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. 2020 Jan 15;28(3):963–974. doi: 10.1016/j.ymthe.2020.01.014

High Glucose Induces Mesangial Cell Apoptosis through miR-15b-5p and Promotes Diabetic Nephropathy by Extracellular Vesicle Delivery

Yi-Chun Tsai 1,2,3,4,5, Mei-Chuan Kuo 2,4, Wei-Wen Hung 6, Ling-Yu Wu 7, Ping-Hsun Wu 1,4,7, Wei-An Chang 7,8, Po-Lin Kuo 7, Ya-Ling Hsu 9,10,∗
PMCID: PMC7054723  PMID: 31991106

Abstract

Diabetic nephropathy (DN) is an increasing threat to human health and is regarded as an important public issue. The pathophysiologic mechanisms of DN are complicated. The initiating molecular events triggering the loss function in mesangial cells (MCs) in DN are not well known. In this cross-disciplinary study, transcriptome analysis of high glucose (HG)-treated mouse MCs (MMCs) using next-generation sequencing and systematic bioinformatics analyses indicated that miR-15b-5p and its downstream target B cell lymphoma 2 (BCL-2) contribute to HG-induced apoptosis in MMCs. HG elevated miR-15b-5p expression, which in turn decreased the translation of BCL-2, leading to MMC apoptosis under HG. Apoptosis of MCs was enhanced in the presence of extracellular vesicles isolated from the urine of type 2 diabetic patients with high levels of miR-15b-5p. Furthermore, increased levels of urinary miR-15b-5p were found in db/db mice and type 2 diabetic patients, and such levels correlated with low baseline kidney function and rapid decline in kidney function during a mean of follow-up period of 2.4 ± 0.1 years. Therefore, miR-15b-5p induced mesangial cells apoptosis by targeting BCL-2 under HG. miR-15b-5p has the potential to predict kidney injury in DN. Blocking the miR-15b-5p epigenetic regulatory network could be a potential therapeutic strategy to prevent mesangial apoptosis in DN.

Keywords: miR-15, BCL-2, mesangial cell apoptosis, extracellular vesicle, diabetic nephropathy

High glucose elevates miR-15b-5p expression, which induces mesangial cell apoptosis by targeting BCL-2. Increased urinary miR-15b-5p levels are correlated with rapid kidney progression and have the potential to predict kidney injury in type 2 diabetic patients, implying that miR-15b-5p could be a therapeutic target for preventing diabetic nephropathy.

Introduction

The incidence of diabetic nephropathy (DN) has continued to increase in recent decades. DN is the major cause of chronic kidney disease (CKD) worldwide, accounting for 30%–40% of renal disease (end-stage renal disease [ESRD]),1,2 and it is associated with high rates of morbidity and mortality in patients with diabetes. The management of diabetes and the burden of diabetic complications are enormous global challenges.

Mesangial cells (MCs) maintain the structure and function of the glomerulus, offer structural stability for capillary loops, and regulate glomerular filtration through their contractility.3,4 The number of MCs has been correlated with the severity of albuminuria, which is a marker of kidney injury.5 High glucose (HG) causes MC apoptosis by triggering an intrinsic pro-apoptotic pathway,6 which has been associated with albuminuria and the deterioration of kidney function.6 Thus, maintaining the number of MCs is an important issue in the treatment of DN.

Previous studies have reported that microRNA (miRNA) dysregulation participates in the pathophysiologic mechanisms of DN, and that miRNAs may potentially be used as biomarkers of kidney injury and predictors of DN progression.7,8 miRNAs are a class of small non-coding RNA molecules that function in RNA silencing and the posttranscriptional regulation of gene expression through binding to target sequences within the 3′ untranslated region (3′ UTR) of a target mRNA.9 miRNAs are abundant in various organisms and have been considered to regulate gene expression and cellular phenotypes. miRNAs have been shown to be involved in various biological processes, and also to modulate the onset or progression of various diseases.10 However, whether miRNAs play a role in the pathogenetic signaling pathway of MC apoptosis in DN has yet to be elucidated.

In this cross-disciplinary study, we hypothesized that miR-15b-5p may regulate HG-mediated signaling pathways and that it could be used to predict the severity of kidney injury in DN. We included in vitro and in vivo models, as well as human studies, to investigate the fundamental mechanisms of miR-15b-5p-induced apoptosis in MCs. The results illustrate that HG causes MC apoptosis through the upregulation of miR-15b-5p, which directly binds to its target BCL-2 (B cell lymphoma 2) in DN.

