Decrease in IL-10 and increase in TNF-α levels in renal tissues during systemic inhibition of nitric oxide in anesthetized mice

Abstract

Earlier, we demonstrated that the inhibition of nitric oxide synthase (NOS) by nitro-l-arginine methyl ester (l-NAME) infusion increases the endogenous production of proinflammatory cytokine, tumor necrosis factor (TNF-α). In the present study, we examined the hypothesis that inhibition of nitric oxide (NO) production leads to the suppression of interleukin (IL)-10 (anti-inflammatory cytokine) generation which facilitates the enhancement of TNF-α production endogenously. Using appropriate enzyme-linked immunosorbent assay kits and immunohistochemical staining, the levels of IL-10 and TNF-α in plasma (P) and in renal tissues (R) were measured in anesthetized mice (C57BL/6; ~10 weeks age; n = 6/group) infused with or without l-NAME (200 μg/min/kg; i.v. for 2 h). Compared to vehicle-treated control mice, l-NAME-treated mice had a lower level of IL-10 (P, 0.3 ± 0.1 vs. 2.6 ± 0.6 ng/mL; R, 0.5 ± 0.1 vs. 3 ± 0.1 ng/mg protein) and a higher level of TNF-α (P, 432 ± 82 vs. undetected pg/mL; R, 58 ± 7 vs. 6 ± 5 pg/mg protein). IL-10 protein expression, present mostly in the distal nephron segments in control mice, was markedly downregulated in l-NAME-treated mice. Compared to control mice, TNF-α expression increased 2.5-fold in renal cortical sections (mostly in the distal nephron segments) in l-NAME-treated mice. Coinfusion of a NO donor, S-nitroso-N-acetyl-penicillamine (SNAP; 25 μg/min/kg) with l-NAME in a separate group of mice prevented these changes in IL-10 and TNF-α induced by l-NAME. IL-10 infusion (0.075 ng/min/g) in l-NAME-treated mice markedly attenuated l-NAME-induced increments in TNF-α. Thus, these results demonstrate that NOS inhibition decreases endogenous IL-10 generation and thus, minimizes its immune downregulating action on the TNF-α production in the kidney.

Keywords: IL‐10; SNAP; TNF‐α; kidney; l‐NAME; nitric oxide synthase.

Figure 1.

Schematic diagram of the experimental protocol followed in the present study. C1‐4, Clearance periods for 30 min duration each. S1‐2, Stabilization periods preceding each drug infusion. l‐NAME, nitro‐l‐arginine methyl ester; SNAP, S‐nitroso‐N‐acetylpenicillamine; IL‐10, interleukin‐10.

Figure 2.

Levels of IL‐10 (interleukin‐10) in plasma (A) and in renal tissue (B) in response to l‐NAME (nitro‐l‐arginine methyl ester; 200 μg/min/Kg) infusion with or without coinfusion of SNAP (S‐nitroso‐N‐acetylpenicillamine; a NO donor; 25 μg/min/Kg) or exogenous IL‐10 (0.075 ng/min/g) in anesthetized mice. Values are means ± SE; n = 6 animals/group. *P <0.05 versus control group; ^#P <0.05 versus l‐NAME group.

Figure 3.

(A–C) Immunoexpression for interleukin‐10 (IL‐10) in renal tissues. IL‐10 protein is expressed in the renal tissue (mainly in the distal tubular cells) in control mice (A). Nitro‐l‐arginine methyl ester (l‐NAME) treatment reduces IL‐10 protein expression in the renal tissue (B). SNAP treatment in the presence of l‐NAME restored renal IL‐10 expression (C). TAL, Thick ascending limb of the loop of Henle; CD, collecting ducts. Microphotograph was taken using a 20× magnification objective. (D) illustrates the mean values of the comparative renal expression for IL‐10 protein in various conditions. Values are means ± SE; n = 6 animals/group. *P <0.05 versus control group; ^#P <0.05 versus l‐NAME group.

Figure 4.

Levels of tumor necrosis factor (TNF‐α) in plasma (A) and in renal tissue (B) in response to nitro‐l‐arginine methyl ester (l‐NAME) infusion with or without coinfusion of SNAP (S‐nitroso‐N‐acetylpenicillamine; a NO donor) or IL‐10 in anesthetized mice. UD, undetected level. Values are means ± SE; n = 6 animals/group. *P <0.05 versus control group (UD considered as zero value for statistical purpose); ^#P <0.05 versus l‐NAME group.

Figure 5.

(A–D) Immunoexpression for TNF‐α in renal tissue. TNF‐α immunoexpression was minimal in the renal tissue slices of control mice (A), markedly present in the renal tissue (mainly in the loop of Henle and distal nephron segments) of l‐NAME‐treated mice (B), but not in mice coinfused with SNAP (C) and IL‐10 (D). TAL, Thick ascending limb of the loop of Henle; CD, collecting ducts. Microphotograph was taken using a 20× magnification objective. (E) illustrates mean values of the comparative renal expression of TNF‐α protein in various conditions. Values are means ± SE; n = 6 animals/group. *P <0.05 versus control group, ^#P <0.05 versus l‐NAME group.