How MiRNA Expression Affect Diabetic Kidney Disease?

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PART Ⅱ: MicroRNA Expression Profiling in Diabetic Kidney Disease

HIROKIISHII,SHOHEI KANEKO,KATSUNORI YANAl,AKINORI AOMATSU,KEIJ HIRAl, SUSUMU OOKAWARA, & YOSHIYUKI MORISHITA

The microRNAs (miRNAs) that can regulate diabetic kidney disease (DKD) have not been fully characterized. The aim of this study was to identify the miRNAs that affect DKD(diabetic kidney disease) and could be used as specific biomarkers or therapeutic agents. First, kidney tissues from two DKD(diabetic kidney disease) mouse models and control mice were screened for differences in miRNA expression by microarray analysis followed by quantitative real-time reverse transcription-PCR. Six miRNAs were differentially expressed from controls in both DKD(diabetic kidney disease) mouse models. Among them, miRNA-125b-5p and miRNA-181b-5p were exclusively downregulated in the DKD(diabetic kidney disease) mouse model. Next, we administered miRNA-181b-5p-mimic to DKD(diabetic kidney disease) mice, which reduced the albuminuria and abnormal mesangial expansion. Pathway analysis and database research revealed that the overexpression of miRNA-181b-5p significantly altered the expression of seven mRNAs in six known signaling pathways in the kidneys of DKD(diabetic kidney disease) mice. Furthermore. the serum level of miRNA-125b-5p was significantly higher in patients with DKD(diabetic kidney disease) (1.89±0.40-fold, P<0.05) compared with patients with other kidney diseases (0.94±0.13-fold)and healthy subjects(1.00±0.19-fold).Serum levels of miRNA-181b-5p were lower in patients with DKD(diabetic kidney disease) (0.30±0.06-fold, P<0.05)compared with patients with other kidney diseases(1.06±0.20-fold)and healthy subjects(1.00±0.16-fold). These results suggest that miRNA-125b-5p and miRNA-181b-5p may represent novel diagnostic biomarkers and that miRNA-181b-5p may represent a therapeutic target for DKD(diabetic kidney disease). (Translational Research 2021;237:31-52)

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RESULTS

Profiling of miRNAs in the kidneys of mouse models of DKD(diabetic kidney disease). Microarray analysis of the kidneys of 36 week old C57BL/6-Ins2Akit/J mice(AKITA mice)and 28 week old C57BLKS/J Tar-+Leprh/+Leprb(db/db mice)was performed.Fifty kinds of miRNAs of a total of 1,881 showed statistically significant differential expression in the kidneys of AKITA (n=4)and agematched control (n=4)mice(Table I, Fig.1).Addition-ally,87 kinds of miRNAs showed statistically significant differential expression in the kidneys of db/db (n=4) and agematched control(n=4)mice (Table ⅡI and Fig.2).Of these,15 kinds of miRNAs (miRNA-20a-5p, miRNA-34a-5p,miRNA-125b-5p,miRNA-129-1-3p, miRNA-142a-3p,miRNA-129b-5p,miRNA-142a-5p, miRNA-146a-5p, miRNA-181b-5p,miRNA-342-3p,miRNA-455-5p, miRNA-223-3p,rmiRNA-652-3p, miRNA-6401, and miRNA-6980-5p)were differentially expressed from controls in both the AKITA and db/db mice.Among these 15 miRNAs,the expression of six miRNAs (two upregulated miRNAs (miRNA-34a-5p and miRNA-129b-1-3p) and four miRNA-142a-3p, (miRNA-125b-5p,miRNA-181b-5p,and miRNA-223-3p))were con-firmed by qRT-PCR analysis(Fig.3).Nine other miR-NAs showed no statistically significant changes by qRT-PCR analysis.

Fig 3. Differentially expressed miRNAs in the kidneys of AKITA and db/db mice, according to qRT-PCR analysis. qRT-PCR was used to determine the expression of miRNA-34a-5p, miRNA-125b-5p, miRNA-129b- 1-3p, miRNA-142a-3p, miRNA-181b-5p, and miRNA-223a-3p in each group (n=6 per group). Wilcoxon rank sum test. * P < 0.05.

