#1918 L-NAME accelerates the progression of diabetic kidney disease in mice

Abstract Background and Aims Diabetic kidney disease (DKD) represents one of the major causes of end stage kidney disease worldwide. Despite DN having such a high incidence, mouse models of DN rarely mimic all aspects of the human disease, besides showing very slow progression and sometimes maintenance issues. For this reason, in this project we aimed to develop an accelerated, reproducible and accessible mouse model of DN. Method For our experiments we chose 9-week-old female and male of the leptin receptor knock-out (BKS.Cg-Dock7m +/+ Leprdb/J) mice as type 2 diabetes model. As these mice are still not prone to develop severe kidney injury despite their diabetic condition, we administered the eNOS inhibitor L-NAME for 6 weeks, in order to suppress nitric oxide production and therefore to aggravate kidney damage. Mice were divided in three groups: one vehicle control group, one receiving 40/mg/kg/day of L-NAME and one receiving 80/mg/kg/day of L-NAME dissolved in drinking water. Blood pressure and glomerular filtration rate (GFR) were measured at different time points, together with glycemia and albuminuria. Weight and water intake were also monitored throughout the study. At the end of the experiment, animals were killed and blood and organs collected for further analysis. Results Mice treated with 80 mg/kg/day of L-NAME showed an average increase of 10 mmHg in systolic blood pressure, with a peak of 30 mmHg increase at week 4 of treatment. On the other hand, 40 mg/kg/day of L-NAME did show any effect on blood pressure. Despite the different effect on blood pressure, three weeks of L-NAME administration caused a significant reduction in GFR at both doses, with 5% reduction for the 40 mg-group and 23% for the 80 mg-group. For the 80 mg-group we observed an even further GFR decline after 5 weeks of treatment, with a 28% reduction. No change in GFR was observed in the controls. This decrease in GFR was accompanied by a 10-fold increase in urinary albumin for both experimental groups already after 3 weeks of treatment, which after 4 weeks slightly declined but remained significantly higher than the controls (p 0.0001). At a histological level, PAS staining of kidney sections revealed that in particular mice treated with 80 mg of L-NAME have enlarged Bowman capsule and glomerular tuft area, besides a reduced glomerular capillary density, coinciding with mesangial expansion. We also stained for overall collagen deposition with picrosirius red staining in order to detect possible fibrosis formation. We then observed that 80 mg of L-NAME accelerates interstitial fibrosis in the cortical area of the kidney, which also overlapped with an increased deposition of collagen I. This increase in collage I was also detected in kidney cortex of mice treated with 40 mg of L-NAME, although their overall collagen deposition and therefore fibrotic tissue did not differ from the control group. The medullar region of kidneys was also analyzed, but none of the two experimental groups revealed significant differences in terms overall collagen or collagen I deposition. Unlike mesangial expansion and fibrosis, L-NAME did not influence microphages infiltration, as revealed by CD68 staining in kidney sections. This suggests that L-NAME treatment does not accelerate kidney damage by triggering or accelerating inflammatory processes. This was also confirmed by qPCR analysis of kidney tissue, where the expression pro-inflammatory markers, such as IL1β, TNFα and MCP-1, did not change in the experimental groups. Conclusion Our results show that L-NAME considerably accelerates the progression of DKD in diabetic mice, with GFR declining and progressive albuminuria already after 3 weeks. Such disfunction is confirmed at 6 weeks by histological analysis, where mesangial expansion and fibrosis were detected. This model can be used to assess the therapeutic potential of novel interventions aimed to slow down DKD.