acidosis was induced by addition of NH 4 Cl to the drinking water. Results Chronic metabolic acidosis leads to an increase in preproET-1 mRNA expression in kidney cortex, proximal tubules, and glomeruli. The increase in preproET-1 expression correlates with the decrease in blood [HCO 3 − ]. ET-1 expression is also increased by acidosis in abdominal aorta, but not in cardiac muscle. Conclusion In the renal proximal tubule, chronic metabolic acidosis induces an increase in preproET-1 expression, providing a mechanism for autocrine regulation of proximal tubule NHE3 activity. This response is not unique to the proximal tubule cell, but is also not ubiquitous.

Role of c-Src and ERK in acid-induced activation of NHE3. Background In the renal proximal tubule, chronic acidosis causes increases in apical membrane NHE3 activity, which serve to increase transepithelial H + secretion and return systemic pH to normal levels. Incubation of cultured renal epithelial cells in acid media activates c-Src. Methods OKP cells were incubated in control (pH 7.4) or acid (7.0) media, and NHE3 activity measured as cytoplasmic pH (pHi) recovery from an acid load using BCECF. c-Src, ERK, and JNK kinase activities were measured by immune complex kinase assays with enolase, MBP, and GST-c-Jun, respectively, as substrates in the in vitro assays. To determine the role of c-Src in acid-induced NHE3 activation, cells were transfected with vector alone or a dominant negative c-Src (c-Src K295M ). Results Expression of dominant negative c-src K295M in OKP cells prevented acid-induced activation of NHE3. Incubation of OKP cells in acid media increased ERK activity and c-fos expression, but did not increase JNK activity. Acidosis in vivo also activated renal cortical c-Src and ERK kinases, whereas incubation of 3T3 cells in acid media activated c-Src but not ERK kinase. Expression of c-src K295M did not affect ERK or c-fos activation by acid incubation. Inhibition of MEK with PD98059 inhibited activation of NHE3 by acid incubation. Conclusions These studies suggest that acidosis activates c-Src and MEK/ERK/ c-fos . While both pathways are necessary for activation of NHE3, they are activated independently.

Nephrotic syndrome is a condition commonly associated with end-stage renal disease secondary to diabetic nephropathy. It is usually associated with long-standing renal insufficiency, microalbuminuria, and overt proteinuria. We present a diabetic patient with acute oliguric renal failure and nephrotic syndrome. At presentation, he had a serum creatinine of 2.3 mg/dl, blood urea nitrogen (BUN) of 69 mg/dl, urinary protein excretion of 10.5 g/24 h, serum albumin of 1.3 g/dl, and a urine output

This year the renal community, the United States, and the world endured two tragedies. The first of these, the events of September 11, will be remembered for many years to come. The magnitude of the devastation and pain inflicted on so many innocent people is difficult for us to grasp. Our thoughts

Background: We previously found that the Na+/H+ exchanger 3 (NHE3) is localized in the apical membrane of the rat renal proximal tubule and thick ascending limb of Henle. In the present study, we examined the direct effect of glucagon on the opossum kidney P (OKP) cell Na+/H+ antiporter, encoded by NHE3. Methods: Na+/H+ antiporter activity was measured as the rate of cell pH recovery from an acid load using 2′,7′-bis(carboxyethyl)-5(6)-carboxyfluorescein. Northern blot and Western blot analyses were performed using OKP NHE3 cDNA and anti-OKP-NHE3 antibodies. Results: Glucagon (1 ng/ml) acutely (1 h) inhibited, but chronically (24 h) activated NHE3 activity in OKP cells. These effects were blocked by either KT5720 or RpcAMP [protein kinase A (PKA) inhibitors], and mimicked by 10–4M dibutyryl-cAMP. Both NHE3 mRNA and protein abundance increased with the 24-hour incubation in glucagon or dibutyryl-cAMP. Cycloheximide did not prevent a significant increase in NHE3 activity at 24 h. We therefore examined NHE3 protein abundance in the surface membrane by the biotinylation mehtod. cAMP or glucagon significantly increased NHE3 protein abundance in the surface membrane when incubated with cycloheximide for 24 h. Conclusions: Glucagon acutely inhibits but chronically activates NHE3 activity in OKP cells via a PKA-dependent pathway. Both protein-synthesis-dependent and -independent mechanisms play important roles in the chronic activation of NHE3.