Characterization of the differentially expressed miRNAs. The expression levels of six differentially expressed miRNAs (miRNA-34a-5p,-125b-5p,-129b-1-3p, -142a-3p,-181b-5p,and -223-3p) in the kidneys of DKD(diabetic kidney disease) mice were measured in the kidneys of other mouse models of kidney diseases by qRT-PCR:a model of renal fibrosis generated by unilateral ureteral obstruction, a model of acute kidney injury(ischemia-reperfusion injury), a model of age-dependent renal disease(senescence-accelerated mice), and a model of immunoglobulin A nephropathy(HIGA/NscSlc mice). The expression levels of miRNA-125b-5p and miRNA-181b-5p did not differ between the mouse models and the corresponding control mice (Fig.4), but the expression levels of the other four miRNAs (miRNA-34a-5p,-129b-1-3p,-142a-3p,and -223-3p)differed (Fig.4). These results suggest that the differ-ential expression of miRNA-125b-5p and miRNA-181b-5p in the kidney may be specific to DKD(diabetic kidney disease).

Fig 4. Comparison of miRNA expression levels in various renal diseases. Expression levels of miRNA-34a-5p, miRNA-125b-5p, miRNA-129b-1-3p, miRNA-142a-3p, miRNA-181b-5p, and miRNA-223-3p in the kidneys of mouse models of kidney disease (n=6 per group). Wilcoxon rank sum test. * P < 0.05. Abbreviations: SAMP, Senescence-Accelerated Mouse Prone; SAMR, Senescence-Accelerated Mouse Resistant; IRI, ischemia-reperfusion injury; UUO, unilateral ureteral obstruction; HIGA, high immunoglobulin A; ns, not signifificant.

Next, we analyzed the expression levels of miRNA-125b-5p and miRNA-181b-5p in the kidney and serum of AKITA mice and db/db mice over time using qRT-PCR analysis. The expression levels of miRNA-125b-5p in the kidneys decreased with the progression of DKD(diabetic kidney disease) in db/db mice and AKITA mice compared with control mice(db/db mice 0.79±0.03-fold at 12 weeks old;P<0.05,0.59±0.03-fold at 28 weeks-old;P<0.01, AKITA mice:0.71±0.04-fold at 12 weeks-old;P<0.01, 0.16±0.03-fold at 36 weeks-old;P<0.05)(Fig.5A).In contrast, the expression levels of miRNA-125b-5p in serum increased to 1.76±0.11-fold(P<0.05) in db/db mice at 28 weeks-old; however,they did not change in AKITA mice (Fig.5A). The expression levels of miRNA-181b-5p in the kidney also decreased with the progression of DKD(diabetic kidney disease) in db/db mice and AKITA mice compared with control mice(db/db mice 0.64±0.03-fold at 28 weeks-old;P< 0.05, AKITA mice: 0.75±0.05-fold at 12 weeks-old;P<0.05 and 0.38±0.07-fold at 36 weeks-old;P<0.05)(Fig. 5B).Additionally, the expression levels of miRNA-181b-5p in serum decreased to 0.63±0.05-fold(P<0.05)in db/db mice at 28 weeks-old; however, they did not show statistically significant changes in AKITA mice (Fig.5B).

Fig 5. Time course analysis of the levels of miRNA-125b-5p and miRNA-181b-5p in the kidney and serum of AKITA and db/db mice. The levels of miRNA-125b-5p in the kidney (upper graph) and serum (lower graph) of db/db and AKITA mice (n=6 per group) (a). The levels of miRNA-181b-5p in the kidney (upper graph) and serum (lower graph) of db/db and AKITA mice (n=6 per group) (b). Wilcoxon rank sum test. * P < 0.05.

In situ hybridization (ISH) analysis showed that miRNA-125b-5p was expressed in the nucleus and cyto-sol of the glomeruli and tubules of the kidneys of db/db mice, but miRNA-181b-5p could not be detected using this method(Fig.6). This may suggest that miRNA-125b-5p is expressed ubiquitously in the kidney but that the renal expression of miRNA-181b-5p is lower than the sensitivity threshold for detection by ISH.

Fig 6. In situ hybridization images for miRNA-181b-5p and miRNA-125b-5p in kidneys from db/db mice. In the kidneys of db/db mice, hybridization with an antisense probe to miR-125b-5p revealed expression in the nucleus and cytosol of glomerular and tubular cells (black grains indicate a positive signal), whereas hybridization to the miR-181b-5p probe did not. Scale bar 50 mm. Abbreviations: RNU6, U6 Small Nuclear 1.

