Figure 3.

Mof deletion in glomerular podocytes results in an inability to respond to stress. (a) Mof^fl/fl;Nphs2-Cre^T/+ mice were crossed with tomato^fl/+>eGFP animals to produce Mof^fl/fl;Nphs2-Cre^T/+;tomato^fl/+>eGFP^T/+ progeny. Tomato is ubiquitously expressed in these mice, with the exception of podocytes, where the Nphs2-Cre splices out the Tomato and activates eGfp expression. (b) In vitro quantification of proliferating Mof-deleted and control glomeruli isolated from 10-day-old mice. Number of days of in vitro culture is indicated. (c) Immunofluorescence staining of isolated glomeruli from Mof^fl/fl;Nphs2-Cre^T/+;tomato^fl/+>eGFP ^T/+ mice and littermate controls. Podocytes can be identified by eGFP expression. Scale bars equal 20 μm. (d) Experimental design of stress induction using Adriamycin. Mice were injected intravenously with 17.5 mg/kg Adriamycin. Albumin to creatinine ratio (ACR) and body weight were measured one day before injection and then weekly up to 6 weeks after injection. (e) Urinary ACR after Adriamycin treatment of Mof^fl/fl;Nphs2-Cre^T/+ and littermate Mof^fl/fl controls. n=39 mice per group. (f) Body weight of mice after a single shot of Adriamycin administered to Mof^fl/fl;Nphs2-Cre^T/+ and littermate Mof^fl/fl controls. n=39 mice per group. (g) Periodic acid–Schiff (PAS) staining of kidney sections from 12-week-old Mof^fl/fl;Nphs2-Cre^T/+ mice and littermate controls 6 weeks after Adriamycin injection. Scale bars are indicated in μm. (h) Transmission electron microscopy of kidney sections from Mof^fl/fl;Nphs2-Cre^T/+ mice 6 weeks after Adriamycin injection. Example of a pyknotic nucleus is marked with (*), black arrow indicates normal foot process, whereas red arrow indicates foot process with significant effacement. Scale bars are indicated in μm. (i) Kidney sections from Mof^fl/fl;Nphs2-Cre^T/+ mice and littermate controls immunostained for Nephrin and activated caspase-3 6 weeks after Adriamycin injection. White arrows indicate positive staining of activated caspase-3. (j) Scanning electron micrographs of GCNs at third larval stage. Genotypes: control—sns-GCN-GAL4/+; Mof KD—sns-GAL4/UAS-Mof-RNAi. Scale bars equal 20 μm. (k) Quantitation of circularity revealing a significant loss of circularity upon RNA interference (RNAi)-mediated Mof knockdown. Data are presented as mean±s.e.m. Data were analyzed using two-way analysis of variance followed by a Student's t-test. Asterisks indicate significance at *P<0.05 and ***P<0.001.