Chronic regulation of the renal Na(+)/H(+) exchanger NHE3 by dopamine: translational and posttranslational mechanisms.

The intrarenal autocrine/paracrine dopamine (DA) system contributes to natriuresis in response to both acute and chronic Na(+) loads. While the acute DA effect is well described, how DA induces natriuresis chronically is not known. We used an animal and a cell culture model to study the chronic effect of DA on a principal renal Na(+) transporter, Na(+)/H(+) exchanger-3 (NHE3). Intraperitoneal injection of Gludopa in rats for 2 days elevated DA excretion and decreased total renal cortical and apical brush-border NHE3 antigen. Chronic treatment of an opossum renal proximal cell line with DA decreased NHE3 activity, cell surface and total cellular NHE3 antigen, but not NHE3 transcript. The decrease in NHE3 antigen was dose and time dependent with maximal inhibition at 16-24 h and half maximal effect at 3 × 10(-7) M. This is in contradistinction to the acute effect of DA on NHE3 (half maximal at 2 × 10(-6) M), which was not associated with changes in total cellular NHE3 protein. The DA-induced decrease in total NHE3 protein was associated with decrease in NHE3 translation and mediated by cis-sequences in the NHE3 5'-untranslated region. DA also decreased cell surface and total cellular NHE3 protein half-life. The DA-induced decrease in total cellular NHE3 was partially blocked by proteasome inhibition but not by lysosome inhibition, and DA increased ubiquitylation of total and surface NHE3. In summary, chronic DA inhibits NHE3 with mechanisms distinct from its acute action and involves decreased NHE3 translation and increased NHE3 degradation, which are novel mechanisms for NHE3 regulation.

The reduction of Na/H exchanger-3 protein and transcript expression in acute ischemia-reperfusion injury is mediated by extractable tissue factor(s).

Ischemic renal injury is a formidable clinical problem, the pathophysiology of which is incompletely understood. As the Na/H exchanger-3 (NHE3) mediates the bulk of apical sodium transport and a significant fraction of oxygen consumption in the proximal tubule, we examined mechanisms by which ischemia-reperfusion affects the expression of NHE3. Ischemia-reperfusion dramatically decreased NHE3 protein and mRNA (immunohistochemistry, immunoblot, and RNA blot) in rat kidney cortex and medulla. The decrease in NHE3 protein was uniform throughout all tubules, including those appearing morphologically intact. In the kidney cortex, a decrease in NHE3 surface protein preceded that of NHE3 total protein and mRNA. Kidney homogenates from rats exposed to mild renal ischemia-reduced cell surface NHE3 protein expression in opossum kidney cells in vitro, whereas homogenates from animals with moderate-to-severe ischemia reduced both total NHE3 protein and mRNA. The decrease in total NHE3 protein was dependent on the proteasomal degradation associated with NHE3 ubiquitylation measured by coimmunoprecipitation. The transferable factor(s) from the ischemic homogenate that reduce NHE3 expression were found to be heat sensitive and to be associated with a lipid-enriched fraction, and did not include regulatory RNAs. Thus, transferable factor(s) mediate the ischemia-reperfusion injury-induced decrease in NHE3 of the kidney.

Reduction of renal triglyceride accumulation: effects on proximal tubule Na+/H+ exchange and urinary acidification.

One main pathophysiological mechanism underlying the increased risk for uric acid nephrolithiasis in humans with the metabolic syndrome is the excretion of unduly acidic urine, in part because of reduced excretion of the main urinary buffer, ammonium. The Zucker diabetic fatty (ZDF) rat, an established rodent model of the metabolic syndrome, has similar urinary abnormalities, attributed in part to lower expression and activity of the principal mediator of proximal tubule ammonium excretion, brush-border membrane Na+/H+ exchanger 3 (NHE3). These defects are associated with renal tubular steatosis in ZDF rats, but the causal relationship between renal steatosis and defective urinary acidification has not been investigated in vivo. We hypothesized that reduction of renal steatosis would commensurately normalize urinary acidification parameters. We treated ZDF rats with thiazolidinediones to reduce nonadipose tissue steatosis. Four weeks of treatment reduced renal triglyceride accumulation and restored urinary acidification parameters in ZDF rats to levels comparable to their lean littermates; urinary acidification was not affected by treatment in lean rats. To further document the direct effects of fat, we showed that functional abnormalities induced by fat loading in a cell culture model of proximal tubule steatosis and lipotoxicity can be reversed by fat removal but not by thiazolidinediones alone. Together, these findings support the causative role of renal steatosis in the pathogenesis of urinary acidification defects, demonstrate reversibility upon lipid removal, and highlight a potential therapeutic strategy for renal abnormalities in the metabolic syndrome.