Results

Exploration of Potential miRNAs Participating in HG-Induced Mouse MC Apoptosis

Accumulating evidence shows that HG induces MC apoptosis and that miRNAs participate in apoptosis induction, which is one of the pathophysiologic mechanisms of DN.11,12 The flowchart of exploration of potential miRNAs induced by HG is shown in Figure 1A. The profile of small RNAs from mouse MCs treated with normal glucose (NG, 5.5 mM) or HG (25 mM) for 48 h were profiled by next-generation sequencing (NGS). Seventy miRNAs with a significant 2-fold change were found in mouse MCs treated with HG compared with those treated with NG. Of 70 miRNAs, 54 were upregulated and 16 were downregulated. After excluding miRNAs with a raw read count ≤10, 32 significant miRNAs were displayed by heatmap (Figure 1B). Among 32 featured miRNAs, 22 with poor conservation of different species were excluded, and 10 were analyzed. miR-15b-5p had the highest expression among the 10 miRNAs in mouse MCs treated with HG compared with those treated with NG. We further analyzed the pathophysiologic function of miR-15b-5p potential targets based on the TargetScan website (version 7.1) using core analysis of Ingenuity Pathway Analysis (IPA) and the Database for Annotation, Visualization and Integrated Discovery (DAVID). IPA revealed that the pathogenetic function of miR-15b-5p was positively correlated with renal cell death and glomerular injury (Figure 1C). Additionally, the biologic process of DAVID also displayed that miR-15b-5p is involved in negative regulation of cell proliferation and apoptotic signaling (Table 1). Furthermore, we used qRT-PCR to validate miR-15b-5p expression in mouse MCs. The miR-15b-5p level was measured in mouse MCs treated with different concentration of glucose (5, 10, 20, and 30 mM, Figure 1D) and different time points (6, 8, and 12 h, Figure 1E). The miR-15b-5p level increased stepwise according to the increase in glucose concentration, and it reached a plateau at 20 and 30 mM. Because 25 mM glucose has been used as the HG condition in previous reports of DN,13,14 we chose glucose concentration of 25 mM in the following experiments. Our results also showed that miR-15b-5p expression increased in mouse MCs treated with HG (25 mM) at 12 h (Figure 1F).

Figure 1.

Figure 1

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Identification of Potential miRNAs Contributing to HG-Induced Mouse MC Apoptosis

(A) Flowchart of identification of potential miRNAs associated with HG-treated mouse MCs for 48 h. (B) The heatmap revealed differentially expressed miRNAs from mouse MCs treated with the NG or HG condition with Z score values. (C) Signaling pathway analysis of miR-15b-5p targets according to IPA core analysis. (D) miR-15b-5p levels in mouse MCs. Mouse MCs were treated with 5.5, 10, 20, and 30 mM glucose. (E) miR-15b-5p levels in mouse MCs. Mouse MCs were treated with 25 mM glucose for 6, 8, and 12 h. (F) miR-15b-5p levels in mouse MCs. Mouse MCs were treated under the NG (5.5 mM) or HG (25 mM) condition for 12 h. miR-15b-5p levels were assessed by qRT-PCR. The bar graph represents the mean ± SEM of at least three independent experiments. RPM, reads per million. *p < 0.05, **p < 0.01 by Student’s t test.

Table 1.

Molecules Associated with Cell Death in Biological Process of DAVID

Term Count p Value Genes Fold Enrichment FDR
Negative regulation of cell proliferation 21 0.02 HMGA1-RS1, PRKCA, B4GALT1, FKTN, NACC2, PTGS2, PPP2R5C, NF1, PTPN14, SMAD3, DLL1, SKI, HMGA1, TRIM35, BTG2, TSC1, BTG1, BCL2, JAK2, AXIN2, CHD5 1.69 36.05
Extrinsic apoptotic signaling pathway via death domain receptors 5 0.02 MOAP1, ROCK2, DEDD, BCL2, NF1 4.42 36.74
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FDR, false discovery rate; HMGA1-RS1, high mobility group AT-hook 1; PRKCA, protein kinase C alpha; B4GALT1, beta-1,4-galactosyltransferase 1; FKTN, fukutin; NACC2, NACC family member 2; PTGS2, prostaglandin-endoperoxide synthase 2; PPP2R5C, protein phosphatase 2 regulatory subunit B'gamma; NF1, neurofibromin 1; PTPN14, protein tyrosine phosphatase non-receptor type 14; SMAD3, SMAD family member 3; DLL1, delta like canonical Notch ligand 1; SKI, SKI proto-oncogene; HMGA1, high mobility group AT-hook 1; TRIM35, tripartite motif containing 35; BTG2, B cell translocation gene 2; TSC1, TSC complex subunit 1; BTG1, B cell translocation gene 1; BCL2: B cell lymphoma 2; JAK2, Janus kinase 2; AXIN2, axin 2; CHD5, chromodomain helicase DNA binding protein 5; MOAP1, modulator of apoptosis 1; ROCK2, Rho associated coiled-coil containing protein kinase 2; DEDD, death effector domain containing; NF1, neurofibromin 1. Gene nomenclature was based on HUGO gene nomenclature committee (HGNC) database.

HG Induces Mouse MC Apoptosis by miR-15b-5p Upregulation

Consistent with our hypothesis, HG caused apoptosis in mouse MCs (Figure 2A). To assess the role of miR-15b-5p in HG-inducing mouse MC apoptosis, a miR-15b-5p mimic and inhibitor were utilized. As shown in Figure 2B, the transfection of miR-15b-5p mimic induced apoptosis in mouse MCs under the NG condition. Conversely, the transfection of miR-15b-5p inhibitor blocked HG-induced apoptosis in mouse MCs (Figure 2C). These results verify our assumption that HG induces apoptosis in MCs through miR-15b-5p upregulation.

