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R J Alpern

Publications and source records attributed to R J Alpern.

At least 37 records · Page 2Linked to original sources
PubMed1997

The clinical spectrum of chronic metabolic acidosis: homeostatic mechanisms produce significant morbidity.

Chronic metabolic acidosis is a process whereby an excess nonvolatile acid load is chronically placed on the body due to excess acid generation or diminished acid removal by normal homeostatic mechanisms. Two common, often-overlooked clinical conditions associated with chronic metabolic acidosis are aging and excessive meat ingestion. Because the body's homeostatic response to these pathologic processes is very efficient, the serum HCO3- and blood pH are frequently maintained within the "normal" range. Nevertheless, these homeostatic responses engender pathologic consequences, such as nephrolithiasis, bone demineralization, muscle protein breakdown, and renal growth. Based on this, the concept of eubicarbonatemic metabolic acidosis is introduced. Even in patients with a normal serum HCO3- and blood pH, it is important to treat the acid load and prevent pathologic homeostatic responses. These homeostatic responses, as well as the mechanisms responsible for their initiation, are reviewed.

Acid-Base Equilibrium↗
PubMed1997

Intracellular acidosis activates c-Src.

The purpose of the present studies was to determine whether acidosis activates protein tyrosine kinase pathways. Incubation of MCT cells, a renal proximal tubule cell line, in acid media caused increased phosphotyrosine content of 60- to 70- and 120-kDa cytosolic proteins. Media acidification induced a twofold increase in c-Src activity that occurred within 30 s. Significant activation occurred with media pH changes as small as 0.07 pH unit accompanied by cell acidification of 0.06 pH unit. Sodium propionate addition, NH4Cl prepulse, and nigericin addition, maneuvers that decrease intracellular pH in the absence of changes in extracellular pH, activated c-Src. Significant activation by sodium propionate was seen with cell pH changes as small as 0.07 pH unit. Sodium orthovanadate, a protein tyrosine phosphatase inhibitor, prevented c-Src activation by media acidification but did not prevent protein tyrosine phosphorylation. In summary, decreased intracellular pH activates c-Src. Acid activation of c-Src represents a novel mechanism of c-Src activation that may be relevant to many cellular responses to acidosis.

Acidosis↗
PubMed1997

Adaptation to low-K+ media increases H(+)-K(+)-ATPase but not H(+)-ATPase-mediated pHi recovery in OMCD1 cells.

Studies in rat and rabbit outer medullary collecting duct of inner stripe origin (OMCDis) suggest that both H(+)-ATPase and H(+)-K(+)-ATPase participate in H+ secretion. However, the relative contributions of these transporters, and, in particular, that of H(+)-K(+)-ATPase to K+ absorption have not been defined precisely. The present study was designed to delineate more clearly the response of these two transporters to hypokalemia and acidosis in a newly developed mouse OMCD1 cell line. In cells grown in normal K+ (5 mM) media, intracellular pH (pHi) recovery was similar either in the presence or absence of K+ in the perfusate (delta pHi/min = 0.014 +/- 0.001 vs. 0.017 +/- 0.003, not significant). The inhibitory effects of Sch-28080 (10 microM) and bafilomycin A1 (10 nM) on pHi recovery were evident only in the presence and absence of K+ in the perfusate, respectively. In cells grown in low-K+ (2.5 mM) media to simulate chronic hypokalemia, pHi recovery was significantly faster than in cells grown in normal K+ media (delta pHi/min = 0.045 +/- 0.01 vs. 0.014 +/- 0.001, P < 0.01) and was inhibited specifically by Sch-28080, not by bafilomycin A1. In contrast, in cells preconditioned to low pH (7.0) to simulate chronic acidosis, the enhanced pHi recovery was abolished by bafilomycin A1 but not by Sch-28080. 86Rb+ uptake, when used as a K+ congener, was inhibited by Sch-28080. The K(m) for 86Rb+ uptake (H(+)-K(+)-ATPase activity) and the 50% inhibitory concentration for Sch-28080 were 270 and 5.0 microM, respectively. These studies provide evidence that, in morphologically homogeneous OMCD1 cells, 1) both H(+)-K(+)-ATPase and H(+)-ATPase participate in pHi regulation, 2) the H(+)-K(+)-ATPase is selectively upregulated by preconditioning in low-K+ media, and 3) conversely, preconditioning in low-pH media stimulates only the H(+)-ATPase. Thus, in OMCDis, the H(+)-K(+)-ATPase and H(+)-ATPase respond selectively and independently to chronic hypokalemia and acidosis, respectively.

Adaptation, Physiological↗
PubMed1997

Metabolic alkalosis.

In summary, the kidney possesses numerous mechanisms that help to prevent metabolic alkalosis. Maintenance of metabolic alkalosis for any length of time means that renal homeostatic mechanisms for HCO3- excretion have been disrupted. Understanding the mechanisms that may perturb the kidney's ability to correct alkalosis will lead to improved clinical approaches to differential diagnosis and treatment of the patient. Although metabolic alkalosis is frequently not dangerous, in certain settings metabolic alkalosis may contribute to mortality and should be treated aggressively.

Alkalosis↗
PubMed1997

Pathogenesis of edema formation in the nephrotic syndrome.

The development of edema in the nephrotic syndrome has traditionally been viewed as an underfill mechanism. According to this view, urinary loss of protein results in hypoalbuminemia and decreased plasma oncotic pressure. As a result, plasma water translocates out of the intravascular space and results in a decrease in intravascular volume. In response to the underfilled circulation, effector mechanisms are then activated that signal the kidney to secondarily retain salt and water. While an underfill mechanism may be responsible for edema formation in a minority of patients, recent clinical and experimental findings would suggest that edema formation in most nephrotic patients is the result of primary salt retention. Direct measurements of blood and plasma volume or measurement of neurohumoral markers that indirectly reflect effective circulatory volume are mostly consistent with either euvolemia or a volume expanded state. The ability to maintain plasma volume in the setting of a decreased plasma oncotic pressure is achieved by alterations in transcapillary exchange mechanisms known to occur in the setting of hypoalbuminemia that limit excessive capillary fluid filtration. The intrarenal mechanism responsible for primary sodium retention is not yet known, but may involve tubular resistance to the natriuretic effect of atrial natriuretic peptide.

Blood Volume↗
PubMed1997

Media acidification inhibits TGF beta-mediated growth suppression in cultured rabbit proximal tubule cells.

Chronic metabolic acidosis induces both hyperplastic and hypertrophic renal growth and is associated with progressive loss of renal function. These studies examine the direct effect of media acidification on the growth of rabbit proximal tubule cells in primary culture. The results demonstrate that media acidification has a direct antiproliferative (hypoplastic) effect on both quiescent and mitogen-stimulated [epidermal growth factor (EGF)-stimulated] cells and does not induce hypertrophy. This direct antiproliferative effect of acid is associated with inhibition of EGF-induced phosphorylation of the retinoblastoma protein (pRB), which maintains pRB activity and inhibits cell cycle progression from G1 to S phase. Transforming growth factor-beta (TGF-beta) alone has an antiproliferative effect in these cells. TGF-beta converts EGF-induced hyperplasia to hypertrophy and inhibits EGF-induced pRB phosphorylation. Media acidification inhibits both the antiproliferative effect of TGF-beta and the ability of TGF-beta to convert EGF-induced hyperplasia to hypertrophy. This activity is associated with inhibition of TGF-beta-mediated retention of pRB in the active, hypophosphorylated state. These results demonstrate that metabolic acidosis has a direct growth-suppressive effect on renal epithelial cells but inhibits the growth-suppressive effects of TGF-beta. Inhibition of the antiproliferative effect of cytokines, such as TGF-beta, may be responsible for acidosis-induced hyperplasia in vivo.

Animals↗
PubMed1996

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.

ATP Citrate (pro-S)-Lyase↗
PubMed1996

Endothelin(B) receptor activates NHE-3 by a Ca2+-dependent pathway in OKP cells.

To examine the mechanisms by which endothelin (ET) regulates the Na/H antiporter isoform, NHE-3, OKP cells were stably transfected with ET(A) and ET(B) receptor cDNA. In cells overexpressing ET(B), but not ET(A) receptors, ET-1 increased Na/H antiporter activity (JNa/H). This effect was inhibited by a nonselective endothelin receptor blocker and by a selective ET(B) receptor blocker but was not inhibited by an ET(A) selective receptor blocker. In ET(B)-overexpressing cells, 10(-8) M ET-1 inhibited adenylyl cyclase, but protein kinase A inhibition and pertussis toxin pretreatment did not affect Na/H antiporter activation by ET-1. ET-1 caused a transient increase in cell [Ca2+], followed by a sustained increase. Increases in cell [Ca2+] were partially inhibited by pertussis toxin. ET-1-induced increases in J(Na/H) were 50% inhibited by clamping cell [Ca2+] low with BAPTA, and by KN62, a Ca-calmodulin kinase inhibitor. Inhibitors of protein kinase C, cyclooxygenase, lipoxygenase, and cytochrome P450 and cyclic GMP were without effect. In ET(A)-overexpressing cells, ET-1 increased cell [Ca2+] but did not increase JNa/H. In summary, binding of ET-1 to ET(B) receptors increases Na/H antiporter activity in OKP cells, an effect mediated in part by increases in cell [Ca2+] and Ca-calmodulin kinase. Increases in cell [Ca2+] are not sufficient for Na/H antiporter activation.

Animals↗
PubMed1996

Role of tyrosine kinase pathways in ETB receptor activation of NHE3.

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.

Animals↗
PubMed1996

Glucocorticoids regulate NHE-3 transcription in OKP cells.

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.

Animals↗
PubMed1996

Chronic metabolic acidosis increases NHE3 protein abundance in rat 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.

Acidosis↗
PubMed1995

Overexpression of csk inhibits acid-induced activation of NHE-3.

Opossum kidney OKP cells express an apical membrane Na+/H+ antiporter that is encoded by NHE-3 (for Na+/H+ exchanger 3) and is similar in many respects to the renal proximal tubule apical membrane Na+/H+ antiporter. Chronic incubation of OKP cells in acid medium for 24 hr increases Na+/H(+)-antiporter activity and NHE-3 mRNA abundance. The increase in Na+/H(+)-antiporter activity was not prevented by H7, a protein kinase C/protein kinase A inhibitor, but was prevented by herbimycin A, a tyrosine kinase inhibitor. Incubation of cells in acid medium increased c-src activity, and this was inhibited by herbimycin A. To determine the role of the src family of nonreceptor protein-tyrosine kinases, Csk (for carboxyl-terminal src kinase), a physiologic inhibitor of these kinases, was overexpressed in OKP cells. In three clones overexpressing csk, acid-induced increases in Na+/H(+)-antiporter activity and NHE-3 mRNA abundance were inhibited. In these clones, inhibition of acid activation of Na+/H(+)-antiporter activity paralleled inhibition of acid activation of c-src. Neither herbimycin A nor overexpression of csk inhibited dexamethasone-induced increases in Na+/H(+)-antiporter activity. These studies show that decreases in pH activate c-src and that the src family nonreceptor protein-tyrosine kinases play a key role in acid activation of NHE-3.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1995

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.

Amino Acid Sequence↗
PubMed1995

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.

Animals↗