Non-genetic mechanisms of diabetic nephropathy

Abstract

Diabetic nephropathy (DN) is one of the most common microvascular complications in diabetes mellitus patients and is characterized by thickened glomerular basement membrane, increased extracellular matrix formation, and podocyte loss. These phenomena lead to proteinuria and altered glomerular filtration rate, that is, the rate initially increases but progressively decreases. DN has become the leading cause of end-stage renal disease. Its prevalence shows a rapid growth trend and causes heavy social and economic burden in many countries. However, this disease is multifactorial, and its mechanism is poorly understood due to the complex pathogenesis of DN. In this review, we highlight the new molecular insights about the pathogenesis of DN from the aspects of immune inflammation response, epithelial–mesenchymal transition, apoptosis and mitochondrial damage, epigenetics, and podocyte–endothelial communication. This work offers groundwork for understanding the initiation and progression of DN, as well as provides ideas for developing new prevention and treatment measures.

Keywords

diabetic nephropathy / immune inflammatory response / epithelial–mesenchymal transition / apoptosis / mitochondrial damage / epigenetics / podocyte–endothelial communication

Introduction

Diabetic nephropathy (DN) is one of the most common and severe microvascular complications in diabetes mellitus patients and is the leading cause of end-stage renal disease (ESRD). Furthermore, approximately 50% of patients with DN develop ESRD [1]. The high incidence of DN in diabetes patients is approximately 30%–40% [2]. Moreover, approximately 70% of type 1 or 2 diabetes patients with clinical manifestations of chronic kidney disease (CKD) will progress to DN [3]. DN is pathologically characterized by mesangial dilatation, glomerulosclerosis, and Kimmelstiel‒Wilson nodules [4]. Its clinical manifestations are characterized by abnormal urinary albumin excretion from microalbuminuria (30–300mg/min) to massive albuminuria (>300 mg/min). Its progression includes the following five steps: an initial hyperfiltration and renal hypertrophy, microalbuminuria post-exercise, persistent microalbuminuria, kidney dysfunction, and end-stage renal failure. The onset of DN is difficult to detect; however, once dominant proteinuria is diagnosed, the progress is irreversible, and the rate of progress to ESRD is approximately 14 times higher than that in other kidney diseases.

With the rapid increase in the prevalence of diabetes over the past few years, the incidence of CKD associated with diabetes has surpassed that associated with glomerulonephritis and has become the leading cause of CKD in urban inpatients in China [5]. The social and economic burden caused by DN increases annually. However, the mechanism is not yet elucidated due to the complex pathogenesis of DN. Therefore, exploring the pathogenesis of DN and finding effective prevention and control measures are the main focus of research. Some of the topics in early studies include advanced glycation end products, polyol pathway activation, protein kinase activity, abnormal lipid metabolism, and hemodynamic changes. New discoveries were recently found regarding the pathogenesis of DN, and the genetic factors were frequently involved. Combined with the latest developments, we systematically reviewed the pathogenesis of DN from the aspects of non-genetic factors, such as immune inflammation, epithelial–mesenchymal transition, apoptosis and mitochondrial damage, epigenetics, and podocyte–endothelial communication. This review aims to further understand the development of DN and provide ideas for designing new prevention and treatment measures.

Immune inflammatory response and DN

Immune inflammatory response is the most important factor in promoting the damage of podocytes and the production of albuminuria in DN. The secretion of IL-6, IL-18, TNF-a, and other inflammatory factors is dysregulated in the early stage of DN. TNF-α amplifies the inflammatory network of cytokines, thereby leading to the deterioration of the DN progress [6–10]. Acai (Euterpe oleracea Mart.) seed extract has antioxidant and anti-inflammatory properties, can reduce renal injury, and prevent renal dysfunction by reducing inflammation and ameliorating the renal filtration barrier [11]. Therefore, exploring the mechanism of cell injury in DN from the perspective of “immune inflammation” and finding new therapeutic targets have become the focus of domestic and overseas researchers [12,13]. For example, the increased expression of innate immune receptors can directly mediate the immune inflammation. Tristetraprolin (TTP) can directly and indirectly reduce the expression of inflammatory factors through its anti-inflammatory effect. Moreover, 1,25-dihydroxyvitamin D3 (VD3) and vitamin D receptors (VDR) exert anti-inflammatory effects through some signaling pathways. The activation of complement systems, such as mannose-binding lectin (MBL), H-ficolin and complement component C3, and membrane attack complex (MAC) may promote renal injury under diabetic conditions.