Figure 2.

Figure 2

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HG Induces Mouse MC Apoptosis by miR-15b-5p Upregulation

(A) HG induced apoptosis in mouse MCs at 48 h. (B) miR-15b-5p mimics induce apoptosis in mouse MCs. Mouse MCs were transfected with either miR-15b-5p mimic (200 nM) or control of mimic (miR-NC, 200 nM). After 24 h post-transfection, cells were cultured under the NG condition for 24 h. Apoptotic cells were assessed by flow cytometry with annexin V/PI stain. (C) miR-15b-5p inhibitor reversed the effect of HG on apoptosis induction in mouse MCs. Mouse MCs were transfected with miR-15b-5p inhibitor (50 nM) or control of inhibitor (anti-miR-NC, 50 nM) for 24 h and then incubated under the NG or HG condition for another 24 h. The bar graph represents the mean ± SEM of at least three independent experiments. *p < 0.05, **p < 0.01 by Student’s t test or ANOVA followed by a post hoc test adjusted with a Tukey correction.

BCL-2 as a Direct Target of miR-15b-5p

Owing to HG causing apoptosis in mouse MCs through modulation of miR-15b-5p, we further investigated the potential downstream target of miR-15b-5p, which participates in HG-mediated mouse MC apoptosis. We used miRmap and TargetScan (version 7.1) to conduct bioinformatics predictions, which indicated that the 3′ UTR of BCL-2 contained the target seed sequence against miR-15b-5p (Figures 3A and 3B). The miRmap scores and conservation phyloP meant the probability of predicted target gene of miR-15b-5p. The species-conserved miR-15b-5p seed sequence in the 3′ UTR of BCL-2 is shown in Figure 3B. Additionally, both DAVID and the IPA database pointed out that BCL-2 was associated with negative regulation of cell proliferation, apoptotic signal pathway, and renal cell death (Tables 1 and 2), meaning that BCL-2 may participate in miR-15b-5p-modulated renal cell apoptosis. miR-15b-5p mimics decreased luciferase activity of BCL-2-3′ UTR plasmid when compared to control mimic transfection, suggesting that miR-15b-5p can directly bind to the 3′ UTR of BCL-2 mRNA (Figure 3C). HG treatment decreased BCL-2 level, and miR-15b-5p mimic mimicked the effect of HG on BCL-2 protein in mouse MCs (Figures 3D and 3E). miR-15b-5p inhibitor increased BCL-2 levels in mouse MCs treated with NG. Additionally, miR-15b-5p inhibitor reversed decreased BCL-2 levels induced by HG in mouse MCs (Figure 3F). Furthermore, expression of BCL-2 at the protein level was decreased in MCs of kidney sections of diabetic db/db mice compared with that of non-diabetic db/m mice (Figure 3G). Immunohistochemistry (IHC) staining also revealed that levels of BCL-2 were lower in MCs of kidney sections of type 2 diabetic patients than in those of upper tract urothelial carcinoma (UTUC) patients with normal kidney function and glomerulus structure (Figure 3H). Thus, HG decreased BCL-2 expression by miR-15b-5p upregulation, leading to MC apoptosis in DN.

Figure 3.

Figure 3

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BCL-2 is a Direct Target of miR-15b-5p in Mouse MCs

(A) Predictive binding score of miR-15b-5p on 3′ UTR of BCL-2 mRNA according to the miRmap database. (B) Schematic representation of sequence alignment of BCL-2 mRNA 3′ UTR based on TargetScan version 7.1. (C) BCL-2 3′ UTR luciferase reporter plasmid was repressed by exogenous miR-15b-5p. HEK293 cells were co-transfected with pGL3-BCL-2-3′ UTR luciferase plasmid/pRL-TK Renilla (8:1) or pGL3-BCL-2-3′ UTR MT luciferase plasmid/pRL-TK Renilla (8:1) with various miRNA mimics (control mimic or miR-15b-5p mimic) by DharmaFECT Duo transfection reagent after 48 h; both firefly and Renilla luciferase activities were quantified using the Dual-Glo luciferase assay system. (D) HG reduced BCL-2 expression at 36 h. (E) miR-15b-5p mimic suppressed BCL-2 expression in mouse MCs under the NG condition. (F) miR-15b-5p inhibitor enhanced BCL-2 expression in mouse MCs under the NG or HG condition. Western blotting was utilized to measure BCL-2 protein expression. (G and H) The expression of BCL-2 in MCs of kidneys in mice (G) and humans (H) is shown. The kidney sections of non-diabetic db/m mice and diabetic db/db mice, as well as those of human donors (UTUC with normal kidney function and normal glomerulus) and patients with DN, were co-stained with BCL-2 (brown) and α-SMA (green) as the marker of MCs. The bar graph represents the mean ± SEM of at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 by Student’s t test or ANOVA followed by a post hoc test adjusted with a Tukey correction.

