Physical health, behavioral and emotional functioning in children of gulf war veterans.

OBJECTIVE: We examined whether the prevalence of medical and behavioral conditions is higher in children of deployed veterans (DVs) versus non-deployed veterans (NDVs) after the 1991 Gulf War. METHODS: We examined 1387 children of 737 veterans. Children ages 2-18 had physical exams and parental reports of physical history and behavior. RESULTS: Physical health was analyzed using GEE models. Behavioral health [total, internalizing, and externalizing behavior problems (TBP, IBP, EBP)] was analyzed with mixed-effects regression models. Analyses were conducted by age group (2-3, 4-11, 12-18), and gender (ages 4-11, 12-18). Children of DVs ages 2-3 had significantly worse dentition (13.9% vs. 4.8%, P = 0.03) and more EBP {least square means (lsmeans) 54.31 vs. 47.59, P = 0.02}. Children of DVs ages 4-11 had significantly more obesity (18.8% vs. 12.7%, P = 0.02). Among children 4-11, male children of DVs had significantly more TBP (lsmeans 70.68 vs. 57.34, P = 0.003), IBP (lsmeans 63.59 vs. 56.16, P = 0.002) and EBP (lsmeans 61.60 vs. 52.93, P = 0.03), but female children did not. For children ages 12-18, male children of DVs had more EBP (lsmeans 63.73 vs. 43.51, P = 0.008), while female children of DVs had fewer EBP (lsmeans 45.50 vs. 50.48, P = 0.02). Veteran military characteristics and mental health, and children's social status and health, including obesity, predicted children's TBP for one or more age groups. CONCLUSIONS: Children of DVs experienced worse dentition, greater obesity, and more behavioral problems compared to NDV children, suggesting adverse health effects associated with parental deployment in need of further exploration.

Clinical and laboratory assessment of distal peripheral nerves in Gulf War veterans and spouses.

BACKGROUND: The prevalence of symptoms suggesting distal symmetric polyneuropathy (DSP) was reported to be higher among deployed veterans (DV) to the Persian Gulf in 1990-1991 than to control non-deployed veterans (NDV). The authors therefore compared the prevalence of DSP by direct examination of DV and their spouses to control NDV and spouses. METHODS: The authors performed standardized neurologic examinations on 1,061 DV and 1,128 NDV selected from a cohort of veterans who previously participated in a national mail and telephone survey. Presence of DSP was evaluated by history, physical examination, and standardized electrophysiologic assessment of motor and sensory nerves. Similar examinations were performed without electrophysiologic tests in 484 DV spouses and 533 NDV spouses. Statistical analyses were performed with appropriate adjustments for the stratified sampling scheme. RESULTS: No differences between adjusted population prevalence of DSP in DV and NDV were found by electrophysiology (3.7% vs 6.3%, p = 0.07), by neurologic examination (3.1% vs 2.6%, p = 0.60), or by the methods combined (6.3% vs 7.3%, p = 0.47). Excluding veterans with non-military service related diseases that may cause DSP did not alter outcomes. DV potentially exposed to neurotoxins from the Khamisiyah ammunition depot explosion did not significantly differ in DSP prevalence compared to non-exposed DV. The prevalence of DSP in DV spouses did not differ from NDV spouses (2.7% vs 3.2%, p = 0.64). CONCLUSIONS: Neither veterans deployed during the Gulf War era nor their spouses had a higher prevalence of DSP compared to NDV and spouses.

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.

ET(B) receptor activation causes exocytic insertion of NHE3 in OKP cells.

Endothelin-1 (ET-1) activates sodium/hydrogen exchanger 3 (NHE3) in opossum kidney clone P (OKP) cells expressing ET(B) receptors. ET-1 (10(-8) M) caused a two- to threefold increase in apical membrane NHE3 (assessed by surface biotinylation), in the absence of a change in total cellular NHE3. A maximal effect was achieved within 15 min. The increase in apical NHE3 was not blocked by cytochalasin D but was blocked by latrunculin B, which also prevented the ET-1-induced increase in NHE3 activity. Endocytic internalization of NHE3, measured as protection of biotinylated NHE3 from the membrane-impermeant, sulfhydryl-reducing agent MesNa was minimal within 35 min and was not regulated by ET-1. Exocytic insertion of NHE3, measured as the appearance of biotinylated NHE3 after the blockade of reactive sites with sulfo-NHS-acetate, was increased in response to ET-1. These studies demonstrate that ET-1 induces net trafficking of NHE3 to the apical membrane that is mediated by enhanced exocytic insertion and is required for increased NHE3 activity.

Chronic metabolic acidosis increases NaDC-1 mRNA and protein abundance in rat kidney.

BACKGROUND: Chronic metabolic acidosis increases, while alkali feeding inhibits, proximal tubule citrate absorption. The activity of the apical membrane Na+/citrate cotransporter is increased in metabolic acidosis, but is not altered by alkali feeding. METHODS: Renal cortical mRNA and brush border membrane protein abundances of sodium/dicarboxylate-1 (NaDC-1), the apical membrane Na+/citrate transporter, were measured. RESULTS: By immunohistochemistry, NaDC-1 was localized to the apical membrane of the proximal tubule. Chronic metabolic acidosis caused an increase in NaDC-1 protein abundance that was maximal in the S2 segment and that increased with time. Metabolic acidosis also increased NaDC-1 mRNA abundance, but this was first seen at three hours and correlated with the severity of the metabolic acidosis. Alkali feeding had no effect on NaDC-1 protein or mRNA abundance. CONCLUSIONS: Chronic metabolic acidosis increases renal cortical NaDC-1 mRNA abundance and apical membrane NaDC-1 protein abundance, while alkali feeding is without effect on NaDC-1.

Acid incubation causes exocytic insertion of NHE3 in OKP cells.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.