In this study, we evaluated the DNA methylation clock and DNA DSBs in urinary shedding cells using blood and urine samples from healthy subjects and CKD patients. The results indicated that accelerated biological aging calculated from DNA methylation clocks and increased DNA DSBs of urinary shedding cells may serve as markers for current renal function and the prediction of future renal function deterioration. We also found that DNA DSBs in urinary shedding cells were associated with accelerated DNA methylation age.
The DNA methylation clock is a biological age calculated from DNA methylation levels, focusing on 71 specific CpGs in Hannum’s clock12 and 391 in PhenoAge13. PhenoAge is a second-generation clock that has been further trained for aging phenotypes and is superior to the first-generation clock in predicting many aging-related diseases and lifespan. In addition, age acceleration (AA), the difference between the DNA methylation clock and chronological age, has been used in many studies as an indicator of biological aging23,24.
To evaluate DNA DSBs in the kidney, we extracted genomic DNA from urinary shedding cells and evaluated it by a quantitative long-distance PCR method that we previously reported7. Open chromatin regions of active transcription, such as promoter regions of cell-specific marker genes, are known to be vulnerable to DNA damage. Therefore, we used primer sets of the promoter region of sodium glucose cotransporter 2 (SGLT2) and nephrin, which are expressed specifically in proximal tubular cells and podocytes, to assess DNA DSBs in each cell.
The analysis revealed a negative correlation between DNA DSBs of the urinary proximal tubular cells and eGFR and that DNA DSBs were significantly increased in advanced CKD. Interestingly, DNA damage in the proximal tubules was more associated with eGFR than that in podocytes. This may be related to the fact that proximal tubular cells are energy-consuming cells that reabsorb solutes, and in general, energy-consuming cells are more likely to accumulate oxidative damage, which is known to be a cause of age-related diseases. As CKD advances, tubulointerstitial fibrosis becomes a defining feature, a characteristic shared by all forms of CKD and renal fibrosis. Importantly, the severity of tubulointerstitial damage has been demonstrated to have a stronger correlation with the reduction in GFR compared to the extent of glomerular injury25.
Furthermore, the extent of DNA damage in urinary proximal tubular cells and podocytes exhibited a negative correlation with eGFR slope. Additionally, the degree of biological aging assessed through age acceleration (AA) also displayed a negative correlation with eGFR and eGFR slope. Moreover, there was a significant correlation between the extent of DNA damage in urinary shedding cells and age acceleration (AA). These findings suggest that the escalation of DNA damage in urinary shedding cells and the acceleration of biological aging calculated from the DNA methylation clock could potentially serve as indicators for current renal function and the prediction of future renal function deterioration. In particular, the DNA methylation clock acceleration demonstrated a strong correlation with eGFR slope and current eGFR, suggesting its potential as a predictive marker for future renal function deterioration. To our knowledge, this is the first report demonstrating the potential of DNA methylation clock progression as a marker for predicting future renal function decline and its association with DNA damage in urinary shedding cells.
Although the association between progression of the DNA methylation clock and increased mortality rate23,24, cancer17, and Alzheimer’s disease16 has been previously reported, the relationship with renal function has not been well studied. Recent studies have reported accelerated aging in CKD and that inflammatory and immunologic profiles may be important in predicting biological aging26. In addition, a large meta-analysis in Europeans, African Americans, and Hispanics reported an association between the progression of DNA methylation age and eGFR or CKD27. The results of our study targeting an Asian population are consistent with the findings previously reported. It is widely acknowledged that kidney function declines with age, and some kidney diseases are known to have a poor renal prognosis with aging28. The correlation between biological aging indicated by the DNA methylation clock and the decrease in eGFR is a reasonable finding. The present study provides new findings that biological aging is associated not only with eGFR but also with eGFR slope, which may be a marker for predicting future renal function. Moreover, recent reports have proposed the possibility that chronic kidney disease promotes aging in a multiorgan disease network29. Notably, this is the first study to demonstrate a link between DNA damage in kidney cells and biological aging. Although this study cannot demonstrate a causal relationship between kidney DNA damage and the DNA methylation clock and systemic aging, it has been reported that DNA damage in podocytes induces DNA methylation changes in blood cells8 and that the DNA methylation clock progresses when systemic DNA damage is induced18, suggesting that DNA damage in kidney cells may be related to systemic aging via peripheral blood DNA methylation changes.
Recent studies have suggested that DNA damage in kidney cells contributes to the progression of renal fibrosis and kidney dysfunction. For instance, Kishi et al. demonstrated that in a mouse model, deletion of ATR, a key regulator of the DNA damage response, specifically in renal proximal tubular epithelial cells (RPTECs) led to increased DNA damage, apoptosis, acute kidney injury, and fibrosis following renal insults such as cisplatin treatment, ischemia–reperfusion injury, and unilateral ureteral obstruction30. They also found that ATR expression inversely correlated with DNA damage in kidney tissues from patients with chronic kidney disease. Furthermore, Gupta et al. recently reported using human kidney organoids that FANCD2, a DNA repair factor involved in homologous recombination, was decreased in tubular cells in the setting of incomplete repair, correlating with the degree of fibrosis31.
Moreover, in our current study, we demonstrated that podocyte-specific deletion of ERCC1, a key factor for DNA single-strand break repair, in mice resulted in severe proteinuria, glomerulosclerosis, and renal failure, accompanied by accumulation of DNA damage32. Restoration of podocyte ERCC1 expression attenuated proteinuria and glomerulosclerosis with reduced DNA damage.
Collectively, these findings highlight the potential of targeting DNA damage pathways, such as via restoring expression of repair factors like KAT5, ATR, FANCD2 and ERCC1 in kidney cells, as a therapeutic approach to slow down the progression of kidney disease in both tubular cells and podocytes.
However, this study has several limitations. In particular, the sample size was small, and long-term renal prognosis was not followed up. Further studies are needed to evaluate the association of DNA DSBs in urinary shedding cells and the DNA methylation clock with eGFR and worsening renal function, which would require a longer observation period in a larger population. Despite these limitations, this study holds significance in demonstrating the correlation between the acceleration of biological aging evaluated by the DNA methylation clock and the increase in DNA damage in kidney cells. It also indicates the possibility that these factors could serve as indicators for current kidney function assessment and as predictive markers for future renal function deterioration. Furthermore, although a causal relationship could not be established, the findings suggest that kidney DNA damage and renal dysfunction might be implicated in the progression of the DNA methylation clock, contributing to systemic aging. The noninvasive detection of kidney DNA damage allows for the possibility of not only predicting kidney outcomes but also predicting systemic aging progression.
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- Source: https://www.nature.com/articles/s41598-024-62405-4