Fig 2: Let-7 upregulation requires KSRP and PI3K/AKT signaling. (A) C3H myotubes were stimulated (INS+) or un-stimulated (INS−) with 100 nM of insulin for 2 h, in the presence or absence of indicated kinase inhibitors. miRNAs were extracted and subjected to RT-qPCR analysis. Relative levels of mature miR-1 and miR-133 are shown on top and bottom panels, respectively. (B and C) C3H myotubes were transfected with siCon or siKSRP. Forty-eight hours later, cells were stimulated or un-stimulated with 100 nM of insulin for 2 h, following by RNA extraction and RT-qPCR analysis. (D) C3H myotubes were stimulated (INS+) or un-stimulated (INS−) with 100 nM of insulin for 2 h, in the presence or absence of indicated kinase inhibitors. RNAs were extraction and subjected to RT-qPCR analysis. Relative levels of let-7 and H19 are shown on top and bottom panels, respectively. All data are presented as mean ± SEM. (n = 3); **P < 0.01. (E) C3H myotubes were stimulated (INS+) or un-stimulated (INS−) with 100 nM of insulin. RNAs were extraction at 2 h (top panel) or 24 h (bottom panel) and subjected to RT-qPCR analysis. Relative RNA levels are shown after normalization against beta-tubulin. Numbers are mean ± SEM. (n = 3); **P < 0.01. (F) Proteins were extracted from C3H myotubes 24 h following stimulation with a single dose of 100 nM insulin (INS+) or no INS stimulation (INS−) and analyzed by western blot using antibodies against LPL (top blot), INSR (middle blot) and TUBB (bottom blot). (G) Quantification of western blot gels from three independent experiments. Numbers are presented as mean ± SEM. (n = 3); **P < 0.01.

Fig 3: Reduced proximal tubule and collecting duct cell heights in InsR KO mice—(A) representative images of periodic acid–Schiff (PAS)-stained sections of kidney cortex (400×); (B) mean glomerular area; (C) mean PT and CCD cell height in WTM, KOM, WTF, and KOF mice; a two-way ANOVA (genotype; sex), followed by a multiple comparisons testing (MCT, Tukey’s), was conducted on the data (n = 9–12/genotype/sex); “A” is significantly higher than “B” by MCT; Glm—glomerulus; CCD—cortical collecting duct; PT—proximal tubule.

Fig 4: Kidney weight, blood pressure, and heart rate in PAX8-targeted InsR KO mice—(A) mean arterial blood pressure (MAP); and (B) heart rate measured by tail cuff plethysmography; (C) kidney wet weight (n = 9–12/genotype/sex); p-values from two-way ANOVA (genotype; sex) are shown within each panel; letters above bars indicate results of MCT (only conducted when a main factor p < 0.05), with “A” assigned to the highest mean and all means not different from it, followed by “B”, etc. Bars with letters “not in common” are significantly different from each other, e.g., “AB” versus “C”.

Fig 5: InsR knockout (KO) localization and degree in PAX8-targeted InsR KO mice—(A) β-galactosidase staining (indicating location of Cre-recombinase activity) in kidney cortex and medulla sections from mice that were carriers of both PAX8 and tet-O-Cre transgenes (left panels) or only PAX8 (right panels) and homozygously floxed for InsR; (B) Western blot of whole-kidney homogenates probed with InsR (β-subunit) antibody in WT and KO mice; (C) band densities of InsR Western blot (n = 6/genotype/sex, normalized to Ponceau staining); letters above bars indicate results of multiple comparison testing (MCT), with “A” significantly (p < 0.05) greater than “B”.