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R J Alpern

Publications and source records attributed to R J Alpern.

At least 19 recordsLinked to original sources
PubMed2000

Renal citrate metabolism and urinary citrate excretion in the infant rat.

BACKGROUND: Although hypercalciuria has the same prevalence in children as adults, children rarely develop renal stones. This may be explained by a greater urinary citrate excretion in infants compared with adults. The present study examines the renal excretion of citrate and renal cortical citrate metabolism in infant and adult rats. METHODS: Adult male and newly weaned infant rats were acclimated to metabolic cages and fed synthetic diets. Urine was collected after two days, and renal cortical citrate metabolism was assayed. RESULTS: Infant rats had a lower plasma [HCO3-] and higher plasma [K+] and had a fourfold higher urinary citrate:creatinine ratio and a twofold higher concentration of citrate in their urine compared with adult rats. This higher urinary citrate excretion was not due to a difference in renal proximal tubular Na/citrate cotransporter activity, nor renal cortical citrate synthase or ATP citrate lyase activities in infants as compared with adults. However, infant rat kidneys had significantly lower mitochondrial aconitase (m-aconitase) activity. Renal cortical citrate concentrations were comparable in infant and adult rats. Manipulation of plasma [K+] to adult levels did not affect the higher urinary citrate excretion in infant rats. CONCLUSIONS: Urinary citrate excretion in infant rats is greater than in adults but does not parallel tissue [citrate]. Thus, this higher urinary citrate is likely due to maturational differences in the proximal tubule, other than Na/citrate cotransport, that directly affect citrate transport.

Aging↗
PubMed1999

Incubation of OKP cells in low-K+ media increases NHE3 activity after early decrease in intracellular pH.

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.

Acids↗
PubMed1999

ETB receptor activation leads to activation and phosphorylation of NHE3.

In OKP cells expressing ETB endothelin 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 ETB receptors but not in cells expressing ETA receptors. 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(-9) M 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. ETB receptor 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.

Animals↗
PubMed1999

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.

Animals↗
PubMed1998

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.

Amiloride↗
PubMed1998

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-SrcK295M. 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-SrcK295M, 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-SrcK295M, similar to that seen in wild-type OKP cells. In addition, cells expressing c-SrcK295M 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.

Angiotensin II↗
PubMed1998

Converting enzyme inhibition causes hypocitraturia independent of acidosis or hypokalemia.

BACKGROUND: Angiotensin II stimulates the proximal tubular Na/H antiporter and increases proximal tubular cell pH. Because intracellular pH may affect urinary citrate excretion and enzymes responsible for renal citrate metabolism, the present studies examined the effect of enalapril, an angiotensin converting enzyme inhibitor, on the activity of renal cortical ATP citrate lyase and urinary citrate excretion. METHODS: Enalapril was given to rats (15 mg/kg/day) for seven days and to humans (10 mg twice daily) for 10 days. Blood and 24-hour urine samples were obtained in both groups. Renal cortical tissue from rats was analyzed for enzyme activity. RESULTS: In rats, enalapril decreased urinary citrate excretion by 88%. The change in urinary citrate was not associated with a difference in plasma pH, bicarbonate nor potassium concentration. However, similar to metabolic acidosis and hypokalemia, enalapril caused a 42% increase in renal cortical ATP citrate lyase activity. When given to humans, enalapril significantly decreased urinary citrate excretion and urine citrate concentration by 12% and 16%, respectively, without affecting plasma pH or electrolytes. CONCLUSIONS: Enalapril decreases urinary citrate in rats and humans. This is due, at least in part, to increases in cytosolic citrate metabolism through ATP citrate lyase in rats similar to that seen with chronic metabolic acidosis and hypokalemia. The effects of enalapril on urinary citrate and renal cortical ATP citrate lyase occur independently of acidosis or hypokalemia but may be due to intracellular acidosis that is common to all three conditions.

Acidosis↗
PubMed1998

Effects of thyroid hormone on the neonatal renal cortical Na+/H+ antiporter.

The neonatal proximal tubule has a lower rate of bicarbonate absorption than that of adults. This is due, in part, to a lower rate of apical membrane Na+/H+ antiporter activity. The purpose of these studies was to examine if thyroid hormone could be a factor in the maturational increase in Na+/H+ antiporter activity. Hypothyroid (0.01% propylthiouracil in drinking water starting at day 14 gestation and throughout the postnatal period), euthyroid, and hyperthyroid (intraperitoneal triiodothyronine, 10 micrograms/100 g body wt, once daily on days 17 to 20 of postnatal life) rats were all studied at 21 days of life. Renal cortical brush border Na+/H+ antiporter activity was 453 +/- 24, 527 +/- 30 and 608 +/- 25 pmol/mg protein in the hypothyroid, euthyroid and hyperthyroid groups, respectively (P < 0.001). Hyperthyroid neonates had approximately twofold greater renal cortical NHE-3 mRNA abundance than euthyroid and hypothyroid neonates (P < 0.05). Brush border membrane NHE-3 protein abundance in hypothyroid and hyperthyroid neonates was one-third and twofold that of euthyroid 21-day-old rats, respectively (P < 0.001). These data are consistent with a potential role of thyroid hormone in the postnatal increase in Na+/H+ antiporter activity.

Animals↗
PubMed1998

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.

Acidosis↗
PubMed1998

Renal Na/H exchanger NHE-3 and Na-PO4 cotransporter NaPi-2 protein expression in glucocorticoid excess and deficient states.

Administration of pharmacologic doses of glucocorticoid in vivo increases renal proximal tubule apical membrane Na/H exchange and decreases Na/PO4 cotransport activity (1). Current data suggest that the NHE-3 and NaPi-2 proteins mediate significant fractions of proximal tubule apical membrane Na/H exchange and Na/PO4 cotransport, respectively. This study examines whether glucocorticoid excess or deficiency affects NHE-3 and NaPi-2 protein abundance and the intrarenal distribution of these transporters. Protein abundance of NHE-3 and NaPi-2 in control rats was compared to rats rendered glucocorticoid-deficient by bilateral adrenalectomy, and to rats receiving pharmacologic doses of dexamethasone using immunoblots and immunohistochemistry. Adrenalectomy had modest effects on NHE-3 protein abundance, but dexamethasone administration to either adrenalectomized or sham-operated rats significantly increased NHE-3 protein abundance in both the proximal tubule and thick ascending limb, but not the thin descending limb. Adrenalectomy increased NaPi-2 protein abundance in the proximal tubule, whereas dexamethasone administration dramatically suppressed NaPi-2 protein on the apical membrane in both adrenalectomized and sham-operated animals. No significant reciprocal increase in subapical NaPi-2 staining was seen in the dexamethasone-treated rats. The present study shows that glucocorticoids regulate proximal tubule apical membrane Na/H exchange and NaPi cotransport by changes in protein abundance of NHE-3 and NaPi-2, respectively.

Adaptor Protein Complex 3↗
PubMed1997

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.

Acidosis↗
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↗