Protective role for netrin-1 during diabetic nephropathy

Abstract

Recent studies implicate neuronal guidance molecules in the orchestration of inflammatory events. For example, previous studies demonstrate a functional role for netrin-1 in attenuating acute kidney injury. Here, we hypothesized a kidney-protective role for netrin-1 during chronic kidney disease, such as occurs during diabetic nephropathy. To study the role of netrin-1 during diabetic nephropathy, we induced diabetes in mice at the age of 8 weeks by streptocotozin (STZ) treatment. Sixteen weeks after STZ treatment, we examined the kidneys. Initial studies in wild-type mice demonstrated robust induction of renal, urinary, and plasma netrin-1 protein levels during diabetic nephropathy. Subsequent genetic studies in mice with partial netrin-1 deficiency (Ntrn1(+/-) mice) revealed a more severe degree of diabetic nephropathy, including more severe loss of kidney function (albuminuria, glomerular filtration rate, histology). We subsequently performed pharmacologic studies with recombinant netrin-1 treatment given continuously via osmotic pump. Indeed, netrin-1 treatment was associated with attenuated albuminuria and improved histologic scores for diabetic nephropathy compared to controls. Consistent with previous studies implicating purinergic signaling in netrin-1-elicited tissue protection, mice deficient in the Adora2b adenosine receptor were not protected. Taken together, these studies demonstrate a functional role for endogenous netrin-1 in attenuating diabetic kidney disease.

Figure 1. Netrin-1 is induced by diabetic nephropathy

Age-, sex-, and weight-matched mice were subjected to streptocotozin (STZ)-induced diabetes for 16 weeks until organs were removed for analysis. Wild-type mice show (a) increased renal netrin-1 concentrations, (b) increased urine netrin-1 excretion related to creatinine and (c) no significant increase in netrin-1 serum concentrations following 16 weeks of diabetes compared to wild-type controls without diabetes measured by ELISA (n=3–4).

Figure 2. Diabetic nephropathy is increased in Ntn-1^+/−mice. (a)

Ntn-1^+/− mice and age-, weight-and gender-matched wildtype mice were subjected to streptozotocin (STZ)-induced diabetes for 16 weeks until measurement of (b) renal netrin-1 content, (c) body weight, (d) kidney weight, (e) drink volume, (f) urine volume, (g) glomerular filtration rate and (h) albumin excretion, (n=4–6 in each group).

Figure 3. Severe histologic injury in Ntn-1^+/− mice with diabetic nephropathy

(a) Extracellular matrix deposition as determined by PAS staining. PAS staining was increased in diabetic wild-type mice and in particular in Ntn-1^+/− mice (arrows). (b) histological scores of PAS staining sections. (c) Glomerular size in all groups. Panels b and c show result of at least 4–10 glomeruli in sections of two to four different mice.

Figure 4. Treatment with recombinant netrin-1 attenuates diabetic nephropathy in wild-type mice

Wild-type mice were subjected to streptozotocin (STZ)-induced diabetes for 16 weeks. (a) Two weeks following STZ injection and confirmation of diabetes (glucose levels above 400 mg/dl) mice were treated with netrin-1 or vehicle via Alzet pump (2µg/mouse/per day). Delivery time of the Alzet pumps was six weeks thus they were replaced at week 10. After 16 weeks of diabetes with or without netrin-1 treatment (b) body weight, (c) kidney weight, (d) drink volume, (e) urine volume and (f) albumin excretion (n=4–6 in each group) were determined.

Figure 5. Treatment with recombinant netrin-1 attenuates histologic injury in wild-type mice with diabetic nephropathy

Wild-type mice were subjected to streptozotocin (STZ)-induced diabetes for 16 weeks. Two weeks following STZ injection and confirmation of diabetes (glucose levels above 400 mg/dl) mice were treated with netrin-1 or vehicle via Alzet pump (2µg/mouse/per day). Delivery time of the Alzet pumps was six weeks thus they were replaced at week 10. (a) Extracellular matrix deposition as determined by PAS staining. PAS staining was attenuated in diabetic wild-type mice with netrin-1 treatment compared to wild-type mice without treatment (arrows). (b) histological scores of PAS staining sections. (c) Glomerular size in all groups. Panels b and c show result of at least 4–10 glomeruli in sections of two to four different mice.

Figure 6. The protective effect of treatment with recombinant netrin-1 is absent in Adora2b^−/− mice

Adora2b^−/− mice were subjected to streptozotocin (STZ)-induced diabetes for 16 weeks. (a) Two weeks following STZ injection and confirmation of diabetes (glucose levels above 400 mg/dl) mice were treated with netrin-1 or vehicle via Alzet pump (2µg/mouse/per day). Delivery time of the Alzet pumps was six weeks thus they were replaced at week 10. After 16 weeks of diabetes with or without netrin-1 treatment (b) body weight, (c) kidney weight, (d) drink volume, (e) urine volume and (f) albumin excretion (n=4–6 in each group) were determined.