Continuous non-mutagenic dna damage in podocytes activates pathogenic memory t cells through altered dna methylation

Objective: Chronic kidney disease (CKD) is a common cause of hypertension and a complication of uncontrolled hypertension. Recent epigenome-wide studies suggested the association of altered DNA methylation in blood cells with kidney function. However, the mechanism and pathological significance has remained to be adequately elucidated. Design and method: To investigate the significance of DNA double-strand breaks (DSBs) in podocytes, we generated podocyte-specific I-PpoI-expressing mice (podocin-Cre ROSA26-STOP- I-PpoI, referred as I-PpoI-mice). I-PpoI is a homing endonuclease which causes non-mutagenic DSBs. Results: I-PpoI-mice showed severe albuminuria at 6 weeks of age and developed nephrotic syndrome. The mice died because of renal failure around 24 weeks of age following rapid deterioration of renal function. MeDIP-seq analysis using isolated podocytes revealed that 5219 DMRs were identified, and 5212 hypermethylated DMRs were detected in I-PpoI mice. No significant correlation between DNA methylation levels and the distance from I-PpoI cutting sites was observed. Single-cell RNA seq analysis using renal cortex revealed a marked expansion of immune cells, especially CD8+ T cells exhibited a KLRG1low IL-7Rhigh memory precursor effector cell phenotype, with high expression of NKG2D receptors in I-PpoI-mice. The expression of murine NKG2D ligands, which are upregulated by DNA damages, increased in I-PpoI-mice podocytes. In addition, the expression of human NKG2D ligands, MICB, in glomeruli was upregulated in patients with various kidney diseases including hypertensive nephropathy. Whole T cell depletion prevented exacerbation of albuminuria and attenuated glomerulosclerosis and fibrosis in I-PpoI mice, and similar results was obtained by NKG2D blocking. Me-DIP seq analysis using peripheral blood cells, which revealed hypermethylated DMRs in I-PpoI mice were enriched in binding sites for the STAT1 transcription factor. I-PpoI-mice showed a significant increase in CD44high memory/activated phenotype cells of peripheral CD8+ T cell populations, which was similar phenotype of STAT1 knockout mice. The chimeric WT mice with bone marrow (BM) cells of I-PpoI-mice exhibited an increase in albuminuria temporarily, whereas the chimeric I-PpoI-mice with BM cells of WT mice prevented renal fibrosis and renal death. Conclusions: Continuous non-mutagenic DSBs altered DNA methylation regardless of the distance from the primary DSB sites podocytes. Podocyte DNA DSBs also altered methylome of blood cells, including CD8+ T cells, leading sustained changes in immune microenvironment of the kidney and also BM. These results suggest the altered immune microenvironment in the kidney may be a therapeutic target preventing exacerbating glomerulosclerosis and renal fibrosis following podocyte DNA damage in CKD.