The sections in this article are: 1 Components of Renal Acid-Base Regulation 1.1 Renal Acid Excretion 1.2 Glomerular Filtration 1.3 Urinary Buffers 1.4 Tubular H+/HCO3− Transport 2 Regulation by Peptide Hormones and Catecholamines 2.1 Signaling Intermediates 2.2 Angiotensin II 2.3 Adrenergic Catecholamines 2.4 Endothelin 2.5 Parathyroid Hormone 2.6 Dopamine 2.7 Atrial Natriuretic Factor 2.8 Insulin 3 Regulation by Steroid Hormones 3.1 Mineralocorticoids 3.2 Glucocorticoids 4 Regulation by Thyroid Hormone

Incubation of opossum kidney clone P (OKP) cells in acid media (pH 6.8) causes activation of Na+/H+ exchanger 3 (NHE3) at 6, 12, and 24 h. OKP cell NHE3 protein abundance was increased by 45% at 24 h of acid incubation but was unaffected at 3–12 h. By contrast, apical membrane NHE3, measured by surface biotinylation, increased approximately twofold at 6, 12, and 24 h, mirroring the increase in activity. Acid incubation caused a 76% increase in exocytic insertion of NHE3 into the apical membrane but had no effect on endocytic internalization at 6 h. Latrunculin B, an inhibitor of microfilament organization, inhibited the acid-induced increases in apical membrane NHE3, exocytic insertion of NHE3, and NHE3 activity at 6 h. These studies demonstrate two mechanisms for acid-induced increases in NHE3 activity. Beginning at 6 h, there is an increase in apical membrane NHE3 that is due to stimulated exocytic insertion and is required for increased NHE3 activity. At 24 h, there is an additional increase in total cellular NHE3.

In OKP cells expressing ETBendothelin receptors, activation of Na+/H+antiporter activity by endothelin-1 (ET-1) was resistant to low concentrations of ethylisopropyl amiloride, indicating regulation of Na+/H+exchanger isoform 3 (NHE3). ET-1 increased NHE3 phosphorylation in cells expressing ETBreceptors but not in cells expressing ETAreceptors. Receptor specificity was not due to demonstrable differences in receptor-specific activation of tyrosine phosphorylation pathways or inhibition of adenylyl cyclase. Phosphorylation was associated with a decrease in mobility on SDS-PAGE, which was reversed by treating immunoprecipitated NHE3 with alkaline phosphatase. Phosphorylation was first seen at 5 min and was maximal at 15–30 min. Phosphorylation was maximal with 10−9M ET-1. Phosphorylation occurred on threonine and serine residues at multiple sites. In summary, ET-1 induces NHE3 phosphorylation in OKP cells on multiple threonine and serine residues. ETBreceptor specificity, time course, and concentration dependence are all similar between ET-1-induced increases in NHE3 activity and phosphorylation, suggesting that phosphorylation plays a key role in activation.

Chronic hypokalemia increases the activity of proximal tubule apical membrane Na+/H+antiporter NHE3. The present study examined the effect of the incubation of OKP cells (an opossum kidney, clone P cell line) in control medium {K+concn ([K+]) = 5.4 mM} or low-K+medium ([K+] = 2.7 mM) on NHE3. The activity of an ethylisopropyl amiloride-resistant Na+/H+antiporter, whose characteristics were consistent with those of NHE3, was increased in low-K+cells beginning at 8 h. NHE3 mRNA and NHE3 protein abundance were increased 2.2-fold and 62%, respectively, at 24 h but not at 8 h. After incubation in low-K+medium, intracellular pH (pHi) decreased by 0.27 pH units (maximum at 27 min) and then recovered to the control level. Intracellular acidosis induced by 5 mM sodium propionate increased Na+/H+antiporter activity at 8 and 24 h. Herbimycin A, a tyrosine kinase inhibitor, blocked low-K+- and sodium propionate-induced activation of the Na+/H+antiporter at 8 and 24 h. Our results demonstrate that low-K+medium causes an early decrease in pHi, which leads to an increase in NHE3 activity via a tyrosine kinase pathway.

