Podocyte specific knockout ( KO ) of the natriuretic peptide clearance receptor ( NPRC ) attenuates diabetic kidney disease ( DKD )

FIGURE 1. (a) NPRC expression was significantly increased in WT Akita mice compared to non‐diabetic WT mice. There was also a significant decrease in NPRC expression in KO Akita mice compared to WT Akita mice. (b, c) In Tdtomato mice lacking Cre recombinase, Tdtomato was expressed in most cell types without significant expression of EGFP. (d, e) Tdtomato was also widely expressed in both NPRC+/+ and NPRC−/− reporter mice, with EGFP expression localized to podocytes in the glomerular tuft. (f, g) EGFP cells were isolated by flow cytometry and used for quantitative RT‐PCR to assess expression of the podocyte marker nephrin and expression of NPRC. Expression of nephrin was equally expressed in NPRC+/+ and NPRC−/− reporter mice. In contrast, expression of NPRC was difficult to detect in NPRC−/− reporter mice. A total of 49 mice were studied in Figure 1a, and a total of 12 fluorescent pictures were examined in figures b, c, d, and e. For flow cytometry, cortices from 3 to 4 mice were combined to isolate EGFP labeled cells.

FIGURE 2. Albuminuria in WT and KO mice. Albuminuria was significantly increased in both WT and KO Akita mice compared to non‐diabetic WT and KO mice. Podocyte specific KO of NPRC significantly reduced albuminuria in KO Akita mice compared to WT Akita mice at both the 16‐ and 20‐week time points. A total of 39 mice were studied at the 16‐week time point, and a total of 36 mice were studied at the 20‐week time point.

FIGURE 3. Renal pathology: (a–d). Representative pictures of non‐diabetic and Akita mice. (e) Number of WT‐ and KO‐Akita mice with mesangial expansion. (f) Severity of mesangial expansion in WT‐ and KO‐Akita mice. No mesangial expansion was detected in non‐diabetic mice. These studies were performed using 20‐week‐old mice. A total of 36 kidney specimens were examined for the histologic studies.

FIGURE 4. Expression of fibrotic markers: (a–c). There was a significant increase in fibronectin (FN) and collagen 1a1 in WT Akita mice compared to non‐diabetic controls by Q‐RT‐PCR. The increase in fibronectin and collagen 1a1 mRNA was significantly reduced in NPRC‐KO Akita mice compared to WT Akita mice. (d–g). The expression pattern was similar for alpha‐SMA and fibronectin proteins, but the differences were not statistically significant. These studies were performed using 20‐week‐old mice. A total of 50 mRNA samples were used for each Q‐RT‐PCR study. A total of 30 protein samples were used for each immunoblotting study. Results for non‐diabetic WT and KO mice were similar, and these data were combined for the studies (controls).

FIGURE 5. Expression of nephrin and podocin. (a) Expression of nephrin mRNA was similar in WT Akita mice, KO Akita mice, and non‐diabetic controls, and no statistically significant differences were noted between the groups. (b) There were no statistically significant differences in podocin mRNA expression in WT Akita mice, KO Akita mice, or non‐diabetic controls. (c, d) There were no statistically significant differences in expression of nephrin protein between WT Akita mice, KO Akita mice, or non‐diabetic controls. These studies were performed using 20‐week‐old mice. A total of 28 mRNA samples were used for each Q‐RT‐PCR study. A total of 20 protein sampled were used for each immunoblotting study. Results for non‐diabetic WT and KO mice were similar, and these data were combined for the studies (controls).