Increased expression of innate immune receptors

Innate immune receptors are involved in the progress of DN. Toll-like receptors (TLRs) link the innate and acquired immunities. Toll-like receptors 2 (TLR2) and 4 (TLR4) are both involved in DN inflammatory responses [14]. Xu et al. stated that the expression levels of TLR2, TLR4, IL-1b, and inflammatory infiltration are significantly increased in the kidneys of diabetic rats. However, when the expression levels of TLR2 and TLR4 are suppressed by drugs, the inflammatory molecules and inflammatory infiltration are significantly decreased in the renal tissue [15]. Only TLR4 deficiency can reduce proteinuria, interstitial fibrosis, and inflammatory reaction caused by diabetes without improving the glomerular lesions. This mechanism is associated with the reduced degree of renal tubular damage and the level of target downregulation.In vitro studies showed that inducing albumin is more potent than increasing glucose for heat shock protein 70 (HSP70), which relies on TLR4 to trigger the production of inflammatory mediators and eventually enhance the inflammatory response of DN. Furthermore, the levels of TLR4 and HSP70 are significantly increased in the dilated renal tubules of patients with DN. Therefore, Jhenget al.proposed a new mechanism as follows: the HSP70-TLR4 axis is partially activated by albumin in renal tubular cells, which is associated with tubulointerstitial inflammation and the induction of existing microalbuminuria in DN progression [16].

Fcg receptors are important in DN progression. Lopez et al. proved that the infiltration of renal inflammatory cells is weakened, and the expression levels of genes related to oxidative stress and fibrosis are significantly decreased in the IgG Fcg immunoreceptor-deficient diabetic mice compared with those in with normal Fcg receptors; hence, they proposed that Fcg receptors participate in the inflammatory process of DN [17]. Herrera et al. found that proteinuria is decreased and podocyte injury is improved when the activation of cytotoxic T lymphocyte-associated antigen4 (CTLA4) Fc receptors to T cells is inhibited [18].

Nucleotide-binding domain and leucine-rich domain receptor (NLRS) are oligomerized into polyprotein complexes upon stimulation to mediate innate immunity. The NLR family and apoptosis inhibitory proteins (NAIPs) recognize the bacterial pathogens, induce NLRC4 (NLR family, CARD domain containing 4) activation and NAIP-NLRC4 inflammasome formation, mediate inflammatory response, and promote DN progression [19].

Weakened anti-inflammatory effects of tristetraprolin

Some RNA binding proteins are involved in the regulation of inflammation in DN. TTP is a highly conserved, AU-rich binding protein that is widely distributed in human and animal cells. TTP can regulate the expression of the associated proteins by binding to the AU-rich region of the mRNA 3′UTR region and degrading the mRNA. TTP may bind directly to the mRNA 3′UTR of inflammatory factors, such as ICAM-1, IL-8 [20], IL-10 [21], IL-17 [22], IL-23 [23], IL-6 [24], and TNF-α [25], and then exert its anti-inflammatory effects by degrading its mRNA. Moreover, TTP may indirectly affect the synthesis of downstream inflammatory factors by binding to the P65 subunit of NF-kB and affecting its nuclear translocation [26,27]. NF-kB signaling pathway is important in the inflammation response in DN. This pathway mediates the increased expression levels of inflammation-associated proteins, such as serum amyloid A (SAA), monocyte chemoattractant protein-1 (MCP-1), intercellular adhesion molecules (ICAM-1), vascular cell adhesion molecules (VCAM-1), advanced glycation end products (AGEs), and interleukin (IL) family. These proteins can cause kidney cell damage and lead to kidney damage in different ways [13]. Liu et al.showed that the TTP levels decrease in high-glucose conditions, whereas those of IL-6, IL-18, and other inflammatory factors increase in the urine and serum of DN patients. Thus, a high-sugar environment can reduce TTP expression and thus participate in the inflammation in DN [8].

