Differential control of COX‐2 expression in macula densa cells under calcineurin inhibition by cyclosporin A

Abstract

ObjectiveCalcineurin inhibitors such as cyclosporin A (CsA) are in use as immunosuppressive drugs to prevent rejection of transplanted organs. Despite positive outcomes, side effects such as decreased glomerular filtration rate and overall functional and structural deterioration may affect the kidney. Among the causes, interactions of vasoactive systems have been considered. We therefore studied how CsA may cause dysregulation of key juxtagomerular signaling components.MethodsWistar rats were divided into 4 groups receiving vehicle, 25mg CsA/kg/d, 5mg candesartan (Cand)/kg/d or CsA + Cand for 3 weeks. Organs were perfusion‐fixed and embedded for morphology, or removed for biochemical and physiological analysis. Cultured macula densa (MD) cells were treated with CsA (5 to 40 μM) and angiotensin II (Ang II; 1 to 5 μM) for 6 or 24 h. Calcineurin isoforms were silenced in MD cells and studied after 72h. Immunoreactive signals of cyclooxygenase‐1 (COX‐1), cyclooxygenase‐2 (COX‐2), nitric oxide synthase (NOS), nuclear factor of activated T‐cells (NFAT) subtypes 1 to 4, and activating p38 mitogen‐activated protein kinase (p38 MAPK) were evaluated. Inhibitor to p38 MAPK (SB203580; 10 μM) was applied in MD cells. Cell parameters were further recorded by quantitative PCR and Western blot.ResultsCsA caused activation of the renin angiotensin system (RAS) with hyperplasia of renin granular cells and upregulation of renin biosynthesis in the in vivo situation. Juxtaglomerular COX‐2 expression was suppressed. An assumed link between NFAT subtypes and COX‐2 signal of MD could not be established. Additional administration of Ang II receptor blocker, candesartan, reversed the CsA‐induced COX‐2 suppression in MD. COX‐1 and NOS expression levels were unaffected by CsA or candesartan. Contrastingly, in cultured MD cells CsA caused a rise in COX‐2 mRNA and protein abundance after 6 or 24 hours; p38 MAPK phosphorylation was increased in parallel by > 40%. Along with this, knockdown of calcineurin isoforms in MD cells increased the expression of COX‐2 by > 50%. Inhibition of p38 MAPK attenuated CsA‐induced COX‐2 upregulation by 40%. AngII substantially decreased the baseline expression of COX‐2 by 25% (6h) and blunted CsA‐induced COX‐2 stimulation by 40% (6h), likely via inhibition of p38 MAPK. All data were significant (minimum p<0.05).ConclusionsIn summary, calcineurin inhibition by CsA in vivo suppresses juxtaglomerular COX‐2 biosynthesis independently of NFAT signaling. Suppression is causally linked to a stimulated RAS. Contrastingly, cultured MD cells show substantial stimulation of COX‐2 biosynthesis upon CsA, involving p38 MAPK. AngII has an inhibitory effect on this cascade, since the effect was blocked by candesartan. This novel, MD cell‐specific regulatory synergism of calcineurin and angiotensin, governing COX‐2 biosynthesis, may serve to address juxtaglomerular dysregulation under CsA treatment.Support or Funding InformationThis study was funded by SFB (Sonderforschungsbereich 1365) in Germany.