Calcineurin inhibitors (CNIs) are immunosuppressive drugs, which are used widely to prevent rejection of transplanted organs and treat autoimmune disease. Hypertension and renal tubule dysfunction, including hyperkalemia, hypercalciuria, and acidosis often complicate their use1,2. These side effects resemble familial hyperkalemic hypertension (FHHt), a genetic disease characterized by overactivity of the renal sodium chloride co-transporter (NCC), and caused by mutations in WNK kinases. We hypothesized that CNIs induce hypertension by stimulating NCC. In wild-type mice, the CNI tacrolimus caused salt-sensitive hypertension and increased the abundance of phosphorylated NCC, and the NCC regulatory kinases WNK3, WNK4, and SPAK. The functional importance of NCC in this response was demonstrated by showing that tacrolimus did not affect blood pressure in NCC knockout mice, whereas the hypertensive response to tacrolimus was exaggerated in mice over-expressing NCC. Moreover, hydrochlorothiazide reversed tacrolimus-induced hypertension. In kidney transplant recipients treated with tacrolimus, fractional chloride excretion in response to bendroflumethiazide was greater than in controls, and renal NCC abundance was also greater, extending these observations to humans. Together, these findings indicate that tacrolimus-induced hypertension is mediated largely by NCC activation, and suggest that inexpensive and well-tolerated thiazide diuretics may be especially effective in preventing the complications of CNI treatment.
Na(+)-K(+)-2Cl(-) cotransporter (NKCC2)-mediated NaCl reabsorption in the thick ascending limb (TAL) is stimulated by AVP via V2 receptor/PKA/cAMP signaling. This process is antagonized by locally produced eicosanoids such as 20-HETE or prostaglandin E(2), which are synthesized in a phospholipase A(2)-dependent reaction cascade. Using microarray-based gene expression analysis, we found evidence for an AVP-dependent downregulation of the calcium-independent isoform of PLA(2), iPLA(2)β, in the outer medulla of rats. In the present study, we therefore examined the contribution of iPLA(2)β to NKCC2 regulation. Immunoreactive iPLA(2)β protein was detected in cultured mTAL cells as well as in the entire TAL of rodents and humans with the exception of the macula densa. Administration of the V2 receptor-selective agonist desmopressin (5 ng/h; 3 days) to AVP-deficient diabetes insipidus rats increased outer medullary phosphorylated NKCC2 (pNKCC2) levels more than twofold in association with a marked reduction in iPLA(2)β abundance (-65%; P < 0.05), thus confirming microarray results. Inhibition of iPLA(2)β in Sprague-Dawley rats with FKGK 11 (0.5 μM) or in mTAL cells with FKGK 11 (10 μM) or (S)-bromoenol lactone (5 μM) for 1 h markedly increased pNKCC2 levels without affecting total NKCC2 expression. Collectively, these data indicate that iPLA(2)β acts as an inhibitory modulator of NKCC2 activity and suggest that downregulation of iPLA(2)β may be a relevant step in AVP-mediated urine concentration.
In the present study we attempted to characterize the renal expression pattern of phospholipase A2 group VI isoforms in response to vasopressin (AVP) treatment. To this end we used microarray analysis, PCR, immunohistochemistry, and Western blot in control rats and in Brattleboro rats (DI rats) treated with the selective V2 AVP receptor agonist desmopressin (dDAVP, 5ng/h; 3d) or vehicle via osmotic minipump. Cultured rat medullary TAL cells (mTAL cells) and the R‐ and S‐enantiomere of the iPLA inhibitor, bromoenol lactone (BEL), were employed to study the effect of iPLA on NKCC2 phosphorylation. Microarray analysis and quantitative real time PCR showed a significant downregulation of iPLA2 group VIA mRNA in the outer medulla of treated animals whereas other iPLA2 isoforms were not affected. Immunohistochemistry demonstrated iPLA2 expression in the entire distal nephron. Western blotting revealed a dominant band at 70kDa which was reduced to 50±7% by dDAVP (p<0.05). Treatment of mTAL cells with 5μM S‐BEL for 1h markedly increased phospho‐NKCC2 abundance and membrane localization without effect on total NKCC2. R‐BEL was less effective. In summary, we have shown that the group VIA‐2 isoform of iPLA2 is expressed in the distal nephron of rat kidney and has an inhibitory effect on NKCC2 phosphorylation. Downregulation of iPLA may therefore be an important mechanism of AVP mediated urine concentration.
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