丹波 嘉一郎

Background. The regulation of the epithelial sodium channel (ENaC) is a key determinant of sodium homeostasis. The effect of cyclic AMP (cAMP) on ENaC activity is tissue-specific, and is controversial when ENaC is expressed in Xenopus oocytes. Methods. The modulation of ENaC by cAMP in oocytes expressing human or rat ENaC was performed with two-electrode voltage clamping. Results. 250μM 8-(4-chlorophenylthio)-adenosine 3′, 5′-cyclic monophosphate (8-CPT-cAMP) added to the bath significantly increased normalized amiloride-sensitive currents within 60s in oocytes expressing human α, β, and γ subunits (5ng cRNA each). The cAMP effect was dose-dependent and was partially inhibited by 200μM Rp-CPT-cAMP, a competitive cAMP antagonist. A transient effect of 8-CPT-cAMP on rat ENaC activity was also observed. Oocytes expressing rat α subunits with γ subunits (which have a putative protein kinase A phosphorylation site) showed similar increases in amiloride-sensitive current with 250μM 8-CPT-cAMP, while oocytes expressing rat α sub-units with β subunits were not activated by 8-CPT-cAMP. Further, rat ENaC (but not human ENaC)-expressing oocytes were not activated by cAMP when oocytes were continuously superfused during electrophysiological recordings, suggesting that rat ENaC activation by cAMP is dependent upon the condition of oocytes during cAMP stimulation. Conclusion. The present results suggest that ENaC expressed in Xenopus oocytes can be activated by cAMP, and that the γ subunit confers sensitivity to cAMP modulation of rat ENaC activity.

Background. The regulation of the epithelial sodium channel (ENaC) is a key determinant of sodium homeostasis. The effect of cyclic AMP (cAMP) on ENaC activity is tissue-specific, and is controversial when ENaC is expressed in Xenopus oocytes. Methods. The modulation of ENaC by cAMP in oocytes expressing human or rat ENaC was performed with two-electrode voltage clamping. Results. 250μM 8-(4-chlorophenylthio)-adenosine 3′, 5′-cyclic monophosphate (8-CPT-cAMP) added to the bath significantly increased normalized amiloride-sensitive currents within 60s in oocytes expressing human α, β, and γ subunits (5ng cRNA each). The cAMP effect was dose-dependent and was partially inhibited by 200μM Rp-CPT-cAMP, a competitive cAMP antagonist. A transient effect of 8-CPT-cAMP on rat ENaC activity was also observed. Oocytes expressing rat α subunits with γ subunits (which have a putative protein kinase A phosphorylation site) showed similar increases in amiloride-sensitive current with 250μM 8-CPT-cAMP, while oocytes expressing rat α sub-units with β subunits were not activated by 8-CPT-cAMP. Further, rat ENaC (but not human ENaC)-expressing oocytes were not activated by cAMP when oocytes were continuously superfused during electrophysiological recordings, suggesting that rat ENaC activation by cAMP is dependent upon the condition of oocytes during cAMP stimulation. Conclusion. The present results suggest that ENaC expressed in Xenopus oocytes can be activated by cAMP, and that the γ subunit confers sensitivity to cAMP modulation of rat ENaC activity.

Cystatin-C is a low-molecular-weight basic protein produced at a stable rate by all nucleated cells. It is freely filtered through the renal glomeruli and primarily catabolized in the proximal tubule cells. Since the serum cystatin-C concentration is not affected by muscle mass nor inflammation, it has been postulated to be an improved marker of glomerular filtration rate (GFR) compared with the serum creatinine level. To evaluate the clinical usefulness in terms of estimation of the glomerular filtration rate (GFR), we compared the serum cystatin-C concentration with other markers of GFR, such as serum levels of creatinine (SCr), a 1-microglobulin (α1MG), and β₂-microglobulin (β₂MG) . Their variations were analyzed based on 2-hour creatinine clearance (2hCCr) as a standard marker of GFR. The logarithmic value of serum cystatin-C level showed a stronger negative correlation (-0.959) with the logarithmic value of 2hCCr than that of other markers (-0.924, -0.942, -0.888 ; SCr, α1MG, β₂MG, respectively) . Although β₂ MG showed the next strongest correlation with 2hCCr, it had a significantly lower sensitivity when M detecting mild reduction of GFR. In addition, serum cystatin-C showed the greatest area under the curve of receiver operating characteristic (ROC) of all GFR markers at both higher (90 ml/min.) and lower (70 ml/min.) cut-off value of 2hCCr. These data suggest that serum cystatin-C is useful for estimating GFR, even if the reduction of GFR is very mild.