Renal cortex neuronal nitric oxide synthase in response to rapamycin in kidney transplantation

Abstract

Decreased renal neuronal nitric oxide synthase (nNOS) is present in various chronic kidney diseases although there is relative little known in chronic allograft nephropathy (CAN). Female sex increases the risk of acute rejection and calcineurin-inhibitor toxicity but decreases the risk of CAN. Rapamycin (RAPA) is an alternative immunosuppress although there is no information whether it is effective in females. We therefore investigated the efficacy of RAPA in both sexes and the impact of RAPA on renal cortex structure and nNOS expression. Male (M) and female (F) F344 kidneys were transplanted into same sex Lewis (ALLO) or F344 (ISO) recipients and treated with 1.6 mg/kg/day of RAPA for 10 days. Grafts were removed for renal histology and endothelial (e)NOS and neuronal (n)NOS protein measurements at 22 weeks. All ALLO rats survived without acute rejection. ALLO F survived with mild proteinuria and CAN at 22 weeks similar to ALLO M, while ISO F had better outcome than ISO M. Cortical nNOSalpha was undetectable in all RAPA groups; however, nNOSbeta transcript and protein were compensatory increased. Both ALLO and ISO F showed higher medullary nNOSalpha but lower cortical eNOS abundance than M groups. In male ALLO RAPA decreased renal cortical nNOSalpha but increased nNOSbeta expression. This may represent compensatory upregulation of nNOSbeta when nNOSalpha-derived NO is deficient.

Figure 1

(A) Glomerulosclerois and (B) Banff score in ISO and ALLO grafts 22 weeks after Tx. *p<0.05 vs. respective ALLO group, ^#p<0.05 vs. respective M group.

Figure 2

Renal cortical (left panel) and medullary (right panel) eNOS protein abundance in ISO and ALLO grafts 22 weeks after Tx. Representative western blot whole membranes show eNOS bands (~155kDa). The molecular weight marker is in the first line. +CT represents positive control. *p<0.05 vs. respective ISO group, ^#p<0.05 vs. respective M group.

Figure 3

(A) Renal cortical (left panel) and medullary (right panel) nNOSα protein abundance in ISO and ALLO grafts 22 weeks after Tx. Representative western blot whole membranes show nNOSα bands (~160 kDa). The molecular weight marker is in the first line.+CT represents positive control. ND represents not detectable. *p<0.05 vs. respective ISO group, ^#p<0.05 vs. respective M group. (B) Renal cortical nNOSα protein abundance in CsA and RAPA treated female rats. *p<0.05 CsA vs. RAPA.

Figure 4

Renal cortical nNOSα (A) and nNOSβ (B) mRNA abundance in CsA and RAPA treated ALLO male rats and controls (n=5 in each group). The r18S (C) was used as an internal standard. *p<0.05 vs. control.

Figure 5

(A) Renal cortical nNOSβ protein abundance in CsA and RAPA treated male rats and controls. Representative western blot whole membranes show nNOSα band (~160 kDa) and nNOSβ band (~140 kDa). Cere represents cerebellum used as positive control for nNOSα. KM represents kidney medulla used as positive control for nNOSβ. (B) Renal cortical nNOSβ protein abundance in CsA and RAPA treated male ALLO rats and controls (n=5 in each group). *p<0.05 vs. control.