Effect of renal lipid accumulation on proximal tubule Na+/H+ exchange and ammonium secretion.

Patients with metabolic syndrome have increased risk of uric acid nephrolithiasis due to lower urinary pH and impaired ammonium excretion. The pathophysiology underlying these urinary changes is unknown. We used two animal models and a cell culture model to study whether the alteration in renal acidification is associated with renal fat infiltration (steatosis). Compared with pair-fed lean control rats, Zucker diabetic fatty rats have higher renal triglyceride content, decreased urinary ammonium and pH, and lower levels of brush border membrane Na(+)/H(+) exchanger-3 (NHE3), a major mediator of ammonium excretion. High-fat feeding in Sprague-Dawley rats results in transient lowering of urinary ammonium and pH, with all parameters returning to normal when the animals resumed eating normal chow. This is consistent with an absence of diet-induced renal steatosis in these animals. To examine the direct effect of fat accumulation, we incubated opossum kidney (OKP) cells with a mixture of long-chain fatty acids and found accumulation of intracellular lipids with concomitant dose-dependent decrease in NHE3 activity, surface biotin-accessible NHE3 protein, and ammonium secretion. A lower dose of fatty acids that leads to intracellular lipid accumulation but does not change baseline NHE3 is sufficient to abolish the stimulation of NHE3 by insulin and to partially block the stimulation of NHE3 by glucocorticoid hormones; acid regulation of NHE3 in lipid-loaded OKP cells is not affected. These findings suggest that renal steatosis decreases ammonium secretion in the proximal tubule, in part by reducing NHE3 activity and by impairing the regulation of NHE3 by specific agonists.

A sperm-specific Na+/H+ exchanger (sNHE) is critical for expression and in vivo bicarbonate regulation of the soluble adenylyl cyclase (sAC).

We previously identified a sperm-specific Na(+)/H(+) exchanger (sNHE) principally localized to the flagellum. Disruption of the sNHE gene in mice resulted in absolute male infertility associated with a complete loss of sperm motility. Here, we show that the sNHE-null spermatozoa fail to develop the cAMP-dependent protein tyrosine phosphorylation that coincides with the functional maturation occurring upon incubation in capacitating conditions in vitro. Both the sperm motility defect and the lack of induced protein tyrosine phosphorylation are rescued by the addition of cell-permeable cAMP analogs, suggesting that cAMP metabolism is impaired in spermatozoa lacking sNHE. Our analyses of the bicarbonate-dependent soluble adenylyl cyclase (sAC) signaling pathway in sNHE-null sperm cells reveal that sNHE is required for the expression of full-length sAC, and that it is important for the bicarbonate stimulation of sAC activity in spermatozoa. Furthermore, both codependent expression and coimmunoprecipitation experiments indicate that sNHE and sAC associate with each other. Thus, these two proteins appear to be components of a signaling complex at the sperm flagellar plasma membrane. We propose that the formation of this complex efficiently modulates intracellular pH and bicarbonate levels through the rapid and effective control of sAC and sNHE activities to facilitate sperm motility regulation.

Dopamine acutely decreases apical membrane Na/H exchanger NHE3 protein in mouse renal proximal tubule.

BACKGROUND: Dopamine is a principal natriuretic hormone in mammalian Na+ homeostasis. Dopamine acutely alters glomerular filtration rate (GFR) and decreases Na+ absorption in both the proximal and distal nephron. Proximal tubule natriuresis is effected through inhibition of the apical membrane Na/H exchanger NHE3. METHODS: We examined whether dopamine directly and acutely decreases apical membrane NHE3 protein using renal tissue in two in vitro systems: renal cortical slices and in vitro perfused single tubules. After incubation with dopamine, NHE3 activity was measured by 22Na flux and NHE3 antigen was measured by immunoblot in apical membrane and total cellular membranes. RESULTS: Direct application of dopamine to either cortical slices or microperfused tubules acutely decreases NHE3 activity and antigen at the apical membrane of the proximal tubule. No change in total cellular NHE3 was detected. CONCLUSION: One mechanism by which dopamine causes natriuresis is via direct and acute reduction of NHE3 protein at the apical membrane via changes in NHE3 protein trafficking.