Activation of Kir4.1/Kir5.1 of DCT is essential for acute calcineurin‐inhibition‐induced stimulation of NCC

Abstract

The inhibition of calcineurin (PP2B) with tacrolimus (FK506) or cyclosporin A (CsA), two frequently used immunosuppressive drugs after organ transplantation, has been shown to stimulate cation‐coupled Cl‐ cotransporters such as NCC and NKCC2. Also, the stimulation of NCC induced by CsA or FK506 may be responsible for PP2B‐inhibition‐induced hypertension and hyperkalemia. The aim of the present study is to test whether acute application of FK506 or CsA also stimulates the basolateral K+ channels in the distal convoluted tubule (DCT) thereby activating NCC expression/activity. We first used patch‐clamp technique to examine the effect of FK506 or CsA on the basolateral Kir4.1/Kir5.1 activity in the DCT. Acutely addition of FK506 (5‐10 uM) stimulates the 40 pS K+ (Kir4.1/Kir5.1 heterotetramer) channel activity in the DCT and increases the NPo from 1.6 to 2.6. Moreover, acutely addition of FK506 (10 uM) increases the whole‐cell K+ currents from 1180 pA to 2050 pA in the DCT cells. As consequence of the stimulation of Kir4.1/Kir5.1, K‐currents (IK) reversal potential (an index of the membrane potential) becomes more negative in FK506 treated DCT than untreated (‐73 mV vs ‐62 mV). To examine whether FK506‐induced stimulation of Kir4.1/Kir5.1 in the DCT requires the FKBP12 (12 kDa FK506 binding protein), we then examined the effect of FK506 on the basolateral K+ channels in the DCT1 of kidney‐tubule‐specific (Ks)‐FKBP12 KO mice. The control and Ks‐FKBP12 KO mice were treated with FK506 (0.75 mg/Kg BW) by peritoneal injection 30 min before experiments. The stimulatory effect of FK506 on Kir4.1/Kir5.1 of the DCT is observed only in the control but is absent in Ks‐FKBP12 KO mice, suggesting the effect of FK506 is due to the inhibition of PP2B. Moreover, we have used the whole‐cell recording technique to examine the effect of CsA on the Ba2+‐sensitive K+ currents in the control and FKBP12‐deficient mice because CsA‐induced inhibition of PP2B requires binding to cyclophilin but not to FKBP12. We observed that CsA treatment (3 mg/Kg BW by peritoneal injection 30 min before experiments) significantly increases the whole‐cell K+ currents in both control and Ks‐FKBP12 KO mice. Fluorescence image shows that CsA treatment for 30 min increased the expression of Kir4.1 and pNCC (Serine 71) in the basolateral membrane and apical membrane, respectively. To determine whether the activation of the basolateral K+ channel activity in the DCT is essential for FK506‐induced stimulation of NCC expression, we next examined the effect of FK506 treatment on the expression of pNCC and tNCC in the control (Kcnj10flox/flox) and in kidney‐tubule‐specific (Ks‐Kir4.1) KO mice. Acute FK506 treatment (peritoneal injection of FK506 at 0.75 mg/Kg BW) robustly increases the expression of pNCC and tNCC. However, the stimulatory effect of FK506 on NCC is blunted in Ks‐Kir4.1 KO mice, suggesting the role of Kir4.1 in mediating PP2B‐inhibition induced stimulation of NCC. We conclude that acute inhibition of PP2B stimulates the basolateral Kir4.1/Kir5.1 activity of the DCT and NCC and that the acute effect of PP2B inhibition on NCC is partially achieves by stimulation of the basolateral Kir4.1/Kir5.1.