Targeting DNA Methylation in Podocytes to Overcome Chronic Kidney          Disease

Kaori Hayashi

doi:10.2302/kjm.2022-0017-IR

Abstract

The number of patients with chronic kidney disease (CKD) is on the rise worldwide, and there is urgent need for the development of effective plans against the increasing incidence of CKD. Podocytes, glomerular epithelial cells, are an integral part of the primary filtration unit of the kidney and form a slit membrane as a barrier to prevent proteinuria. The role of podocytes in the pathogenesis and progression of CKD is now recognized. Podocyte function depends on a specialized morphology with the arranged foot processes, which is directly related to their function. Epigenetic changes responsible for the regulation of gene expression related to podocyte morphology have been shown to be important in the pathogenesis of CKD. Although epigenetic mechanisms include DNA methylation, histone modifications, and RNA-based regulation, we have focused on DNA methylation changes because they are more stable than other epigenetic modifications. This review summarizes recent literature about the role of altered DNA methylation in the kidney, especially in glomerular podocytes, focusing on transcription factors and DNA damage responses that are closely associated with the formation of DNA methylation changes.

Introduction

The prevalence of chronic kidney disease (CKD) is increasing worldwide. About 10% of the world’s population is affected by CKD, and millions die each year because of a lack of access to affordable treatment.¹ CKD was ranked 27th in the list of causes of total deaths worldwide in 1990 but rose to 18th in 2010, which is the second highest degree of increase behind the emergence of HIV and AIDS.²^,³ Moreover, CKD is estimated to become the fifth leading cause of death worldwide by 2040, which may be one of the largest increases in any major cause of death.⁴ Therefore, there is an urgent need for measures that can halt the increased incidence of CKD. In general, the prevalence of CKD increases with age, and, in high-income countries, it is more common in people with obesity, diabetes, and hypertension.⁵ For a long time, the treatment of CKD has focused on inhibition of the renin–angiotensin system to prevent or delay the progression to end-stage renal disease (ESRD). Recently, novel agents, such as sodium-glucose cotransporter 2 (SGLT2) inhibitors, have been used for CKD treatment,⁶^,⁷ but there is still no fundamental cure once it progresses. Therefore, many studies to develop novel strategies for CKD treatment are now being conducted worldwide.

Podocytes, glomerular epithelial cells, are an integral part of the primary filtration unit of the kidney because of their formation of a slit membrane, which forms a barrier for proteinuria.⁸ Various injurious stimuli cause morphological changes in podocytes, such as foot process effacement, which lead to disruption of the slit membrane and subsequent proteinuria (Fig. 1). Severe injury causes podocyte detachment and apoptosis, from which glomerulosclerosis develops. Podocyte damage may be caused by genetic, immunological, infectious (e.g., hepatitis C virus infection), toxic (e.g., from various drugs or metals), or adaptive causes including glomerular hyperfiltration caused by low nephron number or increased body mass.⁹ Recently, the efficacy of SGLT2 inhibitors in slowing the progression of CKD has been demonstrated,⁶ with the primary mechanism of renoprotective action being the inhibition of residual glomerular hyperfiltration that occurs after glomerulosclerosis caused by a variety of factors.¹⁰ Notably, podocytes are terminally differentiated cells that are not replaced post development, similar to neurons; therefore, podocyte loss is a major determinant of progressive CKD with proteinuria, as can occur with diabetes, hypertension, and glomerulopathies.¹¹ It is also known that aging is accompanied by podocyte changes, such as hypertrophy, foot process effacement, decreased expression of podocyte-specific genes, and increased expression of the apoptosis regulator p53. Podocyte aging has been of great interest in recent years.¹²^,¹³ Although recent studies have shown that a subset of parietal epithelial cells can serve as podocyte progenitors in a diphtheria toxin model of acute podocyte ablation in mice, podocyte generation fails in aging kidneys and in response to loss of nephron in disease states.¹⁴ To date, the precise mechanisms of podocyte loss have not been adequately clarified, and appropriate treatments to maintain an adequate number of healthy podocytes are lacking.¹⁵

Fig. 1.

Podocyte damage and pathological consequences.

Podocytes have arranged foot processes and form a slit membrane, a barrier for proteinuria. Various injurious stimuli cause morphological changes in podocytes, such as foot process effacement, leading to disruption of the slit membrane and subsequent proteinuria. Severe injury causes podocyte detachment and apoptosis, from which glomerulosclerosis develops. (A) Transmission electron micrograph of healthy podocytes in 8-week-old mice. Arrowhead: podocyte foot processes. (B) Transmission electron micrograph of damaged podocytes in the Adriamycin nephropathy model of 8-week-old mice. Arrowhead: foot process effacement of podocytes. Scale bars: 1 µm.

