Partial netrin-1 deficiency aggravates acute kidney injury

Abstract

The netrin family of secreted proteins provides migrational cues in the developing central nervous system. Recently, netrins have also been shown to regulate diverse processes beyond their functions in the brain, incluing the ochrestration of inflammatory events. Particularly netrin-1 has been implicated in dampening hypoxia-induced inflammation. Here, we hypothesized an anti-inflammatory role of endogenous netrin-1 in acute kidney injury (AKI). As homozygous deletion of netrin-1 is lethal, we studied mice with partial netrin-1 deletion (Ntn-1(+/-) mice) as a genetic model. In fact, Ntn-1(+/-) mice showed attenuated Ntn-1 levels at baseline and following ischemic AKI. Functional studies of AKI induced by 30 min of renal ischemia and reperfusion revealed enhanced kidney dysfunction in Ntn-1(+/-) mice as assessed by measurements of glomerular filtration, urine flow rate, urine electrolytes, serum creatinine and creatinine clearance. Consistent with these findings, histological studies indicated a more severe degree kidney injury. Similarly, elevations of renal and systemic inflammatory markers were enhanced in mice with partial netrin-1 deficiency. Finally, treatment of Ntn-1(+/-) mice with exogenous netrin-1 restored a normal phenotype during AKI. Taking together, these studies implicate endogenous netrin-1 in attenuating renal inflammation during AKI.

Figure 1. Renal netrin-1 expression in wild-type mice (Ntn1^+/+) or mice with partial netrin-1 deficiency (Ntn1^+/−).

(A) Real-time RT-PCR analysis of the expression of Ntn1 mRNA in the kidneys of Ntn1^+/+ and Ntn1^+/− mice, presented relative to β-actin. Data are representative of four mice in each group (mean ± SD). (B,C) Expression of netrin-1 protein in Ntn1^+/+ or Ntn1^+/− mice. (D,E) Expression of netrin-1 protein in kidneys of Ntn1^+/+ or Ntn1^+/− mice following 30 minutes of ischemia and 2 hours reperfusion (+I) compared to control kidneys without ischemia (−I). Each blot was re-probed for β-actin to control for loading. One representative of three blots is displayed.

Figure 2. Immunohistochemical localization of renal netrin-1 and netrin-1 tissue content and urine concentration following ischemia in vivo.

Immunohistolochemical staining for netrin-1 in kidneys of Ntn1^+/− mice or their respective age-, weight-, and gender-matched littermate controls (Ntn1^+/+) following 30 minutes ischemia and 2 hours reperfusion. (A) Netrin-1 protein is mainly expressed in tubule cells of Ntn1^+/+ mice under basal conditions without ischemia (−I) and (B) is increased following ischemia (+I). (C,D) This increase of netrin-1 expression following ischemia is attenuated in Ntn1^+/− mice. Arrows indicate tubules with netrin-1 expression. (magnification 400×). (E) Renal and (F) urine netrin-1 content were assessed by ELISA (mean ± SD; n = 6–8).

Figure 3. In vitro expression of netrin-1 in HK-2 cells.

(A) Expression of netrin-1 in human renal epithelial cells (HK-2 cells) following exposure to hypoxia (1% O₂) for indicated time periods. One representative blot of three is displayed. (B) Quantification or netrin-1 protein in HK-2 cells relative to β-actin.

Figure 4. Renal function in mice with partial deficiency for netrin-1 (Ntn1^+/−) exposed to ischemic AKI.

Previously characterized Ntn1^+/− mice or their respective age-, weight-, and gender-matched littermate controls (Ntn1^+/+) underwent right nephrectomy and were exposed subsequenctly to AKI induced by 30 minutes of left renal artery ischemia. (A) Glomerular filtration rates (as measured by FITC-inulin clearance) were obtained after 1 hour of reperfusion. (B) Urinary flow rate, (C) urinary potassium excretion, (D) serum creatinine and (E) creatinine clearance were obtained 24 hours following reperfusion. Data are representative of six to eight independent experiments for each experimental condition (mean ± SD).

Figure 5. Histological tissue insure induced by AKI in mice with partial netrin-1 deficiency (Ntn1^+/−) or control mice (Ntn1^+/+).

Renal histology in Ntn1^+/− mice exposed to renal ischemia or age-, weight-, and gender-matched littermate controls (Ntn1^+/+) were subjected to 30 minutes of left renal artery ischemia. Renal histology was obtained after 24 hours of reperfusion. (A–D) Representative H&E staining (400×). Arrow marks destructed tubules. (E) Quantification of histological tissue damage assessed by Jablonski index.

Figure 6. Renal inflammatory changes in Ntn1^+/− mice following ischemia.

Ntn1^+/− mice and their respective age-, weight-, and gender-matched littermate controls (Ntn1^+/+) were subjected to 30 minutes of left renal artery ischemia. (A–D) Neutrophil staining. Arrows indicate neutrophils (magnification 400×). (E) Quantification of neutrophil tissue accumulation by measurement of myeloperoxidase (MPO). (F) TNF-α and (G) interleukin-6 (IL-6) and (H) interleukin-10 (IL-10) were assessed by real-time RT-PCR from renal tissues. Data were calculated relative to ß-actin and are expressed as fold change compared to sham-operated animals without ischemia (−I). Data are representative of four to six independent experiments for each experimental condition (mean ± SD).

Figure 7. Reconstitution of Ntn1^+/− mice with exogenous netrin-1.

Renal function and inflammation in mice with partial deficiency for netrin-1 (Ntn1^+/−) treated with exogenous netrin (5 µg/mouse I.V.) or vehicle prior to 30 minutes of renal ischemia. (A) Glomerular filtration rate (as measured by FITC-inulin clearance) was measured after 1 hour of reperfusion. (B) Quantification of neutrophil tissue accumulation by measurement of myeloperoxidase (MPO) (mean ± SD; n = 6–8).