FIG 11.

(A) TEM of Shp2^fl/fl and Shp2^fl/fl, podocin CreTg/+ mouse glomeruli at 48 h after injection of NTS or control sheep IgG. Original magnification, ×4,800. (B) Numbers of junctions per micrometer of glomerular basement membrane, as seen by TEM 48 h after injection (means ± standard errors of the mean). (C) Quantification of proteinuria in NTS model of podocyte injury. Albumin and creatinine were quantified in urine samples obtained from Shp2^fl/fl,Cre− and Shp2^fl/fl,Cre+ mice following injection of sheep IgG (control) and NTS at various time points. There was a sharp rise in urinary albumin leakage following NTS injection in the Shp2^fl/fl,Cre− mice that resolved over the next 5 to 7 days. There was a milder increase in albuminuria in the Shp2^fl/fl,Cre+ mice following NTS injection that resolved completely by day 3. There was no increase in urine albumin leakage in the mice injected with sheep IgG that served as a control. The data are representative of 4 experiments with 3 mice per group. There was a statistically significant difference (*, P < 0.001) in albuminuria between the NTS-injected Shp2 WT and Shp2 KO animals at the 24-h, 48-h, and 72-h time points. There was a different statistically significant albuminuria between NTS- and sheep IgG-injected WT Shp2 mice at the 24-h, 48-h, and 72-h time points (**, P < 0.001). At 24 h and 48 h, NTS- and sheep IgG-injected Shp2 KO mice had statistically different albuminurias (***, P < 0.001). The data are means ± standard errors of the mean. (D) Glomeruli from Shp2^fl/fl,Cre− and Shp2^fl/fl,Cre+ mouse kidneys perfused with sheep IgG (control) and NTS were isolated using Dynabeads. Glomerular lysates were prepared, resolved using SDS-PAGE, and blotted with the indicated antibodies. Increase in nephrin phosphorylation (Y1193 and Y1217) was abrogated in mice with Shp2 deleted. The two blots shown for total nephrin represent separate experiments in which nephrin phosphorylation was assessed. There was a decrease in Src Y416 phosphorylation and enhanced Y527 phosphorylation in glomerular lysates from Shp2^fl/fl,Cre+ mice following NTS perfusion compared to control and NTS-perfused Shp2^fl/fl,Cre− mice. Shp2 Y542 phosphorylation was also decreased in Shp2^fl/fl,Cre+ glomerular lysates following NTS perfusion compared to control and NTS-perfused Shp2^fl/fl,Cre− mice. The data are representative of 3 mice per group. (Quantification of bands using densitometry from three separate experiments is shown in Fig. S2 in the supplemental material.)