Published papers

Previous studies have shown that renin-angiotensin (Ang) system vaccines may be effective for the treatment of hypertension, but their efficacy for the prevention of renal disease is unclear. The aim of this study was to compare the effects of an Ang II type 1 (AT1) receptor vaccine with an Ang II receptor blocker (ARB) and a vasodilator on blood pressure (BP) and renal injury in the L-NAME nephropathy model. Male spontaneously hypertensive rats (SHRs) were divided into six groups and treated transiently with three injections of vehicle or AT1 receptor vaccine (0.1 mg) at age 4, 6 and 8 weeks, or continuously with candesartan cilexetil (0.1 mg kg(-1) per day) or hydralazine hydrochloride (5 mg kg(-1) per day), then administered NG-nitro-L-arginine methyl ester (L-NAME) from age 18 to 21 weeks to induce renal injury. Vaccination against the AT1 receptor caused a significant increase in AT1 receptor titers, and a sustained decrease in BP. L-NAME treatment resulted in a marked increase in proteinuria in the control groups, which was completely suppressed in the AT1 vaccine-treated group, and glomerular injury scores were also significantly decreased. Real-time RT-PCR and immunofluorescence studies revealed increased renin mRNA, and increased glomerular expression of nephrin. Comparable results were seen in rats treated continuously with the ARB candesartan, but not with hydralazine. These results suggest that transient AT1 vaccination is as effective as continuous treatment with ARB, not only for the attenuation of hypertension, but also for the prevention of L-NAME-induced nephropathy in SHR.

The growing number of patients with chronic kidney disease (CKD) is recognized as an emerging problem worldwide. Recent studies have indicated that deregulation of transcription factors is associated with the onset or progression of kidney disease. Several clinical trials indicated that regression of CKD may be feasible via activation of the transcription factor nuclear factor erythroid-2 related factor 2 (Nrf2), which suggests that transcription factors may be potential drug targets for CKD. Agents stabilizing hypoxia-inducible factor (HIF), which may be beneficial for renal anemia and renal protection, are also now under clinical trial. Recently, we have reported that the transcription factor Kruppel-like factor 4 (KLF4) regulates the glomerular podocyte epigenome, and that the antiproteinuric effect of the renin(-)angiotensin system blockade may be partially mediated by KLF4. KLF4 is one of the Yamanaka factors that induces iPS cells and is reported to be involved in epigenetic remodeling. In this article, we summarize the transcription factors associated with CKD and particularly focus on the possibility of transcription factors being novel drug targets for CKD through epigenetic modulation.

OBJECTIVES: To combat global increases in the prevalence of lifestyle-related diseases and concomitant infectious diseases, we aimed to develop an innovative intranasal vaccine that simultaneously targets both hypertension and pneumonia, is not given by invasive injection, and offers prolonged therapeutic effect and reduced frequency of administration. METHODS: Angiotensin II type 1 receptor-pneumococcal surface protein A (AT1R-PspA) vaccine, consisting of a cationic nanometer-sized hydrogel incorporating AT1R partial peptide conjugated with PspA and cyclic diguanylate monophosphate adjuvant, was created and given intranasally to spontaneously hypertensive rats (SHRs). Antigen-specific antibodies and blood pressure were examined to evaluate immune responses and the antihypertensive effect of the vaccine. To examine the protective effect of antibodies induced by vaccination on pneumococcal infection, sera obtained from immunized SHRs were incubated with a lethal dose of Streptococcus pneumoniae and then administered to mice. RESULTS: Five doses of AT1R-PspA nasal-vaccine-induced AT1R-specific serum IgG antibody production and attenuated the development of hypertension in SHRs in the long term. Both in-vitro and in-vivo studies revealed that responses to angiotensin II were suppressed in vaccinated rats. Mice passively immunized with sera obtained from AT1R-PspA-vaccinated SHRs were protected from lethal pneumococcal infection. CONCLUSION: Intranasal immunization with AT1R-PspA vaccine has the potential to simultaneously attenuate the development of hypertension and protect from lethal pneumococcal infection.

