Urinary measurement of Na+/H+ exchanger isoform 3 (NHE3) protein as new marker of tubule injury in critically ill patients with ARF.

BACKGROUND: It has been shown that apical sodium transporters of the renal tubule can be detected by immunoblotting of urine membrane fraction from rats. We raised the hypothesis that protein levels of the Na+/H+ exchanger isoform 3 (NHE3), the most abundant apical sodium transporter in renal tubule, should be increased in urine of patients presenting with acute renal failure (ARF) with severe tubular cell damage and thus might be a noninvasive marker of acute tubular necrosis (ATN). METHODS: Sixty-eight patients admitted to the intensive care unit were studied prospectively (54 patients with ARF, 14 controls without renal dysfunction). Patients with ARF were divided into 3 subgroups as follows: prerenal azotemia, ATN, and intrinsic ARF other than ATN. Urinary NHE3 protein abundance was estimated from semiquantitative immunoblots of urine membrane fraction samples collected from patients. The amount of urinary NHE3 was compared with the fractional excretion of sodium (FeNa) and urinary retinol-binding protein (RBP). RESULTS: NHE3 was not detected in urine from controls. Levels of urinary NHE3 normalized to urinary creatinine level were increased in patients with prerenal azotemia and 6 times as much in patients with ATN, without overlap (ATN, 0.78 +/- 0.36; prerenal azotemia, 0.12 +/- 0.08; P < 0.001). Conversely, urinary NHE3 protein was not detected in patients with intrinsic ARF other than ATN. Normalized NHE3 level correlated positively with serum creatinine level in patients with tubular injury (R2 = 0.305; P = 0.0003). Values for FeNa and normalized urinary RBP did not discriminate ATN from intrinsic ARF other than ATN and prerenal azotemia, respectively. CONCLUSION: In patients with ARF, urinary NHE3 abundance might be a novel noninvasive marker of renal tubule damage, helping to differentiate prerenal azotemia, ATN, and intrinsic ARF other than ATN.

Angiotensin II stimulates NHE3 activity by exocytic insertion of the transporter: role of PI 3-kinase.

BACKGROUND: Low-concentration angiotensin II (Ang II) stimulates Na+/H+ exchanger 3 (NHE3) activity in renal proximal tubule mainly via angiotensin II type 1 (AT1) receptors. The mechanisms that mediate the increase in NHE3 activity elicited by Ang II remain incompletely settled. METHODS: To assess a potential role of NHE3 trafficking in the Ang II effect, NHE3 activity was measured by H+-driven initial rate of 22Na uptake resistant to 50 micromol/L of the Na+/H+ exchange inhibitor cariporide (HOE642), and sensitive to 300 micromol/L ethyl isopropyl amiloride (EIPA), in a model of cultured proximal tubular cells (MKCC), in which functional apical NHE3 and AT receptors are normally present. Apical expression of NHE3 protein was determined by cell surface biotinylation and immunoblotting. RESULTS: Ang II (10-10 mol/L, 43 minutes) increased NHE3 activity and biotinylated NHE3 protein without any change in total amount of NHE3 protein. Both effects were suppressed by specific AT1 receptor antagonists. When 2-mercaptoethanesulphonic acid (MESNA) was used to cleave biotin from all apical proteins, intracellular biotinylated NHE3 protein remained unchanged after Ang II incubation compared to control. When sulfo-N-hydrosuccinimide (NHS)-acetate was used first to block all apical reactive sites, an increase in biotinylated NHE3 protein was observed following Ang II incubation. To evaluate the role of phosphatidylinositol 3-kinase (PI 3-kinase), the specific inhibitor wortmannin was used. It suppressed Ang II-induced increase in NHE3 activity and trafficking. Furthermore, latrunculin B, inhibitor of actin filament polymerization, prevented both Ang II stimulatory effects. CONCLUSION: Ang II stimulates NHE3 activity, at least in part, by exocytic insertion of the protein into the apical membrane. This effect is mediated by PI 3-kinase and required integrity of actin cytoskeleton.

Differentiated thick ascending limb (TAL) cultured cells derived from SV40 transgenic mice express functional apical NHE2 isoform: effect of nitric oxide.

Studying the apical Na/H exchanger NHE2 is difficult in the intact thick ascending limb (TAL) because of its weak expression and transport activity compared with the co-expressed NHE3. From a mouse transgenic for a recombinant plasmid adeno-SV(40) (PK4), we developed an immortalized TAL cell line, referred to as MKTAL, which selectively expresses NHE2 protein and activity. The immortalized cells retain the main properties of TAL cells. They have a stable homogeneous epithelial-like phenotype, express SV(40) T antigen and exhibit polarity with an apical domain bearing few microvilli and separated from lateral domains by typical epithelial-type junctional complexes expressing ZO1 protein. Tamm-Horsfall protein is present on the apical membrane. MKTAL cells express NHE2 and NHE1 proteins but not NHE3 and NHE4, whereby NHE2 protein is expressed selectively in the apical domain of the plasma membrane. NHE2 contributed about half of the total Na/H exchange activity. mRNAs for the Na-K-2Cl cotransporter-2 (NKCC2) and the anion exchangers AE2 and AE3 were also present. While acute exposure to NO donors did not alter NHE2 activity, chronic exposure inhibited NHE2 activity selectively and down-regulated NHE2 mRNA abundance. In conclusion, MKTAL cells retain structural and functional properties of their in vivo TAL counterparts and express functional NHE2 protein in the apical membrane, which may be inhibited by NO. Thus, MKTAL cells may be an appropriate model for studying the cellular mechanisms of NHE2 regulation.