Predicting exacerbation of renal function by DNA methylation clock and DNA damage of urinary shedding cells: a pilot study.

Recent reports have shown the feasibility of measuring biological age from DNA methylation levels in blood cells from specific regions identified by machine learning, collectively known as the epigenetic clock or DNA methylation clock. While extensive research has explored the association of the DNA methylation clock with cardiovascular diseases, cancer, and Alzheimer's disease, its relationship with kidney diseases remains largely unexplored. In particular, it is unclear whether the DNA methylation clock could serve as a predictor of worsening kidney function. In this pilot study involving 20 subjects, we investigated the association between the DNA methylation clock and subsequent deterioration of renal function. Additionally, we noninvasively evaluated DNA damage in urinary shedding cells using a previously reported method to examine the correlation with the DNA methylation clock and worsening kidney function. Our findings revealed that patients with an accelerated DNA methylation clock exhibited increased DNA damage in urinary shedding cells, along with a higher rate of eGFR decline. Moreover, in cases of advanced CKD (G4-5), the DNA damage in urinary shedding cells was significantly increased, highlighting the interplay between elevated DNA damage and eGFR decline. This study suggests the potential role of the DNA methylation clock and urinary DNA damage as predictive markers for the progression of chronic kidney disease.

Podocyte Ercc1 is indispensable for glomerular integrity.

As life expectancy continues to increase, age-related kidney diseases are becoming more prevalent. Chronic kidney disease (CKD) is not only a consequence of aging but also a potential accelerator of aging process. Here we report the pivotal role of podocyte ERCC1, a DNA repair factor, in maintaining glomerular integrity and a potential effect on multiple organs. Podocyte-specific ERCC1-knockout mice developed severe proteinuria, glomerulosclerosis, and renal failure, accompanied by a significant increase in glomerular DNA single-strand breaks (SSBs) and double-strand breaks (DSBs). ERCC1 gene transfer experiment in the knockout mice attenuated proteinuria and glomerulosclerosis with reduced DNA damage. Notably, CD44+CD8+ memory T cells, indicative of T-cell senescence, were already elevated in the peripheral blood of knockout mice at 10 weeks old. Additionally, levels of senescence-associated secretory phenotype (SASP) factors were significantly increased in both the circulation and multiple organs of the knockout mice. In older mice and human patients, we observed an accumulation of DSBs and an even greater buildup of SSBs in glomeruli, despite no significant reduction in ERCC1 expression with age in mice. Collectively, our findings highlight the crucial role of ERCC1 in repairing podocyte DNA damage, with potential implications for inflammation in various organs.

DNA-damaged podocyte-CD8 T cell crosstalk exacerbates kidney injury by altering DNA methylation.

Recent epigenome-wide studies suggest an association between blood DNA methylation and kidney function. However, the pathological importance remains unclear. Here, we show that the homing endonuclease I-PpoI-induced DNA double-strand breaks in kidney glomerular podocytes cause proteinuria, glomerulosclerosis, and tubulointerstitial fibrosis with DNA methylation changes in blood cells as well as in podocytes. Single-cell RNA-sequencing analysis reveals an increase in cytotoxic CD8+ T cells with the activating/costimulatory receptor NKG2D in the kidneys, which exhibit a memory precursor effector cell phenotype, and the CD44high memory CD8+ T cells are also increased in the peripheral circulation. NKG2D blockade attenuates the renal phenotype caused by podocyte DNA damage. Blood methylome shows increased DNA methylation in binding sites for STAT1, a transcription factor contributing to CD8+ T cell homeostasis. Collectively, podocyte DNA damage alters the blood methylome, leading to changes in CD8+ T cells, which contribute to sustained renal injury in chronic kidney disease.

Plasminogen activator inhibitor 1 is not a major causative factor for exacerbation in a mouse model of SARS-CoV-2 infection.

Coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a global pandemic. Although several vaccines targeting SARS-CoV-2 spike proteins protect against COVID-19 infection, mutations affecting virus transmissibility and immune evasion potential have reduced their efficacy, leading to the need for a more efficient strategy. Available clinical evidence regarding COVID-19 suggests that endothelial dysfunction with thrombosis is a central pathogenesis of progression to systemic disease, in which overexpression of plasminogen activator inhibitor-1 (PAI-1) may be important. Here we developed a novel peptide vaccine against PAI-1 and evaluated its effect on lipopolysaccharide (LPS)-induced sepsis and SARS-CoV-2 infection in mice. Administration of LPS and mouse-adapted SARS-CoV-2 increased serum PAI-1 levels, although the latter showed smaller levels. In an LPS-induced sepsis model, mice immunized with PAI-1 vaccine showed reduced organ damage and microvascular thrombosis and improved survival compared with vehicle-treated mice. In plasma clot lysis assays, vaccination-induced serum IgG antibodies were fibrinolytic. However, in a SARS-CoV-2 infection model, survival and symptom severity (i.e., body weight reduction) did not differ between vaccine- and vehicle-treated groups. These results indicate that although PAI-1 may promote the severity of sepsis by increasing thrombus formation, it might not be a major contributor to COVID-19 exacerbation.