Table 2.

Molecules Associated with Renal Cell Death in the IPA Database

Ingenuity Toxicity Lists −Log p Value Ratio Molecules
Renal necrosis/cell death 2.48 0.0913 NOS1, IRF4, YAP1, FGF2, SMAD3, PTHLH, PTCH1, UNC5B, SALL1, TCF3, SIRT4, BAG4, BCL2, FGF1, VEGFA, CASR, FOXO1, IGF1, KL, IGF1R, PAX2, PPM1A
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NOS1, nitric oxide synthase 1; IRF4, interferon regulatory factor 4; YAP1, Yes associated protein 1; FGF2, fibroblast growth factor 2; SMAD3, SMAD family member 3; PTHLH, parathyroid hormone like hormone; PTCH1, patched 1; UNC5B, unc-5 netrin receptor B; SALL1, spalt like transcription factor 1; TCF3, transcription factor 3; SIRT4, sirtuin 4; BAG4, BCL2 associated athanogene 4; BCL2, B cell lymphoma 2; FGF1, fibroblast growth factor 1; VEGFA, vascular endothelial growth factor A; CASR, calcium sensing receptor; FOXO1, forkhead box O1; IGF1, insulin-like growth factor 1; KL, klotho; IGF1R, insulin-like growth factor 1 receptor; PAX2, paired box 2; PPM1A, protein phosphatase; Mg2+/Mn2+ dependent 1A. Gene nomenclature was based on HUGO gene nomenclature committee (HGNC) database.

Urinary EV miR-15b-5p Levels Are Positively Correlated with Urinary Albumin-to-Creatinine Ratio (ACR), Negatively Correlated with Estimated Glomerular Filtration Rate (eGFR), and Associated with Rapid Decline in Kidney Function in Humans

miRNAs have been reported to be not only present in cells but also excreted to the extracellular environment, including plasma, body fluid, or urine, via apoptotic bodies, microvesicles, or exosomes.15 We used nanoparticle tracking analysis (NTA) to evaluate the diameter of EVs, including microvesicles (50–1,000 nm), exosomes (40–120 nm), or apoptotic bodies (50–2,000 nm),16,17 after we isolated the small particles from urine donors. Screen shots indicate the presence of particles in the urine of healthy individuals and type 2 diabetic patients (Figure 4A) with an average diameter of 178.4 ± 12.5 nm (Figure 4B). Further centrifugation of urine samples followed by immunoblotting revealed the presence of the characteristic exosome-associated proteins, including heat shock protein 70 (HSP70), tumor susceptibility gene 101 (Tsg101), CD9, CD63, and CD81 (Figure 4C). Based on the above findings, both microvesicles and exosomes, but not apoptotic bodies, were present in particles isolated from the urine. We enrolled 38 normal individuals and 85 type 2 diabetic patients in our study (Table S1) and measured miR-15b-5p level derived from EVs in the urine. Type 2 diabetic patients had higher urinary EV miR-15b-5p levels when compared with normal individuals (Figure 4D). Urinary EV miR-15b-5p levels were positively correlated with the urinary ACR (Figure 4E). Study participants with microalbuminuria had higher EV urinary miR-15b-5p levels than did those with normoalbuminuria (Figure 4F). More importantly, we found that a high urinary EV miR-15b-5p level was significantly associated with low eGFR at baseline (Figure 4G). We followed these diabetic patients and observed the speed of the decrease in eGFR (eGFR slope) during a mean follow-up period of 2.4 ± 0.1 years. Type 2 diabetic patients with higher urinary EV miR-15b-5p had a more negative eGFR slope in Spearman analysis (r = −0.30, p = 0.006) (Figure 5H). Furthermore, we defined rapid decline in kidney function as eGFR decline >3 mL/min/1.73 m2 per year.18 After adjusting for well-known risk factors for rapid decline in kidney function, such as age, sex, baseline eGFR, urinary albuminuria, and glycated hemoglobin, the relationship between urinary EV miR-15b-5p and rapid kidney function decline in type 2 diabetic patients was significant in multivariate logistic regression analysis (odds ratio [OR], 1.04; 95% confidence interval [CI], 1.01–1.07; p = 0.02) (Table S2). In accord with the results of the in vitro study, miR-15b-5p participates in the mechanism of HG-mediated kidney injury, and urinary EV miR-15b-5p has the potential to predict kidney progression in clinical diabetic patients.

Figure 4.

