Patricia A. Preisig

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.

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.

Pyk2 activation is integral to acid stimulation of sodium/hydrogen exchanger 3

The present study examines the role of Pyk2 in acid regulation of sodium/hydrogen exchanger 3 (NHE3) activity in OKP cells, a kidney proximal tubule epithelial cell line. Incubation of OKP cells in acid media caused a transient increase in Pyk2 phosphorylation that peaked at 30 seconds and increased Pyk2/c-Src binding at 90 seconds. Pyk2 isolated by immunoprecipitation and studied in a cell-free system was activated and phosphorylated at acidic pH. Acid activation of Pyk2 (a) was specific for Pyk2 in that acid did not activate focal adhesion kinase, (b) required calcium, and (c) was associated with increased affinity for ATP. Transfection of OKP cells with dominant-negative pyk2(K457A) or small interfering pyk2 duplex RNA blocked acid activation of NHE3, while neither had an effect on glucocorticoid activation of NHE3. In addition, pyk2(K457A) blocked acid activation of c-Src kinase, which is also required for acid regulation of NHE3. The present results demonstrate that Pyk2 is directly activated by acidic pH and that Pyk2 activation is required for acid activation of c-Src kinase and NHE3. Given that partially purified Pyk2 can be activated by acid in a cell-free system, Pyk2 may serve as the pH sensor that initiates the acid-regulated signaling cascade involved in NHE3 regulation.

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.

Cellular Mechanisms of Renal Tubular Acidification

To maintain acid–base balance, the renal tubules secrete hydrogen ions into the lumen at a rate equal to the sum of extrarenal acid generation and bicarbonate filtration. In this chapter we review the cellular mechanisms responsible for transepithelial hydrogen secretion along the nephron and describe the regulators of these processes.

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.

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.

Cyclic adenosine monophosphate acutely inhibits and chronically stimulates Na/H antiporter in OKP cells.

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.

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 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 10-9 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.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.

What Makes Cells Grow Larger and How Do They Do It? Renal Hypertrophy Revisited

Hypertrophy, defined as an increase in cell size without an increase in cell number, occurs in a number of conditions, including compensatory renal growth, diabetes mellitus, protein feeding, chronic metabolic acidosis, and chronic potassium deficiency. In vitro cell culture studies have been used to characterize the mechanisms involved in the development of hypertrophy. Two mechanisms have been identified and characterized. One mechanism involves regulation of processes that are also associated with the initial events of the hyperplastic growth process, and is referred as a cell cycle-dependent mechanism. The other mechanism occurs independently of these particular cell cycle processes, but involves regulation of protein degradation by lysosomal enzymes. This latter mechanism is referred to as a cell cycle-independent mechanism. In vivo studies suggest that both compensatory renal hypertrophy following uninephrectomy and diabetes mellitus-induced hypertrophy involve the cell cycle-dependent mechanism.