© 2019 The Author(s) Hishikawa et al. reveal that KAT5-mediated DNA repair is essential for podocyte maintenance and is related to changes in DNA methylation status. Decreased podocyte KAT5 expression may contribute to the pathophysiology of diabetic nephropathy, suggesting a therapeutic target.

© 2018, The Japanese Society of Hypertension. Pre-emptive medicine is a novel medical concept proposed in Japan that aims to precisely predict the onset and progression of diseases and to provide therapeutic interventions during early stages, before symptoms appear. The concept of pre-emptive medicine considers the time-course of a disease in each individual and seeks medical interventions to prevent disease progression. Suitable and promising targets for pre-emptive medicine are non-communicable diseases, including hypertension. Recent advances in genomic analysis, information technology, and artificial intelligence should make this medical concept feasible in the near future. In this review, we focused on pre-emptive medicine for hypertension, referring to concrete plans for the future direction of this research. The ultimate goal of pre-emptive medicine is to completely prevent the onset and progression of hypertension by precisely predicting the elevation of blood pressure and performing interventions to avoid it. The diagnostic processes of hypertension, from the standpoint of pre-emptive medicine, should include the detection of abnormal blood pressure regulation as the earliest manifestation of the disease, the depiction of the present status of hypertension in an individual (“nowcasting”), and a prediction of the future trajectory of the disease (“forecasting”). Novel therapeutic strategies for hypertension, from the standpoint of pre-emptive medicine, should aim for the regression of hypertension through early treatments and the remission of hypertension through intermittent intensive therapies. An efficient modification of lifestyle and therapies, according to the progression of hypertension, should be required. If pre-emptive medicine for hypertension becomes established, it would greatly contribute to the extension of a healthy lifespan, which cannot yet be satisfactorily achieved.

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.

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.

The DNA repair system is essential for the maintenance of genome integrity and is mainly investigated in the areas of aging and cancer. The DNA repair system is strikingly cell-type specific, depending on the expression of DNA repair factors; therefore, different DNA repair systems may exist in each type of kidney cell. Importance of DNA repair in the kidney is suggested by renal phenotypes caused by both genetic mutations in the DNA repair pathway and increased stimuli of DNA damage. Recently, we reported the importance of DNA double-strand break repair in glomerular podocytes and its involvement in the alteration of DNA methylation status, which regulates podocyte phenotypes. In this review, we summarize the roles of the DNA repair system in the kidneys and possible associations with altered kidney DNA methylation, which have been infrequently reported together. Investigations of DNA damage repair and epigenetic changes in the kidneys may achieve a profound understanding of kidney aging and diseases.

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.

AIM: Early intervention before the progression of chronic kidney disease (CKD) is essential to prevent end-stage renal disease (ESRD) and cardiovascular complications. This study evaluated the correlation between metabolic and lifestyle-related factors and the decline of estimated glomerular filtration rate (eGFR) over 1 year in a Japanese population without CKD. METHODS: Subjects who received two consecutive annual health checkups from 2013 to 2015 were involved. Factors associated with eGFR decline were identified using multiple regression models. RESULTS: A total of 2531 subjects aged 58.9+/-11.7 years old were included in this study. Baseline levels of HDL-C and ApoA1 correlated with the eGFR decline over 1 year defined as eGFR reduction rate of 15% or more and/or eGFR at the next year 60 ml/min/m(2) (odds ratio (OR) 0.87 (per 10 mg/dl); 95% CI, 0.80-0.94; p=0.0012, 0.90 (per 10 mg/dl); 0.86-0.96; p=0.0004, respectively). A U-shaped relationship between the eGFR decline and HDL-C or ApoA1 levels was not observed in non-CKD population of this study. Metabolic syndrome was significantly associated with eGFR decline (OR 1.32; 1.04-1.67; p=0.0205), although obesity-related factors did not show a significant correlation with eGFR decline over 1 year. CONCLUSION: Low HDL-C and ApoA1 levels significantly correlated with eGFR decline in a short period of 1 year. Metabolic syndrome also showed a significant association with eGFR decline. This study suggests the importance of hypertension and low HDL-C in the metabolic syndrome effect on eGFR decline rather than obesity in non-CKD population.

