Why some kidney transplant patients treated with mTOR inhibitors develop proteinuria and majority of recipients of other solid organs improve their kidney function remains a conundrum. Intact glomerular filter with podocytes as a functional gate-keeper is key to maintenance of kidney function. We recently identified sex-specific differences in the regulation of mTOR complexes 1 and 2 in cardiomyocytes and loss of cardioprotective phenotype in response to rapamycin restricted to females. Like cardiomyocytes or neurons, podocytes are postmitotic cells with limited regeneration capacity and mechanistic understanding of this cell-type restricted response to rapamycin is of great clinical relevance.
Aim of our study was to investigate intrinsic sex related differences and potential sexual dimorphism upon rapamycin treatment in podocytes. ROSAmT/mG-NHPS2(podocin)Cre mice with membrane targeted GFP restricted to podocytes were treated for 3 weeks with vehicle or rapamycin (1.5 mg/kg body weight i.p. every 3rd day, steady state trough levels: 10-17 ng/ml). Purified podocyte fractions were isolated by microbeads perfusion and FACS sorting. Podocyte specific total RNA was subsequently analysed by whole RNA sequencing.
Neither microalbuminuria nor proteinuria occurred and renal function remained normal in all vehicle and rapamycin treated animals. Histological analysis of kidneys from rapamycin and vehicle mice of both sexes displayed normal tubular and glomerular morphology indicating full maintenance of renal structure and cellular homeostasis. However, RNA sequencing revealed strong intrinsic sex-differences in gene transcription. More than 600 mRNA baseline transcripts were differentially expressed between vehicle treated male and female podocytes including much higher gene expression of substantial ribosomal and mitochondrial genes in female podocytes. Furthermore, podocyte specific proteins, like Nphs2, and stress-inducible transcription factor Atf4 were higher transcribed in females, whereas key components of mTOR signaling cascade such as Tsc1, Tsc2 and rictor displayed higher expression levels in male podocytes. Rapamycin treatment reduced sex differences in gene transcription shifting male transcriptome towards female vehicle phenotype and vice versa. Immunohistochemical stainings confirmed higher levels of podocyte specific TF WT-1 and mTORC2 target AKT on protein level, both responsible for cytoskeletal and cellular maintenance.
We are the first to identify sex specific transcriptional regulation of decisive key factors for podocyte homeostasis already after short term rapamycin exposure without apparent renal functional compromise. These early transcriptome changes may translate into sex-specific susceptibility after long term mTORi exposure and help in therapy personalization. Future studies on podocyte adaptation in acute or chronic kidney stress conditions during mTORi treatment are planned.