Dominant negative c-Src inhibits angiotensin II induced activation of NHE3 in OKP cells. Background Angiotensin II is a potent stimulator of the proximal tubule apical membrane Na/H antiporter, encoded by NHE3. The nonreceptor tyrosine kinase, c-Src, plays a key role in regulation of NHE3 by acidosis in the proximal tubule, and in signaling effects of angiotensin II in vascular smooth muscle. Methods The present studies examined the role of c-Src in mediating angiotensin II-induced NHE3 activation in cultured OKP cells. c-Src was inhibited with herbimycin A, a tyrosine kinase inhibitor, and expression of a dominant negative c-Src, c-Src K295M . Results Herbimycin A blocked angiotensin II induced increases in Na/H antiporter activity. In two clonal cell lines expressing vector alone, angiotensin II increased Na/H antiporter activity, while in three clones expressing c-Src K295M , angiotensin II had no effect. Cyclic AMP and protein kinase A have been proposed to be key mediators in regulation of NHE3 by angiotensin II. 10 -4 m 8-bromo cAMP induced a 40 to 50% inhibition of Na/H antiporter activity in cells expressing c-Src K295M , similar to that seen in wild-type OKP cells. In addition, cells expressing c-Src K295M responded normally to 10 -7 m dexamethasone with a 50 to 80% increase in Na/H antiporter activity. Conclusions These studies demonstrate that c-Src is required for angiotensin II-induced increases in NHE3 activity. Thus, c-Src plays a key role in antiporter activation by acidosis and angiotensin II.

Renal cortical mitochondrial aconitase is regulated in hypo- and hypercitraturia. Background Chronic metabolic acidosis and K + deficiency increase, while alkali feeding decreases proximal tubule citrate absorption and metabolism. The present studies examined the regulation of mitochondrial aconitase (m-aconitase), the first step in mitochondrial citrate metabolism, in these conditions. Methods Rats were fed appropriate diets, and m-aconitase activity and protein abundance measured. Results In chronic metabolic acidosis and chronic K + deficiency, renal cortical m-aconitase activity was increased 17% and 43%, respectively. This was associated with respective 90% and 221% increases in renal cortical m-aconitase protein abundance. With chronic alkali feeding, there was a 12% decrease in renal cortical m-aconitase activity, associated with a 35% decrease in m-aconitase protein abundance. Hepatic m-aconitase activity was not regulated in a similar manner. There was no regulation of citrate synthase, the enzyme responsible for mitochondrial citrate synthesis. Conclusions These studies demonstrate tissue specific chronic regulation of renal cortical m-aconitase activity and protein abundance, which likely contributes to the hypocitraturia and hypercitraturia seen in these conditions. As m-aconitase is the only step in citrate transport and metabolism found to be regulated in alkali feeding, its regulation likely plays a significant role in mediating the hypercitraturia seen in this condition.

Extracellular fluid (ECF) volume is determined by the balance between sodium intake and renal excretion of sodium. Under normal circumstances, wide variations in salt intake lead to parallel changes in renal salt excretion, such that ECF volume is maintained within narrow limits. This relative constancy of ECF volume is achieved by a series of afferent sensing systems, central integrative pathways, and renal and extrarenal effector mechanisms acting in concert to modulate sodium excretion by the kidney.

Chronic metabolic acidosis increases the activity of the proximal tubule apical membrane Na/H antiporter, which is encoded predominantly by the NHE3 isoform. The present studies examined the effect of chronic metabolic acidosis on apical membrane NHE3 protein abundance in rats. Rats subjected to NH4Cl in their drinking water developed a metabolic acidosis, which decreased in magnitude over 14 days. During this time, renal cortical brush-border membrane NHE3 protein abundance, assessed by Western blot, increased progressively (28% at 3 days, 59% at 7 days, and 90% at 14 days). Immunohistochemistry revealed that the acidosis-induced increase in NHE3 abundance occurred in the apical membranes of the S1 and S2 segments of the proximal tubule and the thick ascending limb. NHE3 mRNA abundance was not significantly increased in these animals, whereas phosphoenolpyruvate carboxykinase and glyceraldehyde-3-phosphate dehydrogenase mRNA abundances were significantly increased. These studies demonstrate that the increase in Na/H antiporter activity seen in metabolic acidosis involves an increase in NHE3 protein abundance, which is distributed along the proximal tubule and the thick ascending limb. In addition, these studies suggest that a component of this adaptation is unrelated to changes in NHE3 mRNA abundance.

