Netrin-1 regulates colon-kidney cross talk through suppression of IL-6 function in a mouse model of DSS-colitis

Abstract

Organ cross talk is increasingly appreciated in human disease, and inflammatory mediators are shown to mediate distant organ injury in many disease models. Colitis and intestinal injury are known to be mediated by infiltrating immune cells and their secreted cytokines. However, its effect on other organs, such as the kidney, has never been studied. In the current study, we examined the effect of dextran sulfate sodium (DSS)-colitis on kidney injury and inflammation. In addition, we hypothesized that netrin-1 could modulate colon-kidney cross talk through regulation of inflammation and apoptosis. Consistent with our hypothesis, DSS-colitis induced acute kidney injury in mice. Epithelial-specific overexpression of netrin-1 suppressed both colitis and colitis-induced acute kidney injury, which was associated with reduced weight loss, neutrophil infiltration into colon mucosa, intestinal permeability, epithelial cell apoptosis, and cytokine and chemokine production in netrin-1 transgenic mice colon and kidney. To determine whether netrin-1-protective effects were mediated through suppression of IL-6, IL-6 knockout mice were treated with DSS and acute kidney injury was determined. IL-6 knockout was resistant to colitis and acute kidney injury. Moreover, administration of IL-6 to netrin-1 transgenic mice did not affect the netrin-1-protective effects on the colon and kidney, suggesting that netrin-1 may reduce both IL-6 production and its activity. The present study identifies previously unrecognized cross talk between the colon and kidney, and netrin-1 may limit distant organ injury by suppressing inflammatory mediators and apoptosis.

Keywords: IL-6; acute kidney injury; colitis; netrin-1; organ cross talk.

Fig. 1.

Endogenous and transgene netrin-1 expression in wild-type (WT) and transgenic animal colon treated with water or dextran sulfate sodium (DSS). Netrin-1 expression was determined by immunohistochemical localization and RT-PCR as described in materials and methods. A: immunohistochemical localization of endogenous mouse netrin-1 and transgene chicken netrin-1 in wild-type (WT) and transgenic colon. Netrin-1 staining was seen in epithelial cells. B: regulation of endogenous netrin-1 expression during colitis determined by RT-PCR. DSS treatment downregulated endogenous netrin-1 mRNA expression in both WT and netrin-1 transgenic animal colon. *P < 0.05 vs. water-treated. #P < 0.001 vs. DSS-treated WT. C: transgene expression in different tissues determined by RT-PCR. D: regulation of transgene netrin-1 expression in the colon by DSS. DSS induced transgene expression over water-treated animals. Values are means ± SE; n = 6–8. *P < 0.001 vs. water-treated.

Fig. 2.

Disease activity in mice with netrin-1 (N1) overexpression during DSS-colitis. Gender-, age-, and weight-matched mice with netrin-1 overexpression in the colon and their WT littermates were exposed to DSS (3.5%) for 5 days, followed by euthanasia on day 8 and harvesting of the whole colon by blunt dissection. A: daily weight measurements were obtained for each group of mice. P < 0.05 as measured by ANOVA. *P < 0.05 vs. other groups. B: at harvest, colon weight was measured for each mouse and is displayed as means ± SE. *P < 0.001 vs. other groups. C: spleen weight at harvest was measured for each mouse and is displayed as means ± SE. *P < 0.01 vs. other groups. D: interstitial permeability was determined using FITC-dextran as described in materials and methods. DSS-induced intestinal permeability was suppressed in netrin-1 transgenic animals. *P < 0.05 vs. all other groups; n = 8.

Fig. 3.

Histological changes in mice with netrin-1 overexpression during DSS-colitis. Gender-, age-, and weight-matched mice with netrin-1 overexpression in the colon and their WT littermates were exposed to DSS (3.5%) for 5 days, followed by euthanasia on day 8 and harvesting of the whole colon by blunt dissection. Top: representative histological sections from whole colon of WT and netrin-1 transgenic mice harvested following 8 days of DSS or water. Images were acquired at ×20 using Olympus BX51. Bottom: blinded histological scoring of colonic tissue post-DSS. Values are means ± SE; n = 6–8. *P < 0.001 vs. other groups.

Fig. 4.

Inflammatory cytokine and chemokine expression during DSS-colitis is suppressed by transgenic overexpression of netrin-1. A: inflammatory cytokine and chemokine expression in WT and netrin-1 transgenic animal colon treated with water or DSS determined by RT-PCR. DSS-induced inflammatory mediator expression is suppressed in netrin-1 transgenic animals. Plasma MCP-1 (B) and IL-6 (C) concentration was quantified by ELISA. DSS-induced increase in MCP-1 and IL-6 in plasma is suppressed by netrin-1 overexpression in epithelial cells. *P < 0.05 vs. all other groups. #P < 0.05 vs. water-treated.

Fig. 5.