The effects of miRNA-125b-5p and miRNA-181b-5p on DKD(diabetic kidney disease) in vivo. The expression of miRNA-125b-5p and miRNA-181b-5p was specifically downregulated in DKD(diabetic kidney disease)(AKITA mice and db/db mice),and therefore we next determined the effects of overexpressing each miRNA using miRNA-mimics in db/db mice,to inves-tigate their suitability as therapeutic targets for DKD(diabetic kidney disease). The delivery of miRNA-mimics to the kidneys of db/db mice. To deliver miRNA-mimics to the kidneys of db/db mice, we employed polyethylenimine nanoparticles (PEI-NPs), which are nonviral vectors. To confirm sufficient delivery of miRNA-mimics using PEI-NPs, Cy-3-labeled miRNA-mimic conjugated with PEI-NPs (miRNA-mimic-PEI-NPs)were intravenously injected via a tail vein into db/db mice. The Cy3-labeled miRNA-mimic-PEI-NPs were then identified in the glomerular and tubulointerstitial areas of the kidneys of db/db mice (Fig.7A).

Fig 7. Distribution and expression levels of miRNAs administered with PEI-NPs in mouse kidneys. A miRNA-mimic (red) was injected with PEI-NPs and can be observed in the tubulointerstitial area (green) and glomerular regions of a kidney. Scale bar = 200 mm. (A) Expression of miRNA-181b-5p in kidneys using miRNA-181b-5p-mimic-PEI-NPs (n=3 per group) (B). Wilcoxon rank sum test. * P<0.05. Abbreviations. DAPI, 40 ,6-diamidino-2-phenylindole; FITC, flfluorescein isothiocyanate; PEI-NPs, polyethylenimine nanoparticles. (Color version of fifigure is available online.)

Effects of miRNA-181b-5p overexpression on DKD(diabetic kidney disease) in vivo. A single administration of miRNA-181b-5p-mimic-PEI-NPs via a tail vein led to over two-fold overexpression in the kidneys that was maintained for week (Fig. 7B). To investigate the potential 1 therapeutic effects of miRNA-181b-5p in DKD(diabetic kidney disease), miRNA-181b-5p-mimic-PEI-NPs (3 nmol miRNA-mimic, N/P ratio 6) were intravenously injected once per week into db/db mice from 10 to 20 weeks of age. The administration of miRNA-181b-5p-mimc-PEI-NPs significantly reduced proteinuria（538±44 μg/mg creatinine）vs.control db/db mice（869±84 μg/mg creatinine)and db/db mice administered control-miRNA-PEI-NPs（965±152μg/mg creatinine）at 20 weeks of age(Fig.8A).The administration of miRNA-181b-5p-mimic-PEI-NPs also ameliorated the characteristic histological changes of DKD, such as abnormal glomerular enlargement and mesangial area expansion, in db/db mice (Fig.8B).Furthermore, this halved the expression levels of collagen I and collagen IV in the kidneys of db/db mice(Fig.8C and 8D). These findings show that miRNA-181b-5p-mimic-PEI-NPs have a protective effect against the progression of DKD(diabetic kidney disease).

Fig 8. Effect of the administration of miRNA-181b-5p-mimic-PEI-NPs in db/db mice. Alteration of albuminuria (n=6 per group). (A) Wilcoxon rank sum test. * P<0.05. Histological evaluation of 20 glomeruli per group (3- 4 glomeruli per mouse and six mice per group). Mesangial index was calculated using periodic acid-Schiff (PAS)-stained sections from the kidneys of normal mice (control), untreated db/db mice (db/db), db/db mice administered miRNA-181b-5p-mimic-PEI-NPs (db/db+ miRNA-181b-5p-PEI-NPs), and db/db mice administered control-miRNA-PEI-NPs (db/db+ control-miRNA-PEI-NPs). Wilcoxon rank sum test. * P<0.05; y P<0.05 compared with control (B). Scale bar = 50 mm. Immunohistochemical and qRT-PCR evaluation of collagen I (C) and collagen IV (D) expression (n=6 per group). Collagen I expression in glomerular and tubulointerstitial areas, evaluated using immunohistochemistry (upper panels) and Col1a1 mRNA expression in the kidney, evaluated using real-time PCR (lower graphs) in each group. Collagen IV expression in glomerular and tubulointerstitial areas, evaluated using immunohistochemistry (upper panels) and Col4a1 mRNA expression in the kidney, evaluated using real-time PCR (lower graphs) in each group. Wilcoxon rank sum test. * P<0.05. Scale bar 50 mm. Abbreviations: PEI-NPs, polyethylenimine nanoparticles; UACR, urine albumin-to-creatinine ratio.