Figure 4

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Urinary Extracellular Vesicular (EV) miR-15b-5p Is Positively Correlated with Kidney Dysfunction in Humans and Mice

Nanoparticle tracking analysis (NTA) and immunoblotting analysis of microvesicles in human urine are shown. (A) Screenshots of NTA of EVs in the urine samples from donors. Image size recorded by video is 100 × 100 μm. (B) Size and particle distribution plots of EVs in the urine samples from donors using NTA. (C) Western blotting was utilized to measure the characteristic surface markers of exosomes in the urine samples from donors. (D) Urinary EV miR-15b-5p levels were higher in type 2 diabetic patients (n = 85) compared to healthy individuals (n = 38). (E) Urinary EV miR-15b-5p levels were positively correlated with the urinary albumin-to-creatinine ratio (ACR). (F) High urinary EV miR-15b-5p levels were correlated with the severity of urinary ACR in human participants. (G) Urinary EV miR-15b-5p levels were negatively associated with estimated glomerular filtration rate (eGFR) in human participants. (H) High EV urinary miR-15b-5p levels were positively correlated with greater eGFR decline during the follow-up period (eGFR slope) in type 2 diabetic patients. (I) Urinary EV miR-15b-5p levels were higher in db/db mice (n = 5) compared to db/m mice (n = 5). (J) Urinary EV miR-15b-5p was positively correlated with urinary ACR in mice. EV miR-15b-5p in the urine of humans and mice was isolated and then assessed by qRT-PCR. Urine albumin was measured using an immunoturbidimetric assay, and urine creatinine was determined by the enzymatic method. Serum creatinine was measured by the compensated Jaffé (kinetic alkaline picrate) method. eGFR was calculated using the following equation: eGFR = 186 × Serum creatinine−1.154 × Age−0.203 × 0.742 (if female). The bar graph represents the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 by Student’s t test or ANOVA followed by a post hoc test adjusted with a Tukey correction, and p value of the correlation was analyzed by Spearman analysis.

Figure 5.

Figure 5

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Urinary EV miR-15b-5p Enhances Apoptosis in MCs

(A) The flowchart shows the experiment of investigating the effect of urinary extracellular vesicles (EVs) on mouse MCs. (B) Urinary EVs derived from type 2 diabetic patients increase mouse MC apoptosis. Mouse MCs were treated with urinary EVs derived from normal individuals (n = 5) and type 2 diabetic patients (n = 3) for 48 h. (C) Urinary EV miR-15b-5p levels were positively correlated with the proportion of mouse MC apoptosis. Apoptotic cells were assessed by flow cytometry with annexin V/PI stain. The bar graph represents the mean ± SEM. **p < 0.01 by Student’s t test, and the p value of the correlation was analyzed by Spearman analysis.

Urinary EV miR-15b-5p Levels Are Positively Correlated with ACR in Mice

Urinary miR-15b-5p levels in db/m mice (n = 5) and db/db mice (n = 5) were measured, and we found that db/db mice had higher EV miR-15b-5p levels compared to those of db/m mice in the urine (Figure 4I), and urinary EV miR-15b-5p levels were positively correlated with urinary ACR in mice (Figure 4J).

Urinary EV miR-15b-5p enhances apoptosis in MCs

Our results manifested that miR-15b-5p modulates HG-induced apoptosis in MCs. Compared to healthy individuals, urinary EV miR-15b-5p levels are elevated in type 2 diabetes and can predict kidney function in in vivo models and human study. Furthermore, mouse MCs were used to assess proapoptotic activity of EV miR-15b-5p isolated from the urine of healthy individuals and type 2 diabetic patients (Figure 5A). As shown in Figure 5B, apoptosis of MCs was enhanced in the presence of EVs isolated from the urine of type 2 diabetic patients with high levels of EV miR-15b-5p. The promotive effect of EVs on the enhancement of MC apoptosis was positively correlated with urinary EV miR-15b-5p levels (Figure 5C). Our findings prove that high levels of urinary EV miR-15b-5p effectively increase apoptosis in MCs among type 2 diabetic patients.

Discussion

DN is the leading cause of ESRD worldwide.1 To detect the onset of DN in a more timely manner and prevent it from progressing to ESRD is very important. We conducted this cross-disciplinary study and revealed that miR-15b-5p was upregulated in MCs treated with HG. miR-15b-5p upregulation led to MC apoptosis under NG through targeting BCL-2, while miR-15b-5p inhibition reversed HG-induced MC apoptosis. Elevated levels of miR-15b-5p were found in the urine of db/db mice and type 2 diabetic patients. EVs from urine of type 2 diabetic patients with high levels of miR-15b-5p led to MC apoptosis. We further demonstrated that a high level of urinary EV miR-15b-5p correlated with poor kidney function at baseline and, furthermore, was associated with a rapid decline in kidney function in type 2 diabetic patients after adjusting for well-known risk factors. Therefore, we have obtained new perspectives of the unique regulation of miR-15b-5p, which interprets DN development and predicts kidney progression in type 2 diabetic patients (Figure 6).

Figure 6.

Figure 6

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Illustration of the Mechanism of HG Inducing Apoptosis in MCs through a miR-15b-5p-BCL-2 Loop in DN

Dysregulated miRNAs are shown in the kidneys and have been shown to play a role in the development and progression of DN.11,12 Elucidating the role and regulatory network of miRNAs in the course of DN progression may help to improve diagnostic and prognostic tools, as well as enable physicians to mitigate the impairment of kidney function. Our results of both NGS and bioinformatics analysis verified that miR-15b-5p could potentially be used as a predictive marker of kidney injury. A previous study reported that overexpression of miR-15 induced apoptosis through inhibition of nuclear factor κB in colon cancer cells.19 In addition, miR-15 activation has been shown to suppress anti-apoptotic pathways in the livers of rats under hyperosmotic stress.20 However, the role of miR-15b-5p in the pathogenesis of DN is as yet unknown. In the present study, HG increased miR-15b-5p levels in MCs. miR-15b-5p mimicked HG-inducing MC apoptosis under the NG condition, and inhibition of miR-15b-5p suppressed HG-inducing MC apoptosis by targeting BCL-2. Considering that miR-15b-5p levels were elevated in the urine of type 2 diabetic patients, and that these increased urine levels were associated with high urinary albuminuria, correlated with low eGFR at baseline, and associated with a rapid decline in eGFR, miR-15b-5p may be a key factor in DN progression, and it may therefore be a therapeutic target for DN.

miRNAs are stable and are excreted into the circulation through diverse processes, such as apoptotic bodies, microvesicles, and exosomes.21 miRNAs can be detected and quantified in serum, plasma, or body fluids, and identifying urinary miRNAs may provide a rapid and potentially alternative method to diagnose DN instead of renal biopsy.7,11 Thus, we measured urinary EV miR-15b-5p levels in mice and humans and found that both diabetic mice and type 2 diabetic patients had higher miR-15b-5p levels in EVs in urine, and that high urinary EV miR-15b-5p was correlated with high urinary ACR. Moreover, high urinary EV miR-15b-5p was significantly associated with a rapid decline in kidney function in type 2 diabetic patients after adjusting for traditional risk factors for kidney progression, including age, sex, baseline kidney function, urinary ACR, and clinical sugar control (Table S2). In addition, we detected apoptosis of MCs treated with EVs in the urine of type 2 diabetic patients, and the proportion of MC apoptosis was positively correlated with urinary EV miR-15b-5p level. Taken together, we suggest that miR-15b-5p is a profitable biomarker to predict the severity of kidney injury in DN, and it enables clinical physicians to detect early signs of onset or the adverse progression of DN.

Conclusions

We demonstrate that miR-15b-5p modulates HG-induced MC apoptosis, and that it can predict the severity of kidney injury in DN. These findings suggest the potential of future applications of miR-15b-5p to treat and predict the prognosis of DN. We hope that these findings will help to realize the pathogenetic mechanisms and the efficiency of clinical care in DN.

Materials and Methods

Cell Line and Cell Culture

Mouse MCs (CRL1927) and human embryonic kidney (HEK) 293 cells were purchased from the American Type Culture Collection (Manassas, VA, USA). Mouse MCs and HEK293 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with NG (5.5 mM, Gibco, USA) supplemented with 5% and 10% fetal bovine serum (FBS), respectively. All experiments were performed by treating mouse MCs with NG (5.5 mM) or HG (25 mM, Lonza, Switzerland) in DMEM containing 1% FBS.

RNA Sequencing

Mouse MCs were cultured in the NG or HG condition for 48 h. NGS was performed to examine miRNA profiles of MCs. TRIzol reagent (Invitrogen, USA) was utilized to extract total RNA from harvested cells, which were isolated for further RNA preparation and small RNA sequencing (RNA-seq) by Welgene Biotech (Welgene, Taipei, Taiwan). The quality of extracted RNA was evaluated by RNA integrity number (RIN), which was measured using an Agilent Bioanalyzer (Agilent Technologies, USA). Samples were prepared to manufacture the small RNA library and then to execute deep sequencing by an Illumina sample preparation kit. PCR amplification was performed to ligate total RNA with 3′ and 5′ adaptors and to reverse transcription into cDNA. cDNA constructs were separated using 6% polyacrylamide gel electrophoresis, and 18–40 nt RNA fragments (140–155 nt in length with both adapters) were extracted. The sequencing of libraries was performed using an Illumina GAIIx instrument (50-cycle single read), and then the results were processed with Illumina software. The differentially expressed miRNAs between MCs treated with NG or HG were defined at a >2-fold change and >10 reads per million (RPM).

miRmap and TargetScan Bioinformatics Websites

The targets of specific miRNAs were predicted using two bioinformatics websites, miRmap (https://mirmap.ezlab.org) and TargetScan (http://www.targetscan.org) databases. Both miRmap and TargetScan software classify potential specific miRNA targets according to miRmap scores and percentiles of the context++ score, and they indicate the repression strength of a miRNA target.22,23 The miRmap and TargetScan websites can provide miRNA target predictions for different organisms.

