Podocyte-specific Rac1 deficiency ameliorates podocyte damage and proteinuria in STZ-induced diabetic nephropathy in mice
Activation of Ras-related C3 botulinum toxin substrate 1 (Rac1) has been implicated in diverse kidney diseases, yet its in vivo significance in diabetic nephropathy (DN) is largely unknown. In the present study, we demonstrated a podocyte-specific Rac1-deficient mouse strain and showed that specific inhibition of Rac1 was able to attenuate diabetic podocyte injury and proteinuria by the blockade of Rac1/PAK1/p38/β-catenin signaling cascade, which reinstated the integrity of podocyte slit diaphragms (SD), rectified the effacement of foot processes (FPs), and prevented the dedifferentiation of podocytes. In vitro, we showed Rac1/PAK1 physically bound to β-catenin and had a direct phosphorylation modification on its C-terminal Ser675, leading to less ubiquitylated β-catenin, namely more stabilized β-catenin, and its nuclear migration under high-glucose conditions; further, p38 activation might be responsible for β-catenin nuclear accumulation via potentiating myocyte-specific enhancer factor 2C (MEF2c) phosphorylation. These findings provided evidence for a potential renoprotective and therapeutic strategy of cell-specific Rac1 deficiency for DN and other proteinuric diseases.TG mice were characterized by immunofluorescence of the kidney cortex, and real-time PCR and western blot analysis of primary cultured podocytes. Immunofluorescence demonstrated a marked decrease in Rac1 staining in TG glomerular podocytes (P < 0.01) without altering synaptopodin expression (P > 0.05) between wild-type WT and TG mice (Fig. 1a), or podocyte numbers (Figure S) (P > 0.05). In primary podocytes, Rac1 expression was significantly reduced in TG mice compared with WT mice on both protein and mRNA levels (Fig. 1b) (P < 0.05). There were no distinguishable differences in hair growth or appearance between WT and TG mice (Figure S); and metabolic data showed neither tendency of spontaneous proteinuria or diabetes, nor differences between two mice strains on weight and SBP levels (Table 1) (P > 0.05).The nephrin promoter-Rac1 shRNA was linearized and microinjected into C57BL/6 mouse embryos that were transferred to the oviduct of pseudopregnant recipient mice. By DNA genotyping, 3 of 19 pups were identified positive (Cyagen Biosciences, Guangzhou, China) and were crossed with C57BL/6 mice to generate mice used in the present study. Forward primer: TTCGGTCGACTAGGGATAACAGG; reverse primer: TAATCCAGAGGTTGATTATCGGAA. PCR products (548 bp) were analyzed by agarose gel electrophoresis. The littermates lacking the transgene were control wild type (WT) mice.This work was funded by National Natural Science Foundation of China (Grant 81400732, 81370834, and 30971381), Shandong Young Scientists Award Fund (2010BSB14076), and Chinese Society of Nephrology Scientific Fund (14050480585). All authors approved the final version of the manuscript.The authors declare that they have no conflict of interest.Edited by J. ChipukElectronic supplementary materialSupplementary Information accompanies this paper at 10.1038/s41419-018-0353-z.Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.