The mechanism of the effects of miRNA-181b-5p-mimic-PEI-NPs on DKD(diabetic kidney disease) in vivo. Gene microarray analysis was performed on kidney tissue from each group: nor-mal mice (n=2), untreated db/db mice (n=2), db/db mice administered miRNA-181b-5p-mimic-PEI-NPs (n=2), and db/db mice administered control-miRNA-PEI-NPs (n=2). miRNA-181b-5p-mimic-PEI-NP administration increased the expression of 24 genes and reduced the expression of 27 genes including Slc25a25 and Grm7, which are predicted to be target mRNAs of miRNA-181b-5p more than four-fold vs. untreated db/db mice(Table Ⅲa and Fig. 9). Control-miRNA-PEI-NP administration increased the expres-sion of one gene and reduced the expression of one gene more than four fold vs untreated db/db mice(data not shown). Furthermore, signaling pathway analysis using microarray followed by qRT-PCR (each group:n=6)showed miRNA-181b-5p-mimic-PEI-NP admin-istration changed the expressions of seven mRNAs (Slc25a25,Gli3,Cryl,Erc2,Nrld,Creb313,Psme3)in six signaling pathways in db/db mice (Fig.10). These results suggest that miRNA-181b-5p influences DKD(diabetic kidney disease) by modulating various signaling pathways.

Fig 9. Heat map of the differentially expressed genes in the kidney. The heat map shows the hierarchical clustering and systemic variations in the expression of genes in kidneys of mice from each group (n=2 per group). Abbreviations: PEI-NPs, polyethylenimine nanoparticles.

Effects of miRNA-125b-5p on DKD(diabetic kidney disease) in vivo. miRNA-125b-5p-mimic-PEI-NP was administered to db/db mice using the same method as for miRNA-181b-5p-mimic-PEI-NPs administration. However, the adminis-tration of miRNA-125b-5p-mimic-PEI-NPs had no effect on the progression of DKD(diabetic kidney disease) in db/db mice(Supplementary materials). These results suggest that miRNA-125b-5p may not influence the progression of DKD(diabetic kidney disease).

Fig 10. Changes in mRNA by the overexpression of miRNA-181b-5p. qRT-PCR analysis (n=6 per group) showed miRNA-181b-5p-mimic-PEI-NP administration changed the expression of seven mRNAs (Slc25a25, Gli3, Cry1, Erc2, Nr1d1, Creb3l3, Psme3) in each group (n=6 per group). Wilcoxon rank sum test. * P<0.05.

The suitability of miRNA-125b-5p and miRNA-181b-5p as diagnostic biomarkers of DKD(diabetic kidney disease). To investigate the utility of miRNA-125b-5p and miRNA-181b-5p as diagnostic biomarkers of DKD(diabetic kidney disease), the serum levels of each were measured in patients with DKD(diabetic kidney disease)(n=25), healthy subjects(n=25),and matched patients with kidney diseases other than DKD(diabetic kidney disease)(n=50) using qRT-PCR. The characteristics of each group are shown in Table IV. The serum level of miRNA-125b-5p was significantly higher(1.89±0.40-fold, P<0.05)and that of miRNA-181b-5p was significantly lower ((0.30±0.06-fold, P<0.05)in patients with DKD(diabetic kidney disease) compared with those with renal diseases other than DKD (miRNA-125b-5p:(0.94±0.13-fold),miRNA-181b-5p:(1.06±0.20-fold))and healthy subjects (miRNA-125b-5p:(1.00±0.19-fold), miRNA-181b-5p:(1.00±0.16-fold))(Fig 11A). These two miRNAs were also capable of discriminating DKD(diabetic kidney disease) from other kidney diseases, according to receiver operating characteristic (ROC) analysis [iRNA-125b-5p: cut-off value 0.78,area under the curve(AUC)0.74, sensitivity 0.86,specificity 0.58, P<0.05; miRNA-181b-5p: cut-off value 0.58,AUC 0.79,sensitivity 0.93, specificity 0.55,P<0.05](Fig.11B).The ratio of the serum levels of miRNA-125b-5p and miRNA-181b-5p (miRNA-125b-5p/miRNA-181b-5p) was significantly higher in patients with DKD(diabetic kidney disease)(7.23±1.50-fold P<0.01)compared with patients with renal diseases other than DKD(diabetic kidney disease)(1.78±0.28-fold) and healthy subjects (1.32±0.25-fold)(Fig.11C). The use of this expression ratio improved the utility of these measurements to differentiate DKD(diabetic kidney disease) from other kidney diseases,according to ROC analysis (miRNA-125b-5p/miRNA-181b-5p: cut-off value 2.05, sensitivity 1.00, specificity 0.73, AUC 0.92,P<0.05)(Fig.1IC).

Figure 11. Utility of the serum levels of miR-125b-5p and miRNA-181b-5p as diagnostic biomarkers for DKD(diabetic kidney disease). The serum levels of miR-125b-5p (left panel) and miRNA-181b-5p (right panel) in healthy subjects (n=25), participants with DKD(diabetic kidney disease) (n=25), and participants with kidney diseases other than DKD(diabetic kidney disease) (n=50) (A). ROC analysis of the serum levels of miRNA-125b-5p (left panel) and miRNA-181b-5p (right panel) for the diagnosis of DKD(diabetic kidney disease) (B). The ratio of the serum levels of miRNA-125b-5p and miRNA-181b-5p (miRNA-125b-5p/miRNA- 181b-5p) in healthy subjects (n=25), participants with DKD(diabetic kidney disease) (n=25), and participants with kidney diseases other than DKD (n=50) (left panel); and ROC analysis of the ratio of the serum levels of miRNA-125b-5p and miRNA-181b-5p (miRNA-125b-5p/miRNA-181b-5p) for the diagnosis of DKD(diabetic kidney disease) (right panel) (C). Wilcoxon rank sum test. * P<0.05. Abbreviations: DKD, diabetic kidney disease.

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Next, the serum levels of miRNA-181b-5p and miRNA-125b-5p and their expression ratio in participants with DKD(diabetic kidney disease)(n=25)or kidney diseases other than DKD(diabetic kidney disease) that were complicated by diabetes mellitus (n=25)were compared. The characteristics of these two groups are shown in Table V. Although the serum levels of miRNA-125b-5p tended to be lower in patients with DKD(diabetic kidney disease) than in those with kidney diseases other than DKD(diabetic kidney disease) that were complicated by diabetes mellitus, this did not reach statistical significance (Fig.12A). The serum level of miRNA-181b-5p was significantly lower in patients with DKD(diabetic kidney disease)(0.37±0.08-fold, P<0.05) compared with patients with kidney diseases other than DKD(diabetic kidney disease) that were complicated by diabetes mellitus(0.94±0.24-fold)(Fig.12A). The ratio of the serum levels of miRNA-125b-5p and miRNA-181b-5p(miRNA-125b-5p/miRNA-181b-5p) was significantly higher(6.65±1.57-fold,P<0.01) in patients with DKD(diabetic kidney disease) than in patients with kidney diseases other than DKD(diabetic kidney disease) that were complicated by diabetes mellitus (2.13±0.51 fold) (Fig. 12B). miRNA-125b-5p/miRNA-181b-5p significantly discriminated participants with DKD(diabetic kidney disease) from those with kidney diseases other than DKD(diabetic kidney disease) that were complicated by diabetes mellitus, according to ROC analysis(cut-off value 3.11, sensitivity 0.77,specificity 0.91,AUC 0.84,P<0.05)(Fig.12B).These findings suggest that the serum levels of miRNA-125b-5p and miRNA-181b-5p and their ratio may be useful diagnostic biomarkers of DKD(diabetic kidney disease).

Fig 12. Utility of the serum levels of miR-125b-5p and miRNA-181b-5p to differentiate DKD(diabetic kidney disease) from other kidney diseases complicated by diabetes mellitus. The serum levels of miR-125b-5p (left panel) and miRNA- 181b-5p (right panel) in participants with DKD(diabetic kidney disease) (n=25) and participants with kidney diseases other than DKD(diabetic kidney disease) that were complicated by diabetes mellitus (n=25) (A). The ratio of the serum levels of miRNA-125b-5p and miRNA-181b-5p (miRNA-125b-5p/miRNA-181b-5p) in participants with DKD(diabetic kidney disease) (n=25) and participants with kidney diseases other than DKD (n=25) (right panel). ROC analysis of the ratio of the serum levels of miRNA- 125b-5p and miRNA-181b-5p (miRNA-125b-5p/miRNA-181b-5p) for the diagnosis of DKD(diabetic kidney disease) (left panel) (B). Wilcoxon rank sum test. * p<0.05. Abbreviations: DKD, diabetic kidney disease.

Abbreviations: BMI= body mass index; DAPI=4,6-diamidino-2-phenylindole; FITC= fluorescein isothiocyanate; DKD= diabetic kidney disease; eGFR= estimated glomerular fltration rate;HDL high-density lipoprotein; HIGA= high immunoglobulin A; IRI= ischemia-reperfusion injury;LDL= low-density lipoprotein; ns= not significant; PEI-NPs= polyethylenimine nanoparticles;RNU6=U6 Small Nuclear 1; SAMP=senescence-accelerated mouse; SAMR=control for the senescence-accelerated mