Current clinical insights into circRNAs and signal transduction in diabetic nephropathy

Diabetic nephropathy (DN) is a chronic kidney disease that occurs in patients with diabetic mellitus. In the United States of America, one in three people with diabetes suffers from DN. Globally, DN occurs in 30%–40% of patients diagnosed with diabetes. Circular ribonucleic acids (circRNAs) are non-coding, closed-loop RNAs that play critical roles in regulating gene expression by sponging microRNAs (miRNAs). Recent studies have implicated circRNAs in the regulation of vari - ous diseases including DN. We delineate circRNA biological networks from the evidence gleaned from clinical studies. Further, we elucidate circRNA-associated signal transduction pathways in the pathogenesis of DN. Taken together, this will facilitate the discovery of novel circRNA and/or miRNA biomarkers for diagnostic and/or therapeutic intervention in DN.


Introduction
Diabetic mellitus, a metabolic disorder, is generally associated with other chronic disorders like peripheral neuropathy, retinopathy, and nephropathy.Diabetic nephropathy (DN) is a critical clinical complication in the kidney that results from diabetes.It has been reported that chronic kidney disease (CKD) or end-stage renal failure occurs due to DN around the world.In the US, one in three people with diabetes suffers from DN [1].Globally, DN occurs in 30%-40% of patients diagnosed with diabetes [2].Some of the major causes of DN include disorders of glucose metabolism and oxidative stress.Renal fibrosis, extracellular matrix accumulation, and proteinuria are some of the clinical characteristics associated with DN [3].
Circular ribonucleic acids (circRNAs) are closed loop noncoding RNAs formed by backsplicing.Gene regulation can be modulated by circRNAs by sponging microRNAs (miRNAs) and regulating nuclear transcription [4].Hence, there has been a resurgence of interest in understanding circRNAs that are involved in the pathogenesis of numerous diseases.The characteristics of circRNAs including wide distribution, stability, and differential expression suggest their potential use as therapeutic and diagnostic biomarkers in the treatment of various diseases including DN [5].
The pathogenesis of DN is complex, an improved understanding of the molecular basis of this disease may help in faster diagnosis and therapeutic intervention.Reports indicate that vitamin K2-7 may be useful in DN and patients on hemodialysis [6][7][8].Indeed, biological networks, whether directed or undirected, are key to identifying novel biomarkers/targets from big data [9].In this review, we elucidate a circRNA biological network from available clinical evidence in DN and delineate circRNA-regulated signal transduction pathways in DN.Indeed, the identification of circRNA biomarkers will facilitate early diagnosis and therapeutic intervention in DN.

Clinical Evidence for CircRNAs in DN
Several upregulated circRNAs have been reported in clinical studies of DN.However, only one downregulated circRNA has been reported in clinical studies of DN till date.A list of circRNAs involved in the modulation of clinical DN are highlighted in Table 1.Further, a biological network of these circRNAs in clinical DN was generated using bioinformatic tools and is illustrated in Figure 1.

Upregulated circRNAs
An et al. [2] reported overexpression of hsa_circ_0003928 in the serum of 30 DN patients.Further, upregulation of AnnexinA2 (ANXA2) and downregulation of miR-151-3p was    can be used as a diagnostic marker.

Downregulated circRNAs
Hu et al. [21] found that hsa_circRNA_102682 was downregulated whereas transforming growth factor β (TGF-β) was upregulated with high levels of homocysteine, creatinine and connective tissue growth factor in serum of 43 DN patients.

Significance of circRNA modulation in DN
It is evident from the above clinical studies that modulation of circRNAs negatively correlates with miRNAs in patients suffering from DN. Also, circRNAs can affect mRNA expression involved in the pathogenesis of DN.The identification of upregulated and downregulated circRNAs in DN reveals an important role for these circRNAs in the pathogenesis of this disease.This throws open a plethora of opportunities for diagnostic and/or therapeutic intervention in DN.Thus, future research in circRNAs implicated in DN can likely facilitate theranostic advances in the management of this disease.