Decreased expression of 1,25-dihydroxyvitamin D3 (VD3) and its receptor (VDR)

VD3 and VDR may exert anti-inflammatory and immunomodulatory effects and disturb the balance between inflammation and anti-inflammation in DN. Yanget al. proved that the mechanism is related to the cytoskeleton rearrangement caused by the cross-talk of signal transducers and transcriptional activator 5-vitamin D receptors (STAT5-VDR) in human THP-1 monocytes [28]. Moreover, Zhang et al. reported that VDR could exert anti-inflammatory effects by promoting pro-inflammatory macrophages (M1), which are induced by high glucose levels to transform into anti-inflammatory macrophages (M2) via the vitamin D receptor-peroxisome proliferator-activated receptor-g (VDR-PPARg) signaling pathway [29]. This mechanism was further validated in the VDR-deficient mouse models by Wanget al. and PPARg-deficient mouse models by Toffoli et al.[30,31]. Xie et al. proposed that VDR can delay the progress of fibrosis in the renal interstitium by downregulating p38 MAPK (mitogen-activated protein kinase family of subfamily), collagen protein 3 (COL3), and collagen 4 (COL4) [32]. Guan et al. proposed that VDR can exert a renal protective effect by inhibiting renin–angiotensin system (RAS) and reducing proteinuria [33]. The reduced expression levels of VDR and VD3 in patients with DN may cause the inflammatory response by the TLR4/NF-kB p65 signal transduction pathway and thus are associated with the development of DN [34].

Activation of the complement system

The complement system is an important part of the immune system and plays a key role in the clearance of microbes and damaged cells by the phagocytic cells and antibodies. The following are the three pathways that activate the complement system: classical, alternative, and lectin pathways [35]. No pathogen-induced complement activation of classical and alternative pathways is found in the pathogenesis of DN. However, the lectin pathway, which is highly regarded in the pathogenesis of inflammation, binds to glycoproteins, such as mannan and N-acetylglucosamine, through MBL, acts on endothelin, and leads to complement activation and tissue damage [36]. The lectin pathway is activated initially when MBL binds to the glycan-associated mannose or ficolins bind to N-acetylglucosamine, N-acetylgalactosamine, or N-acetyl-neuraminic acid residues on microbial surfaces. Such binding activates the MBL-associated serine proteases (MASPs) and leads to the cleavage of the complement components, which ultimately generates MAC, the end product of the complement system. The elevated levels of MAC in T1DM suggest the activation of the complement system from MBL, MASP, and C3 to MAC.

Some experimental and clinical studies confirmed the connection between the complement system and DN. Ostergaard et al. proved that compared with wild-type mice, streptozotocin-induced diabetic MBL-knockout mice have less renal damage, such as reduced renal hypertrophy, urinary albumin excretion, and decreased collagen IV expression. Thus, MBL has a potential pathological role for kidneys under diabetic conditions [37]. A subsequent study revealed that the MBL levels in the glomeruli of streptozotocin-induced diabetic mice are twice of those in non-diabetic controls, indicating the direct effect of MBL in DN [38]. Moreover, H-ficolin levels are closely related with risk of progression to microalbuminuria or macroalbuminuria in DN based on a prospective 18-year observational follow-up study [39]. Jenny found that the levels of MASP-1 and MASP-2 are significantly high among T1DM patients [40]. Yang et al. observed the deposition of C3 in glomeruli and glomerular capillaries in the models of T1DM (OVE26 diabetic mouse) and T2DM (KK mice) [41]. Uesugi et al. confirmed the deposition of MAC in diabetic glomeruli, the correlation between the levels of MAC, and the magnitude of mesangial expansion among patients with T1DM [42].

Two main mechanisms can explain the involvement of complement systems and the development of DN. First, the lectin pathway is activated by the binding of PRMs to the proteins glycated in a hyperglycemic state [43]. Second, hyperglycemia can induce the glycation of complement regulatory proteins, which disturbs their regulatory capacity [44]. These phenomena result in the over-activation of complement pathways and facilitate the complement auto-attack, which has a pathogenic role in hyperglycemia-induced renal injury [45].

Other inducing factors

In addition to hyperglycemia, hyperlipidemia, and albuminuria, other inducing factors for DN include diacylglycerol (DAG)-protein kinase C (PKC) pathway, cyclooxygenase, AGEs, polyol pathway, hexosamine pathway, and oxidative stress. These factors interact with one another, thereby causing inflammatory responses and leading to the development of glomerulosclerosis under diabetic conditions [46–48].

The relationship between immune inflammatory response and DN is shown in Fig.1.

Epithelial-mesenchymal transition (EMT) and DN

Podocytes and renal tubular epithelial cells can obtain a mesenchymal property, called epithelial mesenchymal transition (EMT), in a high-glucose condition. EMT is one of the sources of matrix-derived fibroblasts that produce large amounts of ECM accumulation [49]. A typical feature of DN is the excessive deposition of ECM proteins in the glomeruli and tubulointerstitium, leading to glomerulosclerosis and interstitial fibrosis, the most characteristic morphological changes of DN [50].

EMT of podocytes

As the most important component of the renal filtration barrier, podocytes have a critical role in the development of DN. Moreover, podocyte injury and production of proteinuria are closely related. EMT is an important link in DN podocyte injury. In this process, the expression levels of podocyte proteins, such as nephrin and podocin, are reduced. By contrast, the expression levels of mesenchymal cell marker proteins, such as desmin, fibroblast-specific protein-1 (FSP-1), α-smooth muscle actin (α-SMA), and matrix metalloproteinase 9 (MMP9), are increased. These phenomena result in the dysfunction of podocytes, damage of filtration barrier, leakage of protein, and eventually to glomerular sclerosis, thereby affecting the function of glomerular filtration and promoting the progress of DN [51].

Transforming growth factor (TGF-b) is an important transcriptional regulator of cell transdifferentiation; its expression increases under diabetic conditions. On the one hand, TGF-β can activate E-cadherin through the TGF-β/snail1/E-cadherin signal pathway. On the other hand, TGF-β can induce the expression of transforming growth factor β-induced protein (TGFβIp), which in turn reduces the expression of β-integrin and promotes EMT of podocytes [52].

A key molecule named β-catenin plays a key role in high glucose-induced podocyte transdifferentiation. Glycogen synthase kinase 3β (GSK-3β) shows an increased expression and activity in db/db mice DN model and immortalized mouse podocytes cultured with high glucose; this increase results in the EMT of podocytes by classical GSK-3β/Wnt/β-catenin [53,54]. SB216763, a highly selective small molecule inhibitor of GSK-3β, significantly enhances the podocyte protective effect that depends on the antioxidant effect of nuclear factor erythroid 2-related factor 2 (Nrf2) [55]. However, the expression of β-catenin is not entirely regulated by GSK-3β through the classical Wnt/β-catenin pathway. Connective tissue growth factor (CTGF) can result in podocyte EMT by activating β-catenin under a high-glucose condition [56]. In addition, VD3 and its receptor, VDR, are reduced in high-glucose-cultured podocytes, regulate the nuclear translocation of β-catenin, and participate in the transdifferentiation of podocytes [53]. RAS-related C3 botulinum toxin substrate 1 (Rac1) and its major downstream effector, p21-activated kinase 1 (PAK1), have a key role in cellular events, such as cytoskeletal remodeling, and contributes to EMT. Rac1/PAK1 signaling promotes the high glucose-induced podocyte EMT by activating β-catenin and snail transcription [57].

Derangement of autophagy is also an important mechanism that leads to the transdifferentiation of podocytes. A high-glucose condition results in the reduction of phosphorylated p62 protein and accumulation of p62 protein, the subsequent acceleration of cell exit from cell mitosis, and the enhanced lysosomal dysfunction and derangement of autophagy associated with EMT of podocytes [58].

The relationship between EMT of podocytes and DN is shown in Fig.2.

EMT of tubular epithelial cells

CD36 is a key mediator of reactive oxygen species (ROS) production in CKD [59]. Hou et al.stated that high glucose induces the generation of ROS, which can upregulate CD36 expression, regulate TGF-β1 expression, and activate Smad2 and extracellular signal-regulated kinase (ERK) in HK-2 cells. The inhibition of CD36 alleviates HG-induced EMT by decreasing TGF-β1 expression and activating Smad2 and ERK1/2 [60,61]. Moreover, the increase in TGF-β1 can activate mitogen-activated protein kinase (MAPK) and ERK in proximal tubular epithelial cells, thereby contributing to EMT. However, allicin can regulate the TGF-β1/ERK signaling pathway to inhibit EMT [62].

Duan et al.showed that high glucose can induce the production of oxidative stress and ROS, which activate the nuclear transcription factor specificity protein 1 (Sp1), and this is involved in the EMT of tubular epithelial cell and the accumulation of ECM, thereby promoting the progress of DN [63].

Pang et al. found a new pathogenic pathway for the development of renal fibrosis in DN.