The association between podocyte foot process effacement and proteinuria has been established in animal model studies.¹⁶^,¹⁷ Additionally, in humans, the role of podocytes in the pathogenesis and progression of CKD has been recognized.¹⁸^,¹⁹ Proteinuria is an important clinical diagnostic indicator that is correlated with ultrastructural estimation of podocyte foot process effacement.²⁰ In addition, podocyte hyperplasia and interstitial fibrosis are suggested to be significant predictors of kidney function decline according to a recent report from the TRIDENT (Transformative Research in Diabetic Nephropathy) cohort.²¹ Taken together, these results suggest that podocyte loss and morphological changes are closely related to renal prognosis and that targeting podocytes may be a reasonable approach for better prognosis of CKD.

‘Epigenetic’ regulation represents a flexible and reversible mechanism for modulating gene expression that does not involve changes in the underlying DNA sequence. Therefore, epigenetic alterations cause a change in phenotype without a change in genotype. Epigenetic mechanisms include DNA methylation, histone modifications, and RNA-based regulation, such as noncoding RNAs and microRNAs.²² We focused on DNA methylation changes because they are more stable than other epigenetic modifications. Methylation of cytosine in CpG islands, which are often found in or around the promoter region, usually causes repression of transcription. This review summarizes recent literature about the role of DNA methylation changes in the kidney, especially in glomerular podocytes, focusing on transcription factors and DNA damage responses that are closely associated with the formation of altered DNA methylation.

DNA Methylation and Metabolic Memory in the Kidney

Epigenetic alterations are well documented in studies of diabetic kidney disease (DKD), which is the most common cause of ESRD worldwide. Epigenetic mechanisms can respond to changes in the environment and mediate the persistent long-term expression of DKD-related genes and phenotypes induced by prior hyperglycemia despite subsequent glycemic control, a phenomenon called metabolic memory. Metabolic memory was first described in large clinical trials of diabetes. The landmark Diabetes Control and Complications Trial (DCCT) and the Epidemiology of Diabetes Intervention and Complications (EDIC) study showed that intensive glycemic control at the early stage of type 1 diabetes delays the progression of microvascular complications at the later stage.²³ Similar long-term benefits of intensive glycemic control were also documented in patients with type 2 diabetes.²⁴^,²⁵ One of the mechanisms responsible for metabolic memory is epigenetic alteration. Recent studies using DCCT/EDIC cohorts have indicated that altered DNA methylation in blood cells, especially myeloid cells and hematopoietic stem cells, is significantly associated with complications of diabetes, including retinopathy and nephropathy.²⁶^,²⁷ These studies, together with the results of recent epigenome-wide association studies,²⁸^,²⁹^,³⁰ strongly suggest that DNA methylation changes in peripheral blood cells are related to a decline in renal function, although the precise mechanisms remain unclear.

In addition to blood cells, DNA methylation changes in kidney cells are implicated in kidney function. Cytosine methylation of kidney tubules was examined in human DKD using Illumina Infinium 450 K arrays or whole-genome bisulfite sequencing,³¹^,³² and the results showed that kidney methylation differences have the potential to be functionally important.

Transcription Factors and DNA Methylation inPodocytes

Similar to the report of DNA methylation changes in tubules described above, the association of podocyte DNA methylation changes with renal function has been indicated by researchers worldwide, including our group,³³^,³⁴^,³⁵^,³⁶^,³⁷^,³⁸^,³⁹ although human epigenome-wide studies have not yet been performed. We have previously demonstrated that the transcription factor Kruppel-like factor 4 (KLF4) regulates podocyte gene expression through epigenetic mechanisms in a gene-specific manner, which leads to a sustained change in podocyte phenotype.³⁴ The activated renin–angiotensin system in CKD leads to decreased expression of KLF4, which results in decreased KLF4-binding to the nephrin gene promoter region, causing increased DNA methylation through increased DNA methyltransferase 1 (DNMT1) binding. Increased DNA methylation of the promoter region causes reduced nephrin expression, disruption of the slit membrane, and proteinuria.³⁵ KLF4 is known to be involved in epigenetic remodeling at the early stage of reprograming as a Yamanaka factor inducing iPS cells.⁴⁰^,⁴¹ The fact that the combination of several transcription factors could induce iPS cells suggests the immense potential of transcription factors as gene-specific epigenetic regulators. Following our reports, the importance of podocyte KLF4 in various pathways on renal integrity has been reported. Podocyte KLF4 negatively regulates signal transducer and activator of transcription 3 (STAT3)-induced glomerular epithelial cell proliferation⁴² and maintains parietal epithelial cell quiescence in the kidney.⁴³ KLF4 is expressed primarily in epithelial and endothelial cells, and endothelial KLF4 in the kidney has also been shown to act in a renoprotective manner.⁴⁴^,⁴⁵ These results suggest the diverse action of KLF4 as a transcription factor, in addition to its epigenomic regulatory function, and indicate that it is a clear target for renal protection. Currently, hypoxia-inducible factor prolyl hydroxylase (HIF-PH) inhibitors are attracting attention as novel agents for renal anemia in CKD. HIF is also a transcription factor that acts in response to hypoxia and is suggested to be linked to epigenetic regulation.⁴⁶^,⁴⁷^,⁴⁸ Collectively, targeting transcription factors has the potential to alter the renal epigenome and is expected to have many possibilities of sustained effects for CKD therapy.

Although various transcription factors and epigenomic modifiers are involved in rewriting epigenetic marks, DNA methyltransferases are responsible for DNA methylation in podocytes. DNMT1 in podocytes has been reported to be involved in many pathological conditions, including diabetic nephropathy, and is reported to be associated with urinary protein levels through podocyte phenotypical changes.³⁶^,³⁷^,³⁹ Conversely, the expression of TET2, a DNA demethylase, has been recognized to have a possible therapeutic effect on podocytes.³⁸ Another report states that DNMT1 suppression in the kidney leads to protection against renal aging.⁴⁹ Therefore, inhibition of promoted DNA methylation in podocytes has the potential to be a new therapeutic approach for podocyte protection.

DNA Damage Repair and Podocyte DNA Methylation

Although the number of podocyte double-strand breaks (DSBs) in vivo could not be counted, DNA DSBs occur in any given cell to the order of 10 to 50 per cell per day, depending on the cell cycle and tissue.⁵⁰ DNA damage repair is one of the causes of changes in chromatin structure and DNA methylation status. It is known that DNA DSBs induce de novo DNA methylation of the repaired segment.⁵¹^,⁵²^,⁵³ Therefore, podocyte DNA methylation changes may be closely associated with the environment of DNA damage repair in podocytes.

Generally, DNA damage can be caused by various stresses, including exogenous stresses, such as ultraviolet radiation and chemicals, and endogenous stresses, such as reactive oxygen species, stress hormones, DNA replication errors, spontaneous reactions, and mechanical stress.⁵⁴^,⁵⁵ Because podocytes are not divided or regenerated, the accumulation of podocyte DNA damage may not cause cancer but, rather, contribute to podocyte loss through apoptosis, which may be related to renal aging.⁵⁶ Podocyte DNA DSBs are increased in various glomerular diseases, including diabetic nephropathy.⁵⁷ Glomerular DNA damage in IgA nephropathy patients is associated with a decline in kidney function,⁵⁸ and urine-derived cells isolated from patients suffering from diabetes and hypertension showed increased levels of DNA DSBs.⁵⁹ However, congenital defects of DNA repair factors are associated with various degrees of renal involvement.⁶⁰ These results indicate that acquired environmental factors as well as genetic defects are involved in podocyte DNA damage. Recently, we demonstrated that the expression of the DNA DSB repair factor lysine acetyltransferase 5 (KAT5) in podocytes is reduced in both mouse models and humans with diabetic nephropathy, leading to attenuated DNA repair in podocytes despite increased induction of DNA damage caused by high-glucose conditions.⁵⁷ Increased DNA DSBs were associated with increased DNA methylation in podocytes, leading to silenced promoter activity and decreased expression of podocyte critical genes, such as nephrin. KAT5 in proximal tubular cells acts in cytoprotection via promoted DNA repair, similar to podocytes, but it is interesting that loss of proximal tubular cell KAT5 does not alter renal function at baseline. In addition, KAT5 is involved in the epigenetic regulation of gene expression specific to tubular function.⁶¹ Knockdown of KAT5 in podocytes caused nephrotic syndrome and death from renal failure, whereas knockdown of KAT5 in proximal tubular epithelial cells resulted in almost normal kidney function at baseline. Similar to KAT5, differential expression of DNA repair factors in various types of kidney cells was indicated in a recent single-cell RNA-seq analysis of the kidney, suggesting that DNA damage repair may be conducted in a cell type-specific manner.⁶²

Various genes have different DNA methylation states in different cells, which is responsible for their cellular characteristics, at least in part. Phenotypes can differ because of changes in the DNA methylation status of genes that play important roles in regulating the functions of the cells. Focusing on DNA damage repair may reveal the gene-specific mechanisms of DNA methylation changes because previous reports suggest that DNMT1 is recruited to DNA DSB sites, contributing to epigenetic gene silencing through DNA methylation.⁵²^,⁶³ Furthermore, our reports showed that CKD is associated with promoted DNA methylation in podocyte marker genes, including nephrin, where chromatin is open and gene transcription is active.³⁴^,³⁵ Taken together, gene-specific DNA methylation may be explained in part by chromatin accessibility. Because opened chromatin regions are vulnerable to DNA damage induction,⁶⁴^,⁶⁵ DNMT1 recruitment to DNA damage sites followed by DNA DSBs may occur more frequently than recruitment to closed chromatin regions. Although DNA demethylation of closed chromatin regions may occur in pathological conditions, both DNA damage and repair occur in opened chromatin regions more frequently; therefore, mechanisms other than DNA damage may be involved in DNA demethylation of disease states. Understanding DNA damage repair in podocytes may be necessary for developing renoprotective therapies via epigenetic regulation in podocytes.

Clinical Application and Future Perspectives

The detection of DNA damage and epigenetic events, including DNA methylation, during the early stages of CKD could be valuable for estimating renal prognosis and prompting treatment to prevent progression to ESRD (Fig. 2). We have reported that DNA methylation and DNA DSBs in the glomeruli of IgA nephropathy patients were associated with annual decline in the estimated glomerular filtration rate (eGFR).⁵⁸ Additionally, podocyte DNA DSBs estimated by examining urine-derived cells were increased in early-stage CKD in patients with diabetes and hypertension.⁵⁹ Besides podocytes, it is reported that the DNA methylation pattern of proximal tubule-specific loci in urine sediment is a potential marker of kidney function decline in diabetes.⁶⁶^,⁶⁷ DNA damage and DNA methylation changes have been suggested as useful markers of kidney function and prognosis, and further research is needed for the accurate and non-invasive assessment of these changes, which is quite different among cell types.

Fig. 2.

Targeting podocyte DNA damage and DNA methylation for CKD treatment.

Detection of DNA damage and DNA methylation during the early stages of CKD could be valuable for estimating renal prognosis and for prompt treatment to prevent progression to end-stage renal disease. Restoring the podocyte epigenome may prolong the ‘healthy lifespan’ of podocytes.

Using podocyte epigenome in a therapeutic strategy for CKD, the advantages of detection of DNA damage and DNA methylation changes may include the following: 1) the possibility of bringing remaining injured podocytes closer to a healthy state, 2) the possibility of a sustained therapeutic effect, 3) the potential to be a renal prognostic marker or a marker of aging, and 4) the potential to contribute to precision medicine. Because of the podocyte nature of a terminally differentiated cell, one of the major therapeutic goals would be to protect the remaining podocytes and return them to a healthy state. If we can improve the epigenetic status of podocytes, we may be able to achieve a sustained therapeutic effect on podocyte function. Epigenetic changes in terminally differentiated cells, such as neurons and podocytes, eventually lead to a decrease in the number of normal cells by promoting cell death, which might be one of the reasons for the aging phenomenon.⁶⁸^,⁶⁹ Therefore, the podocyte epigenome may become not only a renal prognostic marker but also a marker of aging. This may be clinically useful because systemic aging is associated with renal aging, resulting in decreased eGFR and increased risk of kidney injury. Moreover, identification of epigenetic signatures of podocytes might also inform precision medicine approaches. Based on the profiles of DNA damage and epigenetic alterations in podocytes, a system could be developed to evaluate aging and predict renal prognosis and risk of cardiovascular complications, which could be used to select drugs and therapies according to the individual characteristics of a patient.

When considering epigenetic modulations, especially for chronic diseases, site-specific therapy is necessary to avoid serious side effects such as malignancies.⁷⁰ Focusing on transcription factors would be one solution, because many transcription factors are site-specific and are involved in gene-specific epigenetic regulation, such as KLF4.³⁴^,³⁵ Another solution would be to focus on cell type-specific DNA damage repair pathways. Podocyte-specific delivery of epigenetic modifiers may be a candidate for achieving site specificity. It is expected that recent advances in new technologies, such as nanoparticles, to deliver epigenetic modulators specific to the kidney⁷¹^,⁷² or epigenetic editing technology⁷³^,⁷⁴ will be applied in the development of novel therapies to restore the podocyte epigenome. The combination of new technologies and further understanding of the pathophysiology is expected to lead to the development of novel therapies targeting the podocyte epigenome to address the emerging number of CKD patients.

Acknowledgments

I am grateful to the Keio University Medical Science Fund for the Rising Star Award. I also thank all my mentors, collaborators, and colleagues. This study was supported by the FOREST Program of the Japan Science and Technology Agency (Grant Number JPMJFR210 V) and Grants for Scientific Research (22H03091 and 19K08688) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Conflicts of Interest

The author declares that no conflict of interest exists.

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