Abstract
Diabetes and hypertension have become the primary causes of chronic kidney disease worldwide. However, there are no established markers for early diagnosis or predicting renal prognosis. Here, we investigated the expression profiles of DNA repair and DNA methylation factors in human urine-derived cells as a possible diagnostic or renal prognosis-predicting marker. A total of 75 subjects, aged 63.3 ± 1.9 years old, were included in this study. DNA and RNA were extracted from 50 mL of urine samples. We evaluated DNA double-strand breaks (DSBs) by the quantitative long distance-PCR method and performed real-time RT-PCR analysis to analyze the expression of renal cell-specific markers, DNA DSB repair factor KAT5, DNA methyltransferases DNMTs, and demethylation enzymes TETs. In patients with hypertension and diabetes, DNA DSBs of the nephrin gene increased with decreased urine KAT5/nephrin expression, consistent with our previous study (Cell Rep 2019). In patients with hypertension, DNA DSBs of the AQP1 gene were increased with elevated urine DNMTs/AQP1 and TETs/AQP1 expression. Moreover, urine DNMTs/AQP1 expression was significantly correlated with the annual eGFR decline rate after adjustment for age, baseline eGFR, the presence of diabetes and the amount of albuminuria, suggesting a possible role as a renal prognosis predictor.
Highlights
The prevalence of chronic kidney disease (CKD) has increased globally and become recognized as a worldwide health problem[1]
Since it has been reported that DNA double-strand breaks (DSBs) may induce aberrant gene silencing via DNA methylation[11,12], we focused on the expression of DNA methylation modifiers
This study has demonstrated that podocyte DNA DSBs increased with reduced expression of DSB repair factor KAT5 in patients with diabetes, using urine-derived cells
Summary
The prevalence of chronic kidney disease (CKD) has increased globally and become recognized as a worldwide health problem[1]. The association of KAT5 expression with DNA DSB levels and kidney disease progression has not been clarified. Urine examinations are noninvasive procedures, and because they identify changes in gene expression in urine-derived cells[3,4,5,6], they may be useful for the noninvasive assessment of kidney condition and prediction of renal outcomes in early hypertension and diabetes. There are several methods to detect DNA DSBs. The most commonly used DNA DSB marker is phosphorylated histone H2AX (γH2AX), which plays an important role in the recruitment of DNA repair factors to damaged sites[7]. We performed the quantitative long-distance PCR method using DNA samples of urine-derived cells to estimate kidney DNA DSBs because PCR analysis can be performed with small amounts. The quantitative long-distance PCR method for detecting DNA damage is based on the assumption that DNA with fewer damage lesions will amplify to a greater extent than more damaged DNA if equal amounts of DNA from different samples are amplified under identical conditions[8,9]
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