Urotensin II (UII) and its receptor, G protein-coupled receptor 14 (GPR14), are both highly expressed in the kidney tissue of DN patients. UII, combined with its receptors, can activate phospholipidase C (PLC) and the inositol 1,4,5 triphosphate (InsP3) receptor-dependent pathway; release calcium from endoplasmic reticulum (ER) and induce ER stress; cause EMT; and increase the production of extracellular matrix (ECM) in renal tubular epithelial cells [64].

The relationship between EMT of tubular epithelial cells and DN is shown in Fig.3.

Apoptosis and DN

Apoptosis is one of the leading causes of podocyte and renal tubular epithelial cell defects and has a crucial role in the DN pathogenesis. Podocytes are highly important component cells of the glomerular filtration barrier; their defect from apoptosis results in proteinuria and promotes the progress of DN. Therefore, the mechanism of podocyte apoptosis may help us further understand the DN pathogenesis.

ER stress is an important mechanism that leads to diabetic complications, including DN [65]. Excessive ER stress can induce the apoptotic signaling and result in cell injury and death through the mediation of downstream molecules, such as caspase 12 and c-Jun N-terminal kinases (JNK) [66]. Cao et al. proved that excessive ER stress can promote the progress of DN by inducing podocyte apoptosis. This phenomenon is inhibited by ursodeoxycholic acid and 4-phenylbutyrate by suppressing the ER stressin vivo and in vitro [67]. Sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) plays a key role in this mechanism. SERCA2 dysfunction is a potential pathogenesis of diabetic complications, including DN. Impaired activity or expression of SERCA2 triggers ER stress and leads to the disruption of ER Ca²⁺ homeostasis [68]. ER stress and diabetic conditions can be alleviated by increasing SERCA2 function using SERCA2 activators, such as CDN1163 [69]. In vivo and in vitrostudies of Guo et al. proved that astragaloside IV (AS-IV) can inhibit ER stress-induced podocyte apoptosis by restoring SERCA activity, increasing SERCA2 expression, and attenuating renal injury in diabetes [70]. Ca²⁺ is released out of the ER predominantly through the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) [71–74] and ryanodine receptor (RyR) [75–77]. Especially, IP3R and IP3-induced Ca²⁺ release are key mechanisms in apoptosis [78–80].

Patients with DN have increased palmitic acid, the most common saturated free fatty acid in the plasma that is involved in cellular dysfunction and death of humans [81,82]. This fatty acid upregulates the Ca²⁺ level in the cytosol and mitochondria, cytochrome C release, mitochondrial membrane potential (MMP), and depletion of ER Ca²⁺ via the mitochondrial Ca²⁺ uniporter (MCU), thereby inducing apoptosis in podocytes that leads to the aggravation of the proteinuria of DN. MCU inhibitors, such as ruthenium red and Ru360, can block the activity of palmitic acid in mouse podocytes. Thus, Ca²⁺ uptake via mitochondrial uniporters contributes to palmitic acid-induced apoptosis [83].

Tubular injury can predict renal disease progression better than glomerular pathology. Apoptosis plays a critical role in tubular atrophy in diabetic patients. The increased expression levels of apoptosis-related proteins, such as Bax, cytoplasmic cytochrome-c, and caspase-3, are involved in renal tissue injury induced by hyperglycemia. This phenomenon is suggestive of apoptosis. However, the treatment of irbesartan and perindopril can effectively ameliorate renal tissue injury and apoptosis by blocking the RAS in DN and reducing the expression of apoptosis-related proteins [84].

The relationship between apoptosis and DN is shown in Fig. 4.

Mitochondrial damage and DN

Mitochondria are the key organelles of energy metabolism. Hyperglycemia, hyperlipidemia, and albuminuria of diabetic patients can cause dysfunction or structural deletion of electron transport in the mitochondrial oxidation respiratory chain, leading to mitochondrial dysfunction, increased lipid peroxidation, and decreased antioxidant effects, thereby promoting the progress of DN [85]. On the one hand, mitochondrial damage can induce the production of a large number of ROS, and the expression levels of vascular endothelial growth factor (VEGF) and TGF-β are increased by the phosphoinositide 3 kinase/serine-threonine kinase (P13K/Akt) pathway to accelerate glomerular sclerosis and the apoptosis of renal tubular epithelial cells; this phenomenon eventually causes the thickening of the basement membrane, renal fibrosis, and tubulointerstitial accumulation, thereby promoting the progress of DN [86]. On the other hand, NADPH oxidases (Nox) are expressed in a cell-specific manner in the kidney [87,88], wherein the Nox isoform, Nox4, is abundantly expressed and produces substantial renal ROS during mitochondrial damage. Nox-derived ROS are involved in the physiological processes of DN by reducing glucose tolerance and increasing proteinuria and renal fibrosis in high glucose-induced mitochondrial damage [89,90].