BACKGROUND: Cerebrovascular disease is a major cause of mortality and morbidity. Chronic kidney disease (CKD) is prevalent in stroke patients. This study evaluated the correlation between kidney dysfunction and asymptomatic findings on carotid ultrasonography (US) and brain magnetic resonance imaging (MRI) in a Japanese population with health checkups.Methods and Results:In total, 1,716 subjects aged 40-80 years, who received health checkups from January 1 to December 31, 2015, were included. Common carotid artery intima-media thickness (CCA-IMT) and carotid plaques by US, and the presence of old non-lacunar infarctions, lacunar infarctions, white matter lesions (WMLs), cerebral microbleeds (CMBs), and atrophy by brain MRI were evaluated. After adjusting for cardiovascular risk factors, multiple regression analyses revealed that an eGFR ranging from 15 to 44 mL/min/1.73 m(2)was independently associated with CCA plaques and hypoechoic or heterogeneous plaques. Proteinuria was associated with CCA or internal carotid artery plaques, the number of carotid plaques, and the presence of old non-lacunar infarctions and CMBs. CONCLUSIONS: Decreased eGFR and proteinuria were independent risk factors for asymptomatic abnormalities on carotid US and brain MRI, which are surrogate markers for cerebrovascular diseases. Evaluation of these abnormalities may be useful for prevention of symptomatic cerebrovascular events in CKD patients.

Recently it has been shown that epigenetic mechanisms are involved in initiation and progression of caridiovascular and metabolic diseases, including diabetes, obesity, atherosclerosis, heart failure, hypertension and kidney diseases. In these chronic diseases, various exogenous and endogenous stresses cause DNA damage, followed by DNA repair process. Accumulation of DNA damages and impaired repair process can lead to epigenetic changes, which may contribute to onset and progression of diseases. Recently we have shown that therapeutic effect of transcription factor KLF4 (Kruppel-like factor 4) in kidney glomerular epithelial cells (podocytes) on proteinuric kidney diseases through epigenetic mechanisms. Our result suggests the possibility of transcription factors as a target of selective epigenetic therapy. Moreover, we have reported that renin-angiotensin system (RAS) blockers, which are widely prescribed for the treatment of cardiovascular diseases, can restore epigenetic changes through KLF4 in part. These results suggest that activation of RAS causes epigenetic changes in disease states, and elucidation of the precise mechanism may lead to establishment of novel therapeutic target of kidney diseases. In this review we focus on DNA damage repair system and epigenetic modulators in disease states, and speculate a candidate for epigenetic therapy of kidney diseases.

Recently emerging evidence has shown that epigenetic mechanisms are involved in initiation and progression of life-style related diseases, including hypertension. In this article, we reviewed therapeutic effect of renin-angiotensin system (RAS) inhibitors on epigenetic changes in the disease states. We have recently reported that transcription factor Kruppel-like factor 4(KLF4) is expressed in kidney podocytes and modulates podocyte phenotype by gene-selective epigenetic control, and that the effect of RAS inhibitors is mediated by epigenetic modulation through KLF4 in part. It is suggested that the 'memory' or 'legacy' effect of RAS inhibitors previously reported in human studies may be partly due to the epigenetic modulation by these agents, and further studies are necessary for investigation of new therapeutic target.

Proteinuria is a central component of chronic kidney disease and an independent risk factor for cardiovascular disease. Kidney podocytes have an essential role as a filtration barrier against proteinuria. Kruppel-like Factor 4 (KLF4) is expressed in podocytes and decreased in glomerular diseases leading to methylation of the nephrin promoter, decreased nephrin expression and proteinuria. Treatment with an angiotensin receptor blocker (ARB) reduced methylation of the nephrin promoter in murine glomeruli of an adriamycin nephropathy model with recovery of KLF4 expression and a decrease in albuminuria. In podocyte-specific KLF4 knockout mice, the effect of ARB on albuminuria and the nephrin promoter methylation was attenuated. In cultured human podocytes, angiotensin II reduced KLF4 expression and caused methylation of the nephrin promoter with decreased nephrin expression. In patients, nephrin promoter methylation was increased in proteinuric kidney diseases with decreased KLF4 and nephrin expression. KLF4 expression in ARB-treated patients was higher in patients with than without ARB treatment. Thus, angiotensin II can modulate epigenetic regulation in podocytes and ARB inhibits these actions in part via KLF4 in proteinuric kidney diseases. This study provides a new concept that renin-angiotensin system blockade can exert therapeutic effects through epigenetic modulation of the kidney gene expression.

The role of salt intake in the development of hypertension is prominent, but its mechanism has not been fully elucidated. Our aim was to examine the effect of transient salt intake during the prehypertensive period in hypertensive model animals. Dahl salt-sensitive rats and spontaneously hypertensive rats were fed from 6 to 14 weeks with low-salt (0.12% NaCl), normal-salt (0.8% NaCl), high-salt (7% NaCl), or high-sodium/normal-chloride diet and returned to normal-salt diet for 3 months. Rats in the high-salt group saw elevations in blood pressure (BP) not only during the treatment period but also for the 3 months after returning to normal-salt diet. We named this phenomenon salt memory. Renal arteriolar injury was found in the high-salt group at the end of experiment. Dahl salt-sensitive rats were fed from 6 to 14 weeks with high-salt diet with angiotensin receptor blocker, vasodilator, calcium channel blocker, and calcium channel blocker+angiotensin receptor blocker and returned to normal-salt diet. Although BP was suppressed to control levels by vasodilator or calcium channel blocker, elevated renal angiotensin II and renal arteriolar injury were observed, and salt memory did not disappear because of sustained renal arteriolar injury. Calcium channel blocker+angiotensin receptor blocker suppressed renal arteriolar injury, resulting in the disappearance of salt memory. Cross-transplantation of kidneys from Dahl salt-sensitive rats on high salt to control rats caused increase of BP, whereas control kidneys caused reduction in BP of hypertensive rats, inducing the central role of the kidney. These results suggest that renal arteriolar injury through BP and renal angiotensin II elevation plays important roles in the development of salt memory for hypertension.

AIM: Clinical studies have suggested that renin-angiotensin inhibitors are effective for the prevention of atherosclerosis progression, but the results for the regression of established lesions are equivocal. The aim of this study was to examine the effects of different doses of the angiotensin receptor blocker (ARB) candesartan on the regression of atherosclerosis and lipid-induced nephropathy in apolipoprotein E (apoE)-deficient spontaneously hyperlipidemic (SHL) mice. METHODS AND RESULTS: Male SHL were given an atherogenic diet together with salt loading to induce atherosclerosis. The mice were then treated with various doses of candesartan (0-50 mg/kg/d) for 12 weeks. Treatment with high-dose candesartan caused clear regression of atherosclerotic plaques in the aorta, which was not observed with normal-dose candesartan. Biglycan and ACAT1 expression were significantly decreased, and aortic free cholesterol: cholesterol ester ratios were increased in these mice. Treatment of cultured THP-1 macrophages in vitro with candesartan resulted in a similar decrease in ACAT1 expression. In the kidney, glomerular lipid accumulation, mesangial expansion, and albuminuria were significantly regressed after treatment with high-dose candesartan, while biglycan and ACAT1 expressions were decreased. CONCLUSION: These results suggest that regression of established atherosclerosis lesions in ApoE-deficient mice is feasible using high-dose candesartan, by mechanisms involving (i) a decrease in the lipid-retaining proteoglycan biglycan, and (ii) suppression of ACAT1 expression resulting in increased free cholesterol for lipid release.