IPA

The networks and biologic functions of the selected miRNA targets were analyzed using IPA software (Ingenuity Systems, Redwood City, CA, USA), which provides “core analysis” of genes and proteins. Toxicity lists obtained from core analysis can be used to identify the potential pathogenesis of genes and proteins.23

DAVID Bioinformatics Resources

The functions of selected miRNA targets were assessed using the updated DAVID Bioinformatics Resources (https://david.ncifcrf.gov/), which is a public resource that combines many public bioinformatics resources and provides tools that can be used to analyze large lists of genes from a wide range of genomic studies. DAVID Bioinformatics Resources can provide analysis of gene-term enrichment and a general understanding of the biological functions associated with the genes of interest.24,25

RNA Isolation, Reverse Transcription, and qRT-PCR

Total RNA from cells and exosomes in the urine was isolated using TRIzol and TRIzol LS reagent (Life Technologies), respectively. miRNAs were reverse transcribed using a Mir-X miRNA first-strand synthesis kit (catalog #638313, Takara, Japan). SYBR Green was used to analyze quantitative miRNA with the QuantStudio 3 qRT-PCR system (Thermo Fisher Scientific, Foster City, CA, USA). Relative expression levels of the miRNA in cells were normalized to internal control U6. EV RNAs of human and mice urine (5 mL) were isolated using a urine exosome RNA isolation kit (catalog #47200, Norgen Biotek, Canada) after EV purification. Spike-in control cel-miR-39 (Exiqon, Vedbaek, Denmark) was used for normalization of qRT-PCR reactions. Relative expressions were presented using the 2−ΔΔCt method. The primers used are listed in Table S3.

Transient Transfection

miR-15b-5p mimic (200 nM), miR negative control of mimic (miR-NC, 200 nM), miR-15b-5p inhibitor (50 nM), and miR negative control of inhibitor (anti-miR-NC, 50 nM) (GE Healthcare, USA) were transfected into cells using Lipofectamine RNAiMAX transfection reagent (catalog #13778075, Thermo Fisher Scientific, USA) following the manufacturer’s protocols.

Quantitation of Apoptosis

miR-NC, miR-15b-5p, anti-miR-NC, and miR-15b-5p inhibitors were transfected in mouse MCs for 24 h, and then cells were cultured in NG and HG conditions for 48 h. Mouse MCs were stained with both annexin V-conjugated fluorescein isothiocyanate (FITC) and propidium iodide (PI) (catalog #6592, Cell Signaling Technology, USA) for verification of apoptotic cell death at early and late phases. Stained cells were evaluated using BD Accuri C6 (BD, USA) flow cytometry. The percentage of apoptotic cells was counted when the cells were both annexin V+/PI− or annexin V+/PI+ in total cell populations.

Western Blot Analysis

The total protein of mouse MCs was extracted using RIPA (radioimmunoprecipitation assay) lysis buffer (EMD Millipore, USA). The denatured protein was separated by 9%–11% SDS-PAGE electrophoresis and then transferred onto a polyvinylidene fluoride (PVDF) membrane following blocking and immunoblotting by specific primary and secondary antibodies. Antibody against BCL-2 was obtained from Cell Signaling Technology, USA (catalog #3498s). GAPDH antibody (catalog #MAB374) was obtained from Millipore (Burlington, MA, USA), and HSP70, Tsg101, CD9, CD63, and CD81 (catalog #EXOAB-KIT-1-SBI) were obtained from System Biosciences (SBI, CA, USA). The signals of blots were captured using the ProteinSimple/FluorChem Q system (Alpha Innotech, USA). Densitometry of the blots was calculated using ImageJ software (USA).

3′ UTR Luciferase Reporter Assay

HEK293 cells (1 × 104/well) were co-transfected with pGL3-BCL-2-3′ UTR luciferase plasmid/pRL-TK Renilla (8:1) or pGL3- Bcl-2-3′ UTR mutated (MT) luciferase plasmid/pRL-TK Renilla (8:1) with miRNA mimics (control mimic or miR-15b-5p mimic) using DharmaFECT Duo transfection reagent (catalog #T-2010-03, Thermo Fisher Scientific, USA) for 48 h. The activity of firefly and Renilla luciferase were then quantified using the Dual-Glo luciferase assay system (Promega, USA).

Experimental Animals

We purchased 5-week-old, pathogen-free male db/m mice (non-diabetic animal model) and db/db mice (type 2 diabetic animal model)26,27 from the National Laboratory Animal Center in Taiwan. All samples, including blood, urine, and kidney, were collected at the 12th week. The kidneys were fixed in 4% paraformaldehyde for IHC staining. All animal experiments in this study were approved by the Kaohsiung Medical University and Use Committee.

Human Study Participants

Eighty-five type 2 diabetic patients with eGFR ≥30 mL/min/1.73 m2 and 39 healthy volunteers were enrolled from September 2016 to May 2017 and followed until April 2019. Blood and urine samples were taken after a 12-h fast for biochemistry studies. All urine and blood samples were stored in a −80°C freezer. The sections of kidney were obtained from four DN patients scheduled for biopsies and four UTUC patients receiving nephrectomy. This study was approved by the Institutional Review Board of the Kaohsiung Medical University Hospital. All participants provided written informed consent in accordance with the Declaration of Helsinki.

IHC Staining

The kidney tissue sections were fixed in 4% paraformaldehyde for IHC. BCL-2 antibody (1:200, 12789-1-AP, Proteintech, USA) and anti-α smooth muscle actin (α-SMA) antibody (1:150, catalog #ab5694, Abcam) were used in IHC.