Mitochondria dynamics

Mitochondria are organelles that constantly undergo fission and fusion dynamics. When the cells are in the state of damage or stress, their mitochondria will fissure and produce some mitochondrial fragments, which results in mitochondrial damage and leads to cell damage and death [91]. Wang et al.showed that mitochondria are disrupted by activating Rho-associated coil-forming protein kinase 1 (ROCK1) in podocytes and endothelial cells. The mitochondrial fragmentation is associated with the pathogenesis of a variety of renal diseases, including DN [92].

Effects of increased NLRP3

Urinary albumin overload can induce ROS production by activating the cascade of nucleotide binding oligomerization domain-leucine-rich repeats containing pyrin domain 3 (NLRP3)/caspase-1/cytokines. This phenomenon results in mitochondrial dysfunction, apoptosis, and phenotype transformation of cells [93,94]. In patients with proteinuria, the expression of NLRP3 is significantly increased in the renal tubular epithelial cells, and the elevated levels are parallel to proteinuria severity [95]. Thus, mitochondrial dysfunction/NLRP3 inflammatory tubule axis is important in the pathogenesis of proteinuria-induced injury of renal tubular epithelial cells.

Effects of decreased Rap1b

Ras-proximate-1b (Rap1b) can mitigate mitochondrial oxidative stress and ameliorate mitochondrial dysfunction in renal tubular epithelial cells induced by CCAAT/enhancer-binding protein β-PPAR-γ coactivator-1α(C/EBP-β-PGC-1α) signal transduction pathway, which ameliorates renal tubular epithelial cell injury in DN. However, Rap1b expression is significantly decreased in DN patients, thereby resulting in mitochondrial catalase and superoxide dismutase accumulation and oxidative stress aggravation, promoting the progress of DN [96].

The relationship between mitochondrial damage and DN is shown in Fig.5.

Epigenetics and DN

Epigenetics recently emerged with an increasingly powerful role in the occurrence and development of DN. High-glucose stimulation can cause epigenetic modifications, such as non-coding RNA regulation, DNA methylation, histone modifications, and chromatin remodeling. When exposed to the diabetic environment, persistent and stable epigenetic changes can be acquired during development or as adaptation to the metabolic fluctuations associated with diabetes.

Non-coding RNA regulation

Non-coding RNAs, including microRNAs (miRNAs) and long-chain non-coding RNAs (lncRNAs), refer to RNAs that do not encode proteins. MicroRNAs (miRNAs) are ubiquitous endogenous non-coding RNAs of 19–25 nucleotides in length that regulate genes in eukaryotes and inhibit the expression of target genes. miRNAs are involved in various diseases, including DN, through the post-transcriptional regulation of target genes. Hyperglycemia can alter the expression of miRNA, leading to endothelial cell dysfunction, renal fibrosis, podocyte transdifferentiation, and other pathophysiological processes. A diabetic condition downregulates miR-26a in glomeruli and renal tubules but upregulates miR-27a in glomerular mesangial cells. The former enhances the TGF-β/CTGF signaling pathway, whereas the later inhibits peroxisome proliferator-activated receptorg (PPARg) and activates TGF-β/Smad3 signaling pathway. Both phenomena promote CTGF, fibronectin (FN), collagen I, and other fibrosis of the key media expression and ECM accumulation for glomerular and renal tubular fibrosis, thereby promoting the progress of DN [97]. miR-135a is significantly upregulated in DN patients and thus can reduce calcium depletion-induced calcium into cells by inhibiting the expression of cationic channel C subfamily member 1 (TRPC1) in transient receptors and promoting microalbuminuria and renal fibrosis; thus, the progress of DN is promoted. When miR-135a is knocked down, the level of TRPC1 recovers, and the synthesis level of FN and collagen I decreasesin vivo [98]. miR-21, miR-451-5p, and miR-16 are the key regulators of glomerular sclerosis and can promote the production of inflammatory and fibrotic mediators to induce fibrosis of the glomeruli and tubules and result in DN [99]. miR-126, miR-155, and miR-146a are highly expressed in vascular endothelial cells and are important in maintaining vascular integrity and angiogenesis. These miRNAs are also highly expressed in glomeruli and periosteal endothelial cells but are downregulated in DN patients; hence, the structural damage of endothelial cells promotes the progress of DN [100,101]. Moreover, hyperglycemia can reduce the expression of miR-346 in podocytes. On the one hand, miR-346 upregulates glycogen synthase kinase 3β (GSK-3β) to promote podocyte transdifferentiation [102]. On the other hand, GSK-3β can reduce the signal protein SMAD3/4 expression to induce glomerular histological changes and thus weaken the renal function [103].