Signal Transduction in DN
In this section, we discuss the potential role of circRNAs involved in regulating key proteins that play significant roles in the pathogenesis of DN.CircRNAs and their targets in regulation of DN as well as associated signaling pathways are depicted in Figure 2 and summarized in Table 2 for the benefit of the reader.
Liu et al. [24] reported the overexpression of circular homeodomain-interacting protein kinase 3 (circHIPK3, hsa_ circ_0000284) in high glucose (HG)-treated human podocyte cell line (HPC).It was also found that circHIPK3 promotes the growth of fused in sarcoma (FUS) on the ectodysplasin A2 receptor (EDA2R) promoter, upregulated the expression of the EDA2R and activated apoptotic signaling.It was concluded that circHIPK3/FUS/EDA2R can be used as potential targets for diabetic kidney disease.Further, a study performed by Wang et al. [25] in HG-treated Human kidney cells 2 (HK-2 cells) demonstrated the upregulation of circ_0008529.It was concluded that suppression of circ_0008529 inhibited inflammatory injury by targeting miR-485-5p/Wnt2B pathway in HK-2 cells.Hence, circ_0008529 plays a significant role in DN progression.In addition, Chen et al. [26] studied the role of circ_000166 in DN renal fibrosis in HG-induced HK-2 cells which showed upregulation of circ_000166.Also, it was demonstrated that miR-296 can be regulated by circ_000166 as competing endogenous RNA (ceRNA) resulted in overexpression of sodium-glucose cotransporter 2 (SGLT2).Therefore, suppression of circ_000166 can lead to a decrease in DN by regulating circ_000166/miR-296/SGLT2 signaling pathway.
Ye et al. [27] demonstrated upregulation of hsa_circ_0001162 in HG-treated HPC.In addition, mechanistic studies revealed observed in DN patients indicating the association of hsa_ circ_0003928 and ANXA2 in the progression of DN.Also, Wang et al. [10] reported upregulation of circ_0000064 and Rho-associated coiled-coil-containing kinase 1 (ROCK1) along with downregulation of miR-532-3p in serum samples from 37 DN patients.ROCK1 enhances albuminuria that aggravates the progression of DN.Sun et al. [3] found overexpression of circRNA F-box/WD repeat-containing protein 12 (circ-FBXW12, also known as hsa_circ_0123996) in serum of 23 DN patients.Moreover, Lin-28 homolog B (LIN28B), a key regulator in DN, was upregulated and miR-31-5p was downregulated in DN patients indicating a negative correlation between miR-31-5p and LIN28B.A similar study was performed by Dong et al. [11] to investigate circular ribonucleic acid nucleoporin 98 (circNUP98, also known as hsa_circ_0000274) wherein circNUP98 was overexpressed in serum of 33 DN patients.It was also concluded that circNUP98 can be used as a diagnostic biomarker for DN.Yun et al. [5] found overexpression of circular actin-related protein 2 (circ-ACTR2, hsa_circ_0008529) and high-mobility group AT-hook 2 (HMGA2) in 27 DN samples (kidney tissues).It was reported that HMGA2 can increase the risk of nephropathy in type 2 diabetes (T2D) patients.Likewise, overexpression of circ_0037128 and nuclear factor of activated T cells 5 (NFAT5) was observed in 45 kidney tissue samples taken from DN patients which was investigated by Feng et al. [12] It was also suggested that NFAT5 led to the progression of DN.A study performed by Zhang et al. [13] in 20 patients with early type 2 DN showed upregulation of circ_0001831 and circ_0000867 which concluded that they can be used as diagnostic markers in DN patients.Qiu et al. [14] found overexpression of circular tousled-like kinase 1 (circTLK1, hsa_circ_0004442) in serum of 30 DN patients.
Zhu et al. [15] reported upregulation of hsa_circ_0125310 and downregulation of miR-422a in kidney tissues from 32 DN patients.Lie et al. [4] found overexpression of circ_0003928 and histone deacetylase 4 (HDAC4), with lower expression of miR-506-3p in 41 DN patients (serum samples).A study performed by Wang et al. [16] showed upregulation of circ_0123996 and downregulation of miR-149-5p in kidney tissues collected from 30 T2D with DN patients.Zhuang et al. [17] reported overexpression of circular collagen alpha-2(I) chain (circCOL1A2, hsa_circ_0081108) in plasma samples of 22 DN patients.Serum/glucocorticoid-regulated kinase 1 (SGK1) was upregulated in DN patients that can contribute to pathogenesis of DN.Further, it was also observed that miR-424-5p was downregulated indicating a negative correlation between miR-424-5p and circCOL1A2.
Dong et al. [11] found overexpression of circNUP98 in HG-treated HMCs.It was also observed that silencing of circNUP98 led to suppression of inflammation, fibrosis and oxidative stress by targeting miR-151-3p-HMGA2 pathway.Thus, circNUP98/miR-151-3p/HMGA2 can be targeted to ameliorate DN.An et al. [2] reported increased levels of hsa_circ_0003928 and ANXA2 along with decreased levels of miR-151-3p in HG-treated HK-2 cells.Suppression of circ_0003928 decreased the levels of interleukin-1β (IL-1β), IL-6, tumor necrosis factor-α (TNF-α), and reactive oxygen species (ROS).Further, it was concluded that hsa_circ_0003928 can inhibit miR-151-3p, and miR-151-3p suppressed ANXA2 suggesting circ_0003928 can be used as a potential target for treating DN.A study performed by Zhuang et al. [33] showed