Quantification of Urinary ACR and eGFR in Humans and Mice

Levels of urinary albumin were assessed using an immunoturbidimetric assay with Tina-quant Albumin Gen.2 (ALBT2, Roche, USA). Concentrations of urine creatinine were examined using the enzymatic method (creatinine plus ver.2, CREP2, Roche, USA). Normoalbuminuria was defined as urinary ACR <30 mg/g; microalbuminuria was defined as urinary ACR ≥30 and <300 mg/g; macroalbuminuria was defined as urinary ACR ≥300 mg/g. Serum creatinine was measured using the compensated Jaffé (kinetic alkaline picrate) method in a Roche/Integra 400 analyzer (Roche Diagnostics, Mannheim, Germany) using a calibrator traceable to isotope-dilution mass spectrometry.28 eGFR was calculated using the equation of the four-variable Modification of Diet in Renal Disease (MDRD) study (eGFR = 186 × Serum creatinine−1.154 × Age−0.203 × 0.742 [if female]).29

Measurement of Kidney Progression in Humans

Participants were contacted at outpatient clinics at 3-month intervals to ascertain their clinical status. Rapid decline in kidney function was defined as an eGFR decline >3 mL/min/1.73 m2 per year (eGFR slope).18 The eGFR slope was calculated by the regression coefficient between eGFR and time in units of mL/min per 1.73 m2 per year based on all eGFR values available from enrollment to the end of the observation period. At least three eGFR values were required to estimate the eGFR slope.

Isolation of EVs from the Urine of Mice and Humans

The EVs derived from urine were purified by a urine exosome RNA isolation kit (catalog #47200, Norgen Biotek, Canada) to isolate RNAs in EVs of human and mouse urine (5 mL) following the manufacturer’s protocol.

NTA

NTA used ZetaView PMX120 (Particle Metrix, Germany) to measure the size distribution of nano particles isolated from urine. 100 nm of polystyrene standard beads (Thermo Fisher Scientific) was used to align the focus and camera/laser positions. The temperature was controlled at 25°C. All samples were diluted in phosphate-buffered saline (PBS) to a final volume of 1 mL. Each sample was scanned for 11 cell positions and calculated by Brownian motion. ZetaView software 8.05.10 exported the final data and statistics. The scattered mode detected samples with a 488-nm laser. Fluorescence mode detected fluorescent signals of nano particles with laser at 488 nm and inserted a long wave-pass filter cut-off at 500 nm.

Statistical Analysis

The continuous variables were expressed as mean ± standard error of the mean (SEM) or median (25th, 75th percentile) as appropriate, while categorical variables were expressed as percentages. The chi-square test was used to test differences in the distribution of categorical variables. Skewed distribution continuous variables were log transformed to attain normal distribution. The correlation among continuous variables was examined by Spearman correlation. The significance of differences in continuous variables between groups was tested using Student’s t test or one-way analysis of variance (ANOVA), followed by a post hoc test adjusted with a Tukey correction as appropriate. Multivariate logistic regression models were used to evaluate the association between urinary miR-15b-5p and rapid decline in kidney function as the eGFR decline >3 mL/min/1.73 m2 per year. The multivariate models were adjusted for age, sex, baseline eGFR, urinary ACR, and glycated hemoglobin, which are well-known risk factors for rapid decline in kidney function. Statistical analyses were conducted using SPSS version 18.0 for Windows (SPSS, Chicago, IL, USA) and GraphPad Prism 5.0 (GraphPad, San Diego, CA, USA). Statistical significance was set at a two-sided p value of <0.05.

Author Contributions

Conceptualization, Y.-C.T. and Y.-L.H.; Methodology, Y.-L.H., P.-L.K., and M.-C.K.; Investigation, L.-Y.W.; Writing – Original Draft, Y.-C.T.; Writing – Review & Editing, Y.-L.H.; Funding Acquisition, Y.-C.T.; Resources, W.-W.H, P.-H.W, W.-A.C, M.-C.K, P.-L.K, and Y.-L.H.; Supervision, Y.-L.H.

Conflicts of Interest

The authors declare no competing interests.

Acknowledgments

The authors thank the staff of the Department of Pathology, Kaohsiung Municipal Ta-Tung Hospital, and the Cohort Research Center and Center for Research Resources and Development, Kaohsiung Medical University, Kaohsiung, Taiwan. This study was supported by grants from the Ministry of Science and Technology, Taiwan (MOST; 108-2314-B-037-035-MY3) and Kaohsiung Medical University Hospital, Kaohsiung, Taiwan (KMUH; 107-7R25).

Footnotes

Supplemental Information can be found online at https://doi.org/10.1016/j.ymthe.2020.01.014.

Supplemental Information

Document S1. Tables S1–S3
mmc1.pdf (215.2KB, pdf)
Document S2. Article plus Supplemental Information
mmc2.pdf (2.7MB, pdf)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Document S1. Tables S1–S3
mmc1.pdf (215.2KB, pdf)
Document S2. Article plus Supplemental Information
mmc2.pdf (2.7MB, pdf)

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