lncRNA has recently become a research hotspot. It does not encode protein; however, lncRNA is involved in the expression of genes at multiple levels of regulation and in the occurrence of DN. The mechanism of lncRNA in DN is mainly focused on endothelial and mesangial cells. lncRNA plasmacytoma transforming gene 1 (PVT1) and lung adenocarcinoma metastasis-related transcription factor-1(MALAT1) may be associated with kidney diseases [104,105]. The expression of lncRNA in human mesangial and endothelial cells is significantly upregulated by hyperglycemia, which could be regulated by the expression levels of FN1, collagen IV, TGF-β1, and type 1 plasminogen activator inhibitor (PAI-1) [106].

DNA methylation

DNA methylation is the most widely studied epigenetic mechanism that regulates the expression of genes and is important in chromosomal stability. Different levels of DNA methylation are found in the whole genome sequencing of DN and CKD patients. DNA methylation is also affected by uremic components associated with renal failure. Hyperhomocysteinemia appears in CKD and ESRD patients with increased S adenosine homocysteine. The methylation levels of DNA extracted from saliva vary in the different stages of DN development. Significant differences in the methylation of at least two CpG sites were found in both groups [107]. Thus, DNA methylation levels can be used to distinguish between diabetic ESRD and renal complications without diabetes. This finding suggests that DNA methylation differences may play an important role in predicting disease susceptibility and progression.

Histone modifications

Histone modification is one of the important mechanisms in epigenetics, including histone acetylation, methylation, phosphorylation, ubiquitination, glycosylation, ADP ribosylation, and carbonylation. Among them, histone acetylation and methylation are dominantly reported. However, ubiquitination and other aspects are rarely investigated in current studies. Histone acetyltransferase (HAT) and histone deacetylase (HDAC) are bi-directional reversible regulators of histone and non-histone N-terminal domains. Acetylation and deacetylation are closely related to the occurrence of DN. The important macromolecule protein CBP (CREB binding protein), GCN5 (histone acetylated ammonia synthesis of universal control protein 5), p/CAF (p300/CREB binding protein related factors), and P300 can promote inflammation in HAT. Different types of HDAC have different roles. HDAC1 and HDAC5 can inhibit TGFβ1-induced gene expression and affect the expression of inflammatory factor. HDAC2 can promote the progress of fibrosis and HDAC4 and promote podocyte injury, both of which are involved in the progress of DN through different mechanisms [108]. H3 histone lysine can be methylated (H3Kme) under diabetic condition to induce the expression of TGF-β1 in renal mesangial cells and increase the expression levels of CTGF, collagen⁃α1, PAI-1, and other ECMs related genes. H3K36 can also promote glomerular fibrosis, enhance the expression of pro-inflammatory cytokines, promote the accumulation of ECM, and thus promote the progress of DN. However, both H3K9 and H3K27 can inhibit fibrosis factor, pro-inflammatory genes, and superoxide dismutase expression, thereby reducing the inflammatory response and fibrosis and thus delaying the progress of DN [109]. Both the increased ubiquitination of H2A histone and decreased ubiquitination of H2B histone can promote the progress of DN [110].

Chromatin remodeling

Chromatin remodeling is a mechanism of epigenetics that mainly refers to the process of replication and recombination of gene expression, chromatin packaging status, nucleosomes in the histone, and the changes in corresponding DNA molecules under specific conditions. The changes in the activity of histone kinase MSK2 can cause reshaping of chromatin, which in turn affects the metabolic activity of podocytes and promotes the progression of DN. miR-93, a metabolic/epigenetics switch associated with the metabolic status of diabetes and chromatin remodeling, is a modulator of podocyte nucleosomal dynamics that can affect the metabolism of podocytes and thus promote the progress of DN [111].