downregulation of circHIPK3 in HG-treated HK-2 cells.Further studies demonstrated that circHIPK3 inhibited miRNA-326 and miR-487a-3p.Also, it was observed that overexpression of miRNA-326 or miR-487a-3p led to downregulation of silent information regulator sirtuin 1 (SIRT1) which eventually led to DN progression.Niu et al. [34] used HG-treated HK-2 cells and observed upregulation of circ_0008529 and suppressor of mothers against decapentaplegic 2 (Smad2), and downregulation of miR-185.It was further concluded that circ_0008529 regulated the expression of Smad2 through miR-185-5p, circ_0008529 can be used as a potential target in DN treatment.Zhou et al. [35] reported overexpression of circ_0060077 in HG-treated HK-2 cells.Knockdown of circ_0060077 led to suppression of oxidative stress, fibrosis, and inflammation.Furthermore, it was observed that silencing of circ_0060077 led to upregulation of miR-145-5p that helped in the recovery of HG-treated HK-2 cells via targeting vasorin (VASN).
that hsa_circ_0001162 upregulated matrix metallopeptidase 9 (MMP9) by inhibiting miR-149-5p thus promoting HG-induced podocyte injury.Therefore, it was concluded that silencing of hsa_circ_0001162 can be a potential therapeutic target in DN.Interestingly, Song et al. [28] reported overexpression of hsa_circRNA_012448 in HG-treated HK-2 cells.Also, suppression of hsa_miR-29b-2-5p and overexpression of glycogen synthase kinase 3β (GSK3β) was observed in HK-2 cells.GSK3β plays a critical role in oxidative stress, leading to the progression of DKD.Further, HG induced HK-2 cells treated with dapagliflozin demonstrated downregulation of GSK3β.It was concluded that DN can be treated by targeting hsa_circRNA_012448/hsa_miR-29b-2-5p/GSK3β pathway.
Liu et al. [4] found upregulation of circ_0003928 and HDAC4 and downregulation of miR-506-3p in HG-treated HK-2 cells.HDAC4 promotes apoptosis and oxidative stress that helps in progression of DN.Silencing of circ_0003928 led to suppression of HDAC4.It was concluded that circ_0003928 sponged miR-506-3p that led to upregulation of HDAC4.
Feng et al. [12] found overexpression of circ_0037128 in HG-induced HK-2 cells.Further studies demonstrated that circ_0037128 inhibited miR-497-5p and that miR-497-5p targeted the protein expression of NFAT5.It was concluded that DN can be regulated by miR-497-5p/NFAT5 signaling pathway.In order to determine the role of circ-FBXW12 in DN, a study was performed by Sun et al. [3] in human mesangial cells (HMCs).It was observed that circ-FBXW12 was overexpressed and suppression of circ-FBXW12 decreased cell proliferation, oxidative stress, and extracellular matrix (ECM) production.In addition, it was also demonstrated that the knockdown of circ-FBXW12 led to upregulation of miR-31-5p further suppressing LIN28B.Therefore, it was concluded that miR-31-5p/LIN28B signaling pathway can be a potential target in treating DN.
A study performed by Li et al. [29] found upregulation of circ_0000064 and Wnt2B and downregulation of miR-424-5p in HG-treated HMCs.Also, it was observed that silencing of circ_0000064 suppressed ECM accumulation and inflammatory response in HG-induced HMCs.Further, it was concluded that silencing of circ_0000064 upregulated miR-424-5p led to inhibition of Wnt2B.Similarly, Wang et al. [10] reported overexpression of circ_0000064 in HK-2 cells.It was summarized that circ_0000064 upregulated the expression of ROCK1 by sponging miR-532-3p, circ_0000064 can be used as a potential target for treating DN.Circ_0068087 was upregulated in an experiment carried out by Feng et al. [30] in HG-induced HK-2 cells.Circ_0068087 silencing led to the suppression of fibrosis, inflammation, and oxidative stress.Knockdown of circ_0068087 led to downregulation of ROCK2 by miR-106a-5p suggesting a potential therapy for DN through targeting miR-106a-5p/ROCK2 pathway.
Yun et al. [5] reported overexpression of circ-ACTR2 in HG-treated HMCs.Circ-ACTR2 inhibition led to decrease they are specific and abundantly available in tissues and biological fluids that renders them useful as biomarkers.However, there are also limitations including lack of clarity in understanding the exact mechanisms of circRNAs involved in pathogenesis of DN.Also, the degradation mechanisms of circRNAs have not yet been characterized.Indeed, there is limited clinical evidence till date of circRNAs used as biomarkers as there are various factors to be considered like patients' disease condition, comorbidities and racial/ ethnic diversity.Further, interaction of circRNAs with noncoding RNAs other than miRNAs remain to be explored [38].
However, clinical evidence suggests that modulation of circRNAs plays important roles in the pathogenesis of DN.
Our circRNA biological network based on clinical evidence may provide a proof-of-concept circRNA signature for theranostic intervention in DN.Moreover, our discussion on circRNA associated signal transduction provides a summary of potential targets for therapeutic intervention in DN.Indeed, exploiting our current knowledge of circRNAs in DN dysregulation may throw open newer avenues for faster diagnosis and appropriate therapeutic intervention for DN patients and prevent/delay progression to end-stage kidney disease.