Endothelin-1 (ET-1) binding to ETB receptors increases the activity of the apical membrane Na+/H+ antiporter (NHE3) of renal proximal tubule and cultured OKP cells. In OKPETB6 cells, a clonal cell line of OKP cells that overexpresses ETB receptors, ET-1-induced increases in Na+/H+ antiporter activity are mediated 50% by Ca2(+)-dependent pathways and 50% by tyrosine kinase pathways. ET-1 induces tyrosine phosphorylation of proteins of 68, 110, 125, 130, and 210 kDa. ET-1-induced tyrosine phosphorylation is mediated by the ETB receptor and is not dependent on increases in cell Ca2+ or protein kinase C. The 68-, 110-, 125-, and 130-kDa phosphoproteins are cytosolic, whereas the 210-kDa phosphoprotein is an integral membrane protein. Immunoprecipitation studies showed that the 68-kDa protein is paxillin and the 125-kDa protein is p125FAK (focal adhesion kinase). Cytochalasin D, which disrupts focal adhesions, prevented ET-1-induced tyrosine phosphorylation of paxillin, p110, p125FAK, and p130 but did not prevent tyrosine phosphorylation of p210 and did not prevent ET-1-induced increases in Na+/H+ antiporter activity. Thus 50% of ETB receptor-induced Na+/H+ antiporter activation is mediated by tyrosine kinase pathways, possibly involving p210. ETB receptor activation also induces tyrosine phosphorylation of focal adhesion proteins, but this is not required for antiporter activation.

OKP cells express NHE-3, an amiloride-resistant Na+/H+ antiporter, which is likely an isoform responsible for apical proton secretion by the proximal tubule. We have previously shown that an amiloride-resistant Na+/H+ antiporter in OKP cells is regulated by dexamethasone, a synthetic glucocorticoid. The purpose of the present study was to examine the mechanism for the glucocorticoid-mediated increase in Na+/H+ antiporter activity. Incubation of OKP cells with 10(-6) M dexamethasone resulted in a two- to threefold increase in NHE-3 mRNA abundance. This increase was seen after 4 h of incubation with dexamethasone, a time course similar to that found for Na+/H+ antiporter activity. To examine the mechanism for the increase in NHE-3 mRNA abundance, mRNA half-life and in vitro transcription experiments were performed. NHE-3 mRNA had a half-life of 8 h in control and dexamethasone-treated cells. The rate of in vitro transcription was 1.8-fold greater when OKP cells were treated with dexamethasone. These data suggest that the glucocorticoid-mediated increase in Na+/H+ antiporter activity is due to an increase in NHE-3 gene transcription.

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 ˜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 ˜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 NaHCO3 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.

With the use of degenerate primers based on conserved amino acid sequences in human, rat, and rabbit Na/H exchanger-3 (NHE-3), a polymerase chain reaction product was obtained from reverse-transcribed OKP (a clonal opossum kidney cell line) mRNA and used to screen an OKP cDNA library. The clone obtained predicted an amino acid sequence that was 86% identical to rat NHE-3, 33% to NHE-1, 35% to NHE-2, and 30% to NHE-4. Expression of the corresponding cRNA in Xenopus oocytes induced 22Na uptake with ethylisopropylamiloride. (EIPA) resistance similar to that of the OKP Na/H antiporter. On RNA blot, the cDNA labeled a 9.5-kb transcript whose abundance was increased 2.2-fold by 24-h incubation of OKP cells at pH 7.0 and 2.5-fold by 24-h incubation at pH 6.8. The acid-induced increase in NHE-3 mRNA was detectable at 12 h and increased further at 24 h. Incubation in acid media caused an increase in EIPA-resistant Na/H antiporter activity that preceded the increase in NHE-3 mRNA. In summary, OKP cells express an NHE-3 transcript that encodes an EIPA-resistant Na/H antiporter and is chronically regulated by acid.

Although renal hypertrophy is often associated with the progressive loss of renal function, the mechanism of hypertrophy is poorly understood. In both primary cultures of rabbit proximal tubules and NRK-52E cells (a renal epithelial cell line), transforming growth factor beta 1 (TGF beta) converted epidermal growth factor (EGF)-induced hyperplasia into hypertrophy. TGF beta did not affect EGF-induced increases in c-fos mRNA abundance or cyclin E protein abundance, but inhibited EGF-induced entry into S, G2, and M phases. EGF alone increased the amount of hyperphosphorylated (inactive) pRB; TGF beta blocked EGF-induced pRB phosphorylation, maintaining pRB in the active form. To determine the importance of active pRB in TGF beta-induced hypertrophy, NRK-52E cells were infected with SV40 large T antigen (which inactivates pRB and related proteins and p53), HPV16 E6 (which degrades p53), HPV16 E7 (which binds and inactivates pRB and related proteins), or both HPV16 E6 and E7. In SV40 large T antigen expressing clones, the magnitude of EGF + TGF beta-induced hypertrophy was inhibited and was inversely related to the magnitude of SV40 large T antigen expression. In the HPV16-infected cells, EGF + TGF beta-induced hypertrophy was inhibited in E7- and E6E7-expressing, but not E6-expressing cells. These results suggest a requirement for active pRB in the development of EGF + TGF beta-induced renal epithelial cell hypertrophy. We suggest a model of renal cell hypertrophy mediated by EGF-induced entry into the cell cycle with TGF beta-induced blockade at G1/S, the latter due to maintained activity of pRB or a related protein.