Podocyte–endothelial communication and DN

Podocyte vascular endothelial growth factor A (VEGFA) levels play an important role in the insulin resistance and development of DN [112,113]; both hyperglycemia and ROS can lead to loss of glomerular glycocalyx [114,115]. Salmon et al. found that VEGFA level is increased and GEC glycocalyx is lost in diabetic glomeruli [116]. Gnudi et al. proposed that VEGFA secreted from podocytes has a potential crosstalk with GEC glycocalyx, which is called VEGFA signaling [117]. In conclusion, an increasing VEGFA level can cause glycocalyx shedding from the GECs during the early phases of diabetes (red arrow ① in Fig. 6).

VEGF-A165b, the inhibitory isoform of VEGFA, maintains the GEC glycocalyx in diabetes. Oltean et al. found that the renal expression of VEGF-A165b is lost in patients with diabetes and progressive nephropathy. Subsequent murine models of DN and isolated human diabetic glomeruli showed that VEGF-A165b can restore the damaged glomerular endothelial glycocalyx and improve the renal function [118]. Recently identified key molecules, such as vascular endothelial growth factor (VEGF)-A165b, VEGF-C, and angiopoietin-1 (ANGPT1), confer a beneficial effect toward glycocalyx maintenance (green arrow ② in Fig. 6).

Moreover, endothelin-1 (ET-1), an endothelial-derived vasoconstrictor, is released by GECs under diabetic conditions and results in shedding of the glycocalyx. Subsequently, this finding was confirmed in transgenic murine models and conditionally immortalized murine podocytes and GECs [119]. Therefore, molecules produced by the endothelium can signal to the podocyte and then back to the glomerular glycocalyx. This phenomenon can be prevented by knocking out the ET-1 receptor specifically in the podocytes. ET-1 receptor antagonists can ameliorate early microalbuminuria in DN [120]. Conversely, VEGF-A165 and endothelin (ET; secreted by GECs and signaling to the endothelin-1 receptor in the podocyte, causing this cell to release heparanase, which then acts on the glomerular glycocalyx to cleave heparin sulfate) promote shedding of the GEC glycocalyx (red arrow ③ in Fig. 6).

Conclusions

The pathogenesis of DN is extremely complicated and each step has unique characteristics and cross-links to form a complex regulatory network. For example, the decrease in VD3 and VDR can cause inflammation and promote the EMT of podocytes in DN patients. Some types of damage in mitochondria may cause apoptosis of podocytes and renal tubular epithelial cells. p62/Sequestosome 1 enhances EMT by affecting the pathological conditions of autophagy in podocytes [58]. Mitochondrial dysfunction activates the NLRP3/caspase-1/cytokine cascade response and induces apoptosis and EMT of renal tubular epithelial cells. Epigenetics, such as non-coding RNA, can cause inflammatory response by regulating the expression of inflammatory cytokines and thus promote the progress of DN.

The research about pathogenesis of DN is currently on-going. The aspects of apoptosis, EMT, and anti-inflammatory proteins, such as TTP, GSK-3b, and VDR, recently become the focus of research. Some mechanisms may be potential therapeutic targets, which are feasible and may be coming soon. For instance, treatments using irbesartan and perindopril can prevent renal tissue injury and apoptosis effectively by blocking the generation of excessive RAS and proteins associated with apoptosis. Vitamin D, an inexpensive and easy-to-administer drug that ameliorates DN, can be used to weaken the inflammation and prevent the EMT of podocytes. Some well-known traditional Chinese medicines include astragaloside IV (AS-IV) and Moutan Cortex (MC). AS-IV can normalize insulin sensitivity and glucose tolerance, ameliorate renal inflammation and glomerulosclerosi, and improve the renal function. The mechanism is related to the regulation of SERCA2 expression, which inhibits ER stress-induced podocyte apoptosis and attenuates renal injury. The terpene glycoside component of MC exerts favorable anti-inflammatory properties in DN treatment.

Some of these mechanisms are still not thoroughly explored. As a result, worldwide experts are far from agreeing on the treatment programs of DN, and the treatment effects are not satisfactory. Therefore, unremitting efforts of medical researchers are needed to provide novel insights for the prevention and treatments of DN.

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