Significance of podocyte DNA damage and glomerular DNA methylation in CKD patients with proteinuria.

The number of chronic kidney disease (CKD) patients is increasing worldwide, and it is necessary to diagnose CKD patients in earlier stages to improve their prognosis. Previously, in a study using human samples, we reported that DNA methylation and DNA damage in podocytes are potential markers for kidney function decline in IgA nephropathy; however, these candidate markers have not been adequately investigated in other glomerular diseases. Here, we report that the association of podocyte DNA damage and DNA methylation with eGFR decline and proteinuria differs depending on the type of glomerular disease. Patients diagnosed with minor glomerular abnormality (MGA, n = 33), membranous nephropathy (MN, n = 9) or diabetic nephropathy (DN, n = 10) following kidney biopsy at Keio University Hospital from 2015 to 2017 were included. In MGA patients, both podocyte DNA damage and glomerular DNA methylation were associated with the severity of proteinuria. In DN patients, podocyte DNA double-strand breaks (DSBs) and glomerular DNA methylation were associated with an eGFR decline. When patients with urinary protein levels of more than 1 g/gCr were examined, fewer podocyte DNA DSBs were detected in MN patients than in MGA patients, and the level of glomerular DNA methylation was lower in MN patients than in MGA or DN patients. These results indicate that investigating podocyte DNA DSBs and DNA methylation changes may be useful for understanding the pathogenesis of CKD with proteinuria in humans. This study suggested the association of podocyte DNA damage and subsequent DNA methylation with proteinuria in minor glomerular abnormalities (MGA) patients and those with eGFR declines in diabetic nephropathy (DN) patients, respectively.

Vaccination against connective tissue growth factor attenuates the development of renal fibrosis.

There is a critical need for efficient treatment of chronic kidney disease (CKD). Renal fibrosis is a final common pathway to end-stage renal disease independent of the underlying etiology, and connective tissue growth factor (CTGF) is a well-recognized profibrotic factor in fibrosis of various organ systems. Here, we developed a novel peptide vaccine against CTGF to attenuate the development of renal fibrosis. Three inoculations with this CTGF vaccine at 2-week intervals elicited antibodies specifically binding to human full-length CTGF, and the antigen-specific serum IgG antibody titers were maintained for > 30 weeks. The efficacy of the CTGF vaccine on renal fibrosis was evaluated in adenine-induced CKD and unilateral ureteral obstruction (UUO) murine models. In adenine-induced CKD model, immunization with the CTGF vaccine attenuated renal interstitial fibrosis. Vaccinated mice showed low levels of serum creatinine and urea nitrogen and low urine albumin-creatinine ratio compared with vehicle-treated mice. In UUO model, the CTGF vaccination also suppressed the onset of renal fibrosis. In an in vitro study, CTGF vaccine-elicited IgG antibodies efficiently suppressed CTGF-induced- and transforming growth factor-ß-induced α-smooth muscle actin expression in kidney fibroblasts. These results demonstrate that the CTGF vaccine is a promising strategy to attenuate the development of renal fibrosis.

Efficacy of dexmedetomidine on peritoneal dialysis catheter insertion.

PURPOSE: Dexmedetomidine (DEX) is a sedative agent with minimal respiratory and hemodynamic effects. The present study aimed to evaluate its effectiveness in peritoneal dialysis (PD) catheter insertion. METHODS: This single-center retrospective study included patients who underwent PD catheter insertion under spinal anesthesia in our hospital between January 2016 and December 2020. Patients were divided into the DEX and non-DEX groups according the use of DEX. After 1:1 propensity score matching to adjust for age, sex, body mass index, mean blood pressure (BP), and Charlson comorbidity index, we compared operation-related outcomes, including peak numerical rating scale (NRS), occurrence of nausea, vital signs, or operative time between the two groups. RESULTS: Of a total of 44 patients, 9 patients received DEX, and 35 did not. After propensity score matching, each group consisted of 8 patients. Peak NRS was significantly lower (P = 0.003) in the DEX group compared with the non-DEX group. Maximum mean BP during the operation was also significantly lower in the DEX group compared with the non-DEX group (P = 0.020), with no significant differences in minimum mean BP between the two groups (P = 0.831). The DEX group showed a trend of shortened operative time (P = 0.068). There were no significant differences in the occurrence of nausea (P = 1.000). Moreover, there was no clinically important adverse event associated with use of DEX. CONCLUSION: The use of DEX in PD catheter insertion under spinal anesthesia could safely improve operative analgesia.

DNA repair factor KAT5 prevents ischemic acute kidney injury through glomerular filtration regulation.

The "preconditioning effect" in AKI is a phenomenon in which an episode of ischemia-reperfusion results in tolerance to subsequent ischemia-reperfusion injury. However, its relationship between DNA damage repair has not been elucidated. Here, we show the role of KAT5 in the preconditioning effect. Preconditioning attenuated DNA damage in proximal tubular cells with elevated KAT5 expression. Ischemia-reperfusion (IR) injuries were exacerbated, and preconditioning effect vanished in proximal tubular-cell-specific KAT5 knockout mice. Investigation of tubuloglomerular feedback (TGF) by MALDI-IMS and urinary adenosine revealed that preconditioning caused attenuated TGF at least in part via KAT5. In addition, K-Cl cotransporter 3 (KCC3) expression decreased in damaged proximal tubular cells, which may be involved in accelerated TGF following IR. Furthermore, KAT5 induced KCC3 expression by maintaining chromatin accessibility and binding to the KCC3 promoter. These results suggest a novel mechanism of the preconditioning effect mediated by the promotion of DNA repair and attenuation of TGF through KAT5.

Vaccination Against Receptor for Advanced Glycation End Products Attenuates the Progression of Diabetic Kidney Disease.

Effective treatment of diabetic kidney disease (DKD) remains a large unmet medical need. Within the disease's complicated pathogenic mechanism, activation of the advanced glycation end products (AGEs)-receptor for AGE (RAGE) axis plays a pivotal role in the development and progression of DKD. To provide a new therapeutic strategy against DKD progression, we developed a vaccine against RAGE. Three rounds of immunization of mice with the RAGE vaccine successfully induced antigen-specific serum IgG antibody titers and elevated antibody titers were sustained for at least 38 weeks. In addition, RAGE vaccination significantly attenuated the increase in urinary albumin excretion in streptozotocin-induced diabetic mice (type 1 diabetes model) and leptin-receptor-deficient db/db mice (type 2 diabetes model). In microscopic analyses, RAGE vaccination suppressed glomerular hypertrophy and mesangial expansion in both diabetic models and significantly reduced glomerular basement membrane thickness in streptozotocin-induced diabetic mice. Results of an in vitro study indicated that the serum IgG antibody elicited by RAGE vaccination suppressed the expression of AGE-induced vascular cell adhesion molecule 1 and intracellular adhesion molecule 1 in endothelial cells. Thus, our newly developed RAGE vaccine attenuated the progression of DKD in mice and is a promising potential therapeutic strategy for patients with DKD.

Effects of High Glucose and Lipotoxicity on Diabetic Podocytes.

Glomerular podocytes are highly differentiated cells that cover glomerular capillaries from the outside and have a characteristic morphology with numerous foot processes. The formation of slit membranes between the foot processes serves as a final filtration barrier for urine filtration from the blood. Podocyte damage causes disruption of the slit membrane, subsequent proteinuria and finally glomerulosclerosis, which is a common pathway in various types of chronic kidney disease (CKD). In recent years, there has been an increase in diabetes, due to rapid lifestyle changes, which is the main cause of CKD. Therefore, understanding the effect of diabetic status on podocytes is of great importance to establish a strategy for preventing CKD progression. In this review, we summarize altered glucose and lipid metabolism in diabetic podocytes and also discuss the reversibility of the changes in podocyte phenotype.

DNA damage and expression of DNA methylation modulators in urine-derived cells of patients with hypertension and diabetes.

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.

Association of glomerular DNA damage and DNA methylation with one-year eGFR decline in IgA nephropathy.

Accumulation of DNA double-strand breaks (DSBs) is linked to aging and age-related diseases. We recently reported the possible association of DNA DSBs with altered DNA methylation in murine models of kidney disease. However, DSBs and DNA methylation in human kidneys was not adequately investigated. This study was a cross-sectional observational study to evaluate the glomerular DNA DSB marker γH2AX and phosphorylated Ataxia Telangiectasia Mutated (pATM), and the DNA methylation marker 5-methyl cytosine (5mC) by immunostaining, and investigated the association with pathological features and clinical parameters in 29 patients with IgA nephropathy. To evaluate podocyte DSBs, quantitative long-distance PCR of the nephrin gene using laser-microdissected glomerular samples and immunofluorescent double-staining with WT1 and γH2AX were performed. Glomerular γH2AX level was associated with glomerular DNA methylation level in IgA nephropathy. Podocytopathic features were associated with increased number of WT1(+)γH2AX(+) cells and reduced amount of PCR product of the nephrin gene, which indicate podocyte DNA DSBs. Glomerular γH2AX and 5mC levels were significantly associated with the slope of eGFR decline over one year in IgA nephropathy patients using multiple regression analysis adjusted for age, baseline eGFR, amount of proteinuria at biopsy and immunosuppressive therapy after biopsy. Glomerular γH2AX level was associated with DNA methylation level, both of which may be a good predictor of renal outcome in IgA nephropathy.