Inflammatory cell infiltration (A) in the colon and colonic cell apoptosis (B) following DSS-colitis in WT and netrin-1 transgenic mice. Gender-, age-, and weight-matched mice with netrin-1 overexpression and their WT controls were exposed to DSS (3.5%) for 5 days, and tissue was stained with anti-neutrophil antibody and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). A: immunohistochemical localization of neutrophils in water- or DSS-treated colon from WT and netrin-1 transgenic animals was carried out as described in materials and methods. Neutrophils are indicated by arrows. Quantitative data for neutrophil infiltration are given below. DSS-induced neutrophil infiltration was significantly suppressed in netrin-1 transgenic animals. *P < 0.05 vs. all other groups. B: TUNEL staining in water- or DSS-treated colon from WT and netrin-1 transgenic animals was carried out as described in materials and methods. TUNEL-positive nuclei are seen as dark blue. Quantitative data for TUNEL-positive nuclei in five ×40 fields are given below. DSS induced a large increase in apoptotic cells, which were significantly suppressed in netrin-1 transgenic animals. *P < 0.05 vs. all other groups; n = 6–8.

Fig. 6.

Netrin-1 overexpression protects kidney from DSS-induced dysfunction. Serum creatinine was measured at 8 days after DSS treatment in WT and netrin-1 transgenic animals. Values are means ± SE. *P < 0.01 vs. all other groups; n = 6.

Fig. 7.

Endogenous and transgene netrin-1 expression in WT and transgenic animal kidney treated with water or DSS. Netrin-1 expression was determined by immunohistochemical localization and RT-PCR as described in materials and methods. A: immunohistochemical localization of transgene chicken netrin-1 in netrin-1 transgenic kidney. Netrin-1 staining was seen in proximal tubular epithelial cells. B: regulation of endogenous netrin-1 expression during colitis determined by RT-PCR. DSS treatment did not alter endogenous netrin-1 mRNA expression in both WT and netrin-1 transgenic animal kidney. *P < 0.05 vs. water-treated. #P < 0.001 vs. DSS-treated WT. C: regulation of transgene netrin-1 expression in colon by DSS. DSS did not alter transgene expression over water-treated animals. Values are means ± SE; n = 6–8.

Fig. 8.

Tubular injury was quantified as described in materials and methods. DSS treatment induced a large increase in tubular epithelial injury, which was suppressed in netrin-1 transgenic mice. Quantitative data are given at the bottom. Values are means ± SE. *P < 0.001 vs. all other groups; n = 6–8.

Fig. 9.

Neutrophil infiltration and inflammatory cytokine expression in the kidney following DSS-colitis in WT and netrin-1 transgenic mice. Gender-, age-, and weight-matched mice with netrin-1 overexpression and their WT controls were exposed to DSS (3.5%) for 5 days, and tissue was stained with anti-neutrophil antibody. Cytokine expression was quantified by real time RT-PCR. A: neutrophil staining shown as brown color. DSS-induced neutrophil infiltration was significantly suppressed in netrin-1 transgenic animals. B: DSS induced a large increase in inflammatory cytokines and chemokines, which were suppressed in netrin-1 transgenic animal kidney. *P < 0.05 vs. all other groups. C: MPO activity (as a measure of neutrophil infiltration) was measured as described in materials and methods. DSS induced a significant increase in MPO activity in the kidney over water-treated control mouse kidney, which was suppressed in netrin-1 transgenic animal kidney. *P < 0.05 vs. all other groups; n = 5.

Fig. 10.

Netrin-1 transgenic mice are highly resistant to DSS+IL-6 induced acute kidney injury, and IL-6 knockout mice are resistant to DSS-induced acute kidney injury. A: percent body weight changes in response to DSS treatment in netrin-1 transgenic and WT mice. DSS was fed for 9 days, and mice were euthanized on day 11. IL-6 or vehicle was administered (10 ng·mice⁻¹·day⁻¹) starting on day 2 and lasting until day 11. *P < 0.05 vs. all the groups. B: serum creatinine in WT control and DSS-treated, and control, vehicle-, and IL-6-treated netrin-1 transgenic animals. DSS induced a significant increase in serum creatinine, suggesting renal dysfunction, which was suppressed in netrin-1 transgenic mice. Moreover, IL-6 administration does not cause acute kidney injury in netrin-1 transgenic mice, suggesting that netrin-1 may suppress both IL-6 production and activity *P < 0.01 vs. all other groups. C: percent body weight changes in response to DSS treatment in WT and IL-6 knockout mice. DSS was fed for 7 days, and mice were euthanized on day 9. *P < 0.05 vs. DSS-treated IL-6 knockout mice. D: serum creatinine in WT and IL-6 knockout mice treated with DSS or water. DSS induced acute kidney injury as seen by increased serum creatinine (*P < 0.001 vs. water-treated) whereas IL-6 knockout mice were resistant to acute kidney injury as seen by a significant reduction in serum creatinine (#P < 0.05 vs. WT DSS-treated); n = 6–8. E–J: hematoxylin- and eosin-stained colon tissue section. E: WT water treated. F. netrin-1 transgenic DSS treated. G: IL-6 knockout water treated. H: WT DSS treated. I: netrin-1 DSS+IL-6 treated. J: IL-6 knockout DSS treated. WT DSS-treated mice colon showing damage in epithelial cell lining, glands, and large amount of white cell infiltration, which was suppressed in netrin-1 transgenic and IL-6 knockout mice.