Conclusions and Future Perspectives
Despite recent advances in the therapeutic management of DN, there are still numerous challenges to be addressed.Augmenting our understanding of the molecular mechanisms of DN and the key roles played by circRNAs in regulating DN is essential to improve the prognosis of DN.Non-coding RNAs including circRNAs involved in DN play critical role in gene regulation by sponging miRNA.Also, circRNAs are involved in various signaling pathways that can be used as potential targets for therapeutic treatment in DN.
Numerous studies suggest that various circRNAs including circ_0001831/circ_0000867 [13] and circANKRD36 [20] can be used as diagnostic markers.Epistemological evidence from clinical studies point to a critical role for circRNAs in DN dysregulation.Recent studies demonstrated that various circRNAs including circ_0008529 [25], circ_0001162 [27] and circ_012448 [28] can be used as targets for treatment of DN.There is also a growing body of evidence from in vitro cell line studies and in vivo animal experiments that underscore the importance of circRNAs in DN.
CircRNAs are comparatively more stable than linear RNAs that makes them less resistant to degradation.Moreover, Open Access.© 2023 The author(s), published by De Gruyter on behalf of Scholar Media Publishing.This work is licensed under the Creative Commons Attribution 4.0 International License.*Corresponding author: Dr. Sujit Nair, Viridis Biopharma Pvt. Ltd., Mumbai 400022, India.E-mail: sujit108@gmail.com.

Table 1 to be continued. Serial number CircRNA Sample matrix Number of patients Study type Modulation circBase ID a Reference
a circRNA IDs were accessed from circBase[22].CircRNA, circular ribonucleic acid; DN, diabetic nephropathy; circANKRD36, circular ankyrin repeat domain 36.UACR: urine albumin creatinine ratio.

Table 1 .
Modulation of circRNAs in DN patients.

Table 2
to be continued.

Table 2 :
circRNAs and their targets in regulation of diabetic nephropathy.