Recent reports have shown that low birth weight infants have a higher incidence of adult hypertension. These observations have stimulated a number of studies designed to evaluate the mechanisms of this phenomenon. In this study, fetal growth retardation was induced by treating pregnant rats with dexamethasone. After birth, pups whose mothers were treated with dexamethasone had a lower body and kidney weight and a lower number of glomeruli than control pups. Immunohistochemistry on treated kidneys demonstrated a marked reduction in the number of cells undergoing mitosis in the cortical nephrogenic zone. In the treated group, body and kidney weight normalized by 60 d of age, but blood pressure was significantly higher compared with controls (130+/-4 versus 107+/-1 mm Hg). In addition, GFR was significantly lower, albuminuria was higher, urinary sodium excretion rate and fractional sodium excretion were lower, and sodium tissue content was higher. In contrast, when pregnant rats were treated with a natural glucocorticoid (hydrocortisone) which is metabolized by the placenta, fetal development and adult blood pressure were normal. In conclusion, we found that high levels of maternal glucocorticoids impair renal development and lead to arterial hypertension in offspring. Even though renal mass eventually normalizes, glomerular damage as well as sodium retention occur and these factors may contribute to the development of hypertension.

Like the gamma-subunit of Na-K-ATPase, the corticosteroid hormone-induced factor (CHIF) is a member of the FXYD family of one-transmembrane-segment proteins. Both CHIF and two splice variants of gamma, gamma(a) and gamma(b), are expressed in the kidney. Immunolocalization experiments demonstrate mutually exclusive expression of CHIF and gamma in different nephron segments. Specific coimmunoprecipitation experiments demonstrate the existence in kidney membranes of the complexes alpha/beta/gamma(a), alpha/beta/gamma(b), and alpha/beta/CHIF and exclude mixed complexes such as alpha/beta/gamma(a)/gamma(b) and alpha/beta/gamma/CHIF. CHIF has been expressed in HeLa cells harboring the rat alpha(1)-subunit of Na-K-ATPase. (86)Rb flux experiments demonstrate that CHIF induces a two- to threefold increase in apparent affinity for cytoplasmic Na (K'(Na)) but does not affect affinity for extracellular K (Rb) ions (K'(K)) or V(max). Measurements of Na-K-ATPase using isolated membranes show similar but smaller effects of CHIF on K'(Na), whereas K'(K) and K'(ATP) are unaffected. The functional effects of CHIF differ from those of gamma. An implication of these findings is that other FXYD proteins could act as tissue-specific modulators of Na-K-ATPase.

The activity of Na(+)-K(+)-ATPase can be regulated by hormones that activate adenosine 3',5'-cyclic monophosphate-dependent protein kinase (PKA). Here, using a site-directed phosphorylation state-specific antibody, we show that hormonal regulation of Na(+)-K(+)-ATPase can occur via phosphorylation of Ser-943 on its alpha-subunit. cDNAs coding for wild-type rat Na(+)-K(+)-ATPase and Na(+)-K(+)-ATPase in which the PKA phosphorylation site Ser-943 was mutated to Ala were stably and transiently transfected into COS cells. In COS cells expressing wild-type Na(+)-K(+)-ATPase the beta-adrenergic agonist isoproterenol (1 microM) significantly increased the level of phosphorylation of the alpha-subunit. Phosphorylation was accompanied by a significant inhibition of the enzyme activity, as reflected by a decrease in ATP hydrolysis and 86Rb+ transport. The effect of isoproterenol was reproduced by the PKA activator forskolin used in combination with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine and was abolished by the specific PKA inhibitor H-89. Okadaic acid, an inhibitor of protein phosphatases 1 and 2A, enhanced phosphorylation and inhibition of Na(+)-K(+)-ATPase induced by isoproterenol. The changes in activity of Na(+)-K(+)-ATPase linearly correlated with the extent of the alpha-subunit of Na(+)-K(+)-ATPase being phosphorylated. When Ser-943 was replaced by alanine, stimulation of the phosphorylation and inhibition of the activity of Na(+)-K(+)-ATPase induced by isoproterenol, alone or in combination with okadaic acid, were not observed. These results indicate that, in intact cells, modulation of the activity of Na(+)-K(+)-ATPase can be achieved by regulation of the state of phosphorylation of Ser-943. Moreover, they provide a biochemical mechanism by which beta-adrenergic agonists can regulate Na(+)-K(+)-ATPase activity.

Corticosteroid hormone-induced factor (CHIF) and the ␥ subunit of the Na,K-ATPase (␥) are two members of the FXYD family whose function has been elucidated recently. CHIF and ␥ interact with the Na ؉ pump and alter its kinetic properties, in different ways, which appear to serve their specific physiological roles. Although functional interactions with the Na,K-ATPase have been clearly demonstrated, it is not known which domains and which residues interact with the ␣ and/or ␤ subunits and affect the pump kinetics. The current study provides the first systematic analysis of structure-function relations of CHIF and ␥. It is demonstrated that the stability of detergent-solubilized complexes of CHIF and ␥ with ␣ and/or ␤ subunits is determined by the trans-membrane segments, especially three residues that may be involved in hydrophobic interactions. The transmembrane segments also determine the opposite effects of CHIF and ␥ on the Na ؉ affinity of the pump, but the amino acids involved in this functional effect are different from those responsible for stable interactions with ␣.

Pyelonephritis is a risk factor for renal tubular epithelial cell damage in children. The inter- and intracellular regulator nitric oxide (NO) plays a role in the modulation of cellular viability in urinary tract infections, but the role of the NO pathway in renal proximal tubular-cell death remains unclear. The present study demonstrates that, in renal epithelial cells undergoing death mediated by Escherichia coli strain ARD6 serotype O6K13H1 (O6), levels of the phosphorylated extracellular signal-regulated kinase (ERK) 1/2 and inducible NO synthase (iNOS) proteins are up-regulated, but levels of endothelial NO synthase are down-regulated. When NO synthase (NOS) activity is inhibited by the specific inhibitor of NOS or mitogen-activated protein kinase kinase, cells are prevented from death. Moreover, down-regulating protein 53 (p53) does not prevent the cells from dying, although p53 is up-regulated in O6-exposed cells. Up-regulation of heme oxygenase (HO)-1 by sodium nitroprusside or by the specific activator hemin inhibits cell death. In conclusion, the activation of ERK mediates O6 toxin-mediated renal cell death via induction of iNOS. Stimulation of HO-1 protects cells against death.

Corticosteroid hormone-induced factor (CHIF) is a short epithelial-specific protein that is independently induced by aldosterone and a high-K+ diet. It is a member of the FXYD family of single-span transmembrane proteins that include phospholemman, Mat-8, and the γ-subunit of Na+-K+-ATPase. A number of studies have suggested that these proteins are involved in the regulation of ion transport and, in particular, functionally interact with the Na+-K+-ATPase. The present study describes the characterization, targeted disruption, and phenotypic analysis of the mouse CHIF gene. The CHIF knockout mice are viable and not distinguishable from wild-type littermates under normal conditions. Under K+ loading, they have a twofold higher urine volume and an increased glomerular filtration rate. Similar but smaller effects are observed in mice fed a low-Na+ diet. Treating K+-loaded mice for 10 days with furosemide resulted in lethality in the knockout mice (17 of 39) but not in the wild-type group (1 of 39). The data are consistent with an effect of CHIF on the Na+-K+-ATPase that is specific to the outer and inner medullary duct, its major expression site.

MTX induces cell swelling and cell death in renal tubular LLC-PK(1) cells. The tubular cell death induced by MTX is time-dependent. Cell death can be prevented by co-incubating with amiloride, thus indicating that the Na(+)/H(+) antiporter and possibly other volume regulatory factors in renal tubular cells are involved in MTX-induced renal failure.

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