Na+/H+ antiporter (NHE-1 isoform) in cultured vascular smooth muscle from the spontaneously hypertensive rat. An increase in Na+/H+ antiporter activity may be involved in hyperproliferation of vascular smooth muscle cells (VSMC) and possibly in the vascular hyperplasia characteristic of hypertension. The present study was designed to examine cell proliferation, Na+/H+ exchange activity, and mRNA levels of the NHE-1 isoform of the Na+/H+ antiporter in cultured aortic VSMC derived from the spontaneously hypertensive rat (SHR) and from normotensive controls, the Wistar/Kyoto rat (WKY). VSMC derived from the SHR grown in early (2 to 6), but not in later (7 to 10) sub-passages, exhibited an increase in [3H]-thymidine incorporation and shorter doubling times as compared to those derived from WKY rats. Na+/H+ exchange activity assayed in the nominal absence of HCO3-/CO2, as the rate of intracellular pH (pHi) recovery after cell acidification was significantly higher in cells from SHR than in those from WKY rats when cells were studied in early sub-passages, but not in cells studied in later sub-passages. In cells grown in early sub-passage, Na+/H+ exchange activity assessed as the initial rate of Na+i accumulation following acute cell acidification was also significantly higher in SHR than WKY cells both in the nominal absence (10.22 ± 1.15 and 6.98 ± 1.17 mmol Na+i/90 seconds, P < 0.05, respectively) and in the presence of HCO3-/CO2 (9.94 ± 1.02 and 5.59 ± 0.86 mmol Na+/90 seconds, P < 0.01, respectively). There were no detectable differences in the level of steady-state Na+/H+ antiporter (NHE-1) mRNA between VSMC from SHR and WKY rats. Our findings indicate that Na+/H+ exchange activity is increased in cultured aortic VSMC derived from SHR as compared to those derived from WKY rats. The higher functional activity of the Na+/H+ antiporter in VSMC from the SHR is due to a post-transcriptional event(s) and may be related to enhanced growth in culture.

Glucocorticoids play an important role in modulating proximal tubule acidification. Chronic systemic administration of dexamethasone increases the rate of bicarbonate absorption in isolated perfused proximal convoluted tubules and Na+/H+ antiporter activity in renal brush-border membrane vesicles. The proximal tubule expresses mRNA corresponding to two known Na+/H+ antiporter isoforms: NHE-3, an amiloride-resistant apical membrane Na+/H+ antiporter; and NHE-1, an amiloride-sensitive Na+/H+ antiporter found on most mammalian cells. Administration of dexamethasone for 1 and 2 days (600 micrograms/kg twice daily and 2 h before animals were killed) increased NHE-3 mRNA abundance 1.3- and 2.5-fold, respectively, but had no effect on NHE-1 mRNA abundance. Aminoglutethimide-induced glucocorticoid deficiency had no effect on NHE-1 or NHE-3 mRNA abundance. Incubation of proximal tubules for 3 h with 10(-5) M dexamethasone increased proximal tubule Na+/H+ antiporter activity from 0.69 +/- 0.04 to 0.92 +/- 0.03 pH units/min (P < 0.01); however, there was no increase in NHE-3 or NHE-1 mRNA abundance. Similarly, there was no effect on NHE-3 or NHE-1 mRNA abundance in rabbit renal cortex 4 h after intravenous administration of 600 micrograms/kg dexamethasone. Thus chronic dexamethasone increases NHE-3 but not NHE-1 mRNA abundance. The acute increase in Na+/H+ antiporter activity induced by dexamethasone occurs by mechanisms independent of changes in NHE-1 and NHE-3 mRNA abundance.

Metabolic alkalosis, isolated or in combination with another abnormality, is the most common acid-base disorder in patients with congestive heart failure. In most cases, it is a result of diuretic therapy, which causes activation of the renin-angiotensin system, chloride depletion, increased distal sodium delivery, hypokalemia, and increased urine acidification, all of which contribute to bicarbonate retention. In addition, the disease state itself results in neurohormonal activation (renin-angiotensin system, sympathetic nervous system, and endothelin) that further amplifies the tendency toward alkalosis. Treatment of metabolic alkalosis is based on the elimination of generation and maintenance factors, chloride and potassium repletion, enhancement of renal bicarbonate excretion (such as acetazolamide), direct titration of the base excess (hydrochloric acid), or, if accompanied by kidney failure, low-bicarbonate dialysis. In congestive heart failure, appropriate management of circulatory failure and use of an aldosterone antagonist in the diuretic regimen are integral to treatment.

One hundred years ago, Flexner emphasized the importance of science in medicine and medical education. Over the subsequent years, science education in the premedical and medical curricula has changed little, in spite of the vast changes in the biomedical sciences. The National Research Council, in their report Bio 2010, noted that the premedical curriculum caused many students to lose interest in medicine and in the biological sciences in general. Many medical students and physicians have come to view the premedical curriculum as of limited relevance to medicine and designed more as a screening mechanism for medical school admission. To address this, the Association of American Medical Colleges and the Howard Hughes Medical Institute formed a committee to evaluate the premedical and medical school science curricula. The committee made a number of recommendations that are summarized in this essay. Most important were that competencies replace course requirements and that the physical sciences and mathematics be better integrated with the biological sciences and medicine. The goal is that all physicians possess a strong scientific knowledge base and come to appreciate the importance of this to the practice of medicine. While science education needs to evolve, Flexner's vision is as relevant today as it was 100 years ago.

Parathyroid hormone, dopamine, alpha-adrenergic catecholamines, and angiotensin II regulate renal Na excretion, at least in part through modulation of acute cyclic (c)AMP-induced proximal tubule Na/H antiporter inhibition. The present studies examined the effect of chronic increases in cell cAMP on Na/H antiporter activity in OKP cells. Whereas 8-bromo cAMP acutely inhibited Na/H antiporter activity, chronic application for 6 h led to a 24% increase in Na/H antiporter activity measured 16-20 h after cAMP removal. This chronic persistent activation of the Na/H antiporter required > 2 h exposure. This effect was not a nonspecific effect of 8-bromo cAMP, in that addition of forskolin or forskolin + 3-isobutyl-1-methylxanthine for 6 h also led to a chronic persistent increase in Na/H antiporter activity. Inhibition of protein synthesis with cycloheximide prevented 8-bromo cAMP-induced Na/H antiporter stimulation. Although 8-bromo cAMP addition decreased cell pH by 0.15-0.20 pH U, Na/H antiporter stimulation could be dissociated from cell acidification. In summary, while cAMP acutely inhibits Na/H antiporter activity, it chronically increases antiporter activity. This chronic activation occurs with exogenous addition or endogenous generation of cAMP. These results imply that for hormones that modulate renal Na excretion and proximal tubule Na/H antiporter activity via cAMP and protein kinase A, acute effects may not predict chronic effects.

The NIH has played a pivotal role in shaping biomedical research into the remarkable success that it is today, through both its intramural program and its generous funding of extramural biomedical science. If you think you know the NIH, you will likely still learn much about it from John A. Kastor’s new book, The National Institutes of Health, 1991–2008, which should be required reading for anyone with an interest in this organization that has been so central to biomedical research. While the book purports to focus on the years 1991–2008, it frequently retreats back in time to critical periods as the NIH evolved into the institution it is today. The book covers the intramural and extramural programs, addresses the complex structure of the NIH, and describes many of the key leaders, here focusing more on recent times. It describes most of the key controversies, though admittedly not with the depth or entertainment value desired by those in search of exciting discord. Kastor is an accomplished medical historian who has diligently uncovered the facts underlying the history of the NIH. As he notes, his goal is to describe “how the NIH operates, its problems, its finances, its politics, and its structure within the federal government.” The book includes interviews with a myriad of individuals, most of whom have strong, well-informed opinions regarding what is right and wrong with the NIH. The major strength of the book is that it provides a broad and thorough description of the NIH, its history, its organization, and the many critical people who have served there. The issues surrounding the Intramural Research Program are perhaps the most interesting. During the Vietnam War, universities did not have academic programs that could compete with those at the NIH, and a whole generation of outstanding young researchers joined the intramural program for training. Whether they went to the NIH for superior training or to avoid service in the Vietnam War, the result was that the intramural program defined future biomedical science through these investigators and through their legacy, the researchers who subsequently trained in their labs. But is there a need for the intramural program today? Universities now have well-developed research programs, there is no draft, and the intramural program is not attracting the scientists it once did. This is compounded by limits on compensation, severe budget cuts, and conflict-of-interest rules that all contribute to make universities a more welcome place for the biomedical researcher. The future of the intramural research program is just one of many controversies that are addressed in Kastor’s book. Should there be a graduate school at the intramural program? Should institute directors yield greater power to the director of the NIH? Was Elias Zerhouni’s Roadmap a heroic attempt to insure that the NIH serves its constituency, the American public, or was it just another assault on the R01-supported investigator? Should the NIH fund institute-directed big science, or should it stay focused on supporting the ideas of researchers through investigator-initiated grants? Should the NIH support clinical research, or should it focus on basic research, leaving the former for industry? Are conflict-of-interest rules overly strict or too lenient? Do they undercut the ability of the NIH to attract the best talent, or are they needed to maintain the trust of the public? Then there are the personal scandals that would be expected to occur at any institution as large as the NIH. Kastor covers all of these controversies in an objective fashion, never expressing personal opinions or bias. Though fair, the evenness of his tone frequently leaves the reader wanting something more lurid. At times the book gets a little dry, especially as it reviews the structure of each of the institutes and centers, but even here there is much useful information, and the descriptions of the institutes with which the reader has personal experience are of interest. The NIH has undoubtedly been a driving force in shaping the biomedical research industry in the United States. However, budgets have been relatively flat for a number of years, the future is uncertain, and the American biomedical research enterprise is at great risk. How Congress funds biomedical research and how the NIH chooses to manage the funds it receives will determine whether the United States maintains its preeminence in this arena. For those interested in following or influencing these events, The National Institutes of Health, 1991–2008 should be required — and largely enjoyable — reading.

Metabolically generated acid is the major physiological stimulus for increasing proximal tubule citrate reabsorption, which leads to a decrease in citrate excretion. The activity of the Na-citrate cotransporter, NaDC-1, is increased in vivo by acid ingestion and in vitro by an acidic pH medium. In opossum kidney cells the acid stimulatory effect and the ability of endothelin-1 (ET-1) to stimulate NaDC-1 activity are both blocked by the endothelin B (ET(B)) receptor antagonist, BQ788. Acid feeding had no effect on brush border membrane NaDC-1 activity in mice in which ET(B) receptor expression was knocked out, whereas a stimulatory effect was found in wild-type mice. Using ET(A)/ET(B) chimeric and ET(B) C-terminal tail truncated constructs, ET-1 stimulation of NaDC-1 required a receptor C-terminal tail from either ET(A) or ET(B). The ET-1 effect was greatest when either the ET(B) transmembrane domain and C-terminal tail were present or the ET(B) C-terminal tail was linked to the ET(A) transmembrane domain. This effect was smaller when the ET(B) transmembrane domain was linked to the ET(A) C-terminal tail. Thus, the acid-activated pathway mediating stimulation of NaDC-1 activity requires a functional ET(B) receptor in vivo and in vitro, as does acid stimulation of NHE3 activity. Since increased NaDC-1 and NHE3 activities constitute part of the proximal tubule adaptation to an acid load, these studies indicate that there are similarities in the signaling pathway mediating these responses.

The United States is on the verge of addressing one of the most important issues facing the country, its health care system. An underlying premise of the need for reform is a belief that all citizens of this great country should have access to excellent health care. To accomplish this, universal health insurance must be a component of any plan. Many controversial issues have arisen in the debate, most relating to how to pay for those presently uninsured. Opponents are concerned that this will require increased taxation and/or decreased support for those presently insured. The only acceptable solution will be one in which the cost of covering the uninsured derives from improved efficiencies in the delivery of health care.

Expression of major stress proteins is induced rapidly in ischemic tissues, a response that may protect cells from ischemic injury. We have shown previously that transcriptional induction ofheat-shock protein 70 by hypoxia results from activation of DNA binding of a preexisting, but inactive, pool of heat shock factor (HSF). To determine the intracellular signals generated in hypoxic or ischemic cells that triggerHSF activation, we examined the effects of glucose deprivation and the metabolic inhibitor rotenone on DNA-binding activity of HSF in cultured C2 myogenic cells grown under normoxic conditions. Whole-cell extracts were examined in gel mobility shift assays using a 39-bp synthetic oligonucleotide containing a consensus heat-shock element as probe. ATP pools were determined by high-pressure liquid chromatography and intracellular pH (pH1) was measured using a fluorescent indicator. Glucose deprivation alone reduced the cellularATP pool to50% ofcontrol levels but failed to activate HSF. However, 2 X 10'M rotenone inducedDNA binding ofHSF within 30 min, in association with a fall inATP to30% ofcontrol levels, and a fall in pHifrom 7.3 to 6.9. Maneuvers (sodium proprionate and amiloride) that lowered pH, to 6.7 without ATP depletion failed to activate HSF. Conversely, in studies that loweredATP stores at normal pH (high K+/nigericin) we found induction ofHSF-DNA binding activity. Our data indicate that the effects ofATP depletion alone are sufficient to induce the DNA binding of HSF when oxidative metabolism is impaired, and are consistent with a model proposed recently for transcriptional regulation ofstress protein genes during ischemia. (J. Clin. Invest. 1992.89:1685

Chronic K depletion (KD) causes hypocitraturia. In the present studies, the effect of KD, induced by a low-K diet for 14 days (serum [K] 4.1 +- 0.1, control vs. 2.2 +/- 0.1 meq/l, KD, P less than 0.01), on renal cortical brush-border membrane (BBM) Na-citrate cotransporter activity was examined. KD significantly decreased fractional citrate excretion (3.90 +/- 0.68 vs. 0.53 +/- 0.10%, P less than 0.01). This was paralleled by a significant increase in the initial linear rate of BBM Na-dependent citrate transport (81 +/- 4 vs. 141 +/- 6 pmol citrate.5 s-1.mg protein-1, P less than 0.01). Kinetic studies varying extravesicular citrate concentration demonstrated that KD increased the maximal activity (Vmax) (152 +/- 17 vs. 296 +/- 14 pmol citrate.5 s-1.mg protein-1, P less than 0.01) with no difference in citrate affinity (118 +/- 23, control vs. 135 +/- 22 microM citrate, KD). Similarly, when extravesicular Na concentration was varied, KD increased the Vmax (132 +/- 9 vs. 206 +/- 6 pmol citrate.5 s-1.mg protein-1, P less than 0.01) with no difference in Na affinity (29 +/- 3, control vs. 28 +/- 1 mM Na, KD). The effect of KD on Na-citrate cotransport was specific in that KD did not alter Na-glucose (84 +/- 12, control vs. 89 +/- 5 pmol glucose.5 s-1.mg protein-1, KD) or Na-proline (87 +/- 3, control vs. 84 +/- 5 pmol proline.5 s-1.mg protein-1, KD) cotransport. In conclusion, KD increases the Vmax of the proximal tubule apical membrane Na-citrate cotransporter.(ABSTRACT TRUNCATED AT 250 WORDS)

A major function of renal tubules is the net transport of acid from the blood into the luminal fluid. To accomplish this, acid (H ions) is secreted from cell to lumen across the apical membrane, and base generated within the cell exits into the interstitium across the basolateral membrane. Because cell pH (pHi) is above electrochemical equilibrium in all cells, acid efflux from cell to lumen requires the input of energy, whereas base efflux from cell to interstitium can occur passively.

An apical membrane Na(+)-H+ antiporter mediates proximal tubule NaCl and NaHCO3 reabsorption. The present studies examined whether chronic changes in dietary NaCl intake lead to an adaptation of the Na(+)-H+ antiporter. Rats were maintained either on a low-salt (LS, 0% NaCl) or a high-salt (HS, 2% NaCl) diet for 4 days. Na(+)-H+ antiporter was assayed using the acridine orange fluorescence method in apical membrane vesicles prepared by Mg2+ aggregation. Rats on LS diet exhibited a higher maximal activity (Vmax) for antiporter activity (0.109 +/- 0.008 s-1) compared with rats on HS diet (0.090 +/- 0.008 s-1; P less than 0.01), whereas Na activity (KNa) was similar (13.1 +/- 1.5 and 14.2 +/- 0.8 mM in HS and LS groups, respectively). The modulation of Na(+)-H+ antiporter activity was amiloride sensitive. Proton permeability, Na(+)-dependent [3H]glucose uptake, and vesicle enrichments were similar in both groups. In addition, the dietary protocols were not associated with any systemic acid-base disturbances, K+ deficiency, or hyperfiltration, conditions that have previously been demonstrated to alter Na(+)-H+ antiporter activity. Acute differences in extracellular fluid volume, induced by 10% body weight isohydric expansion failed to affect Na(+)-H+ antiporter activity. Thus chronic alterations in dietary NaCl intake alter the maximal activity (Vmax) of the Na(+)-H+ antiporter. This adaptation may contribute to decreased NaCl reabsorption in extracellular fluid volume expansion, enhanced NaCl reabsorption in extracellular fluid volume depletion, and enhanced NaHCO3 reabsorption in chronic metabolic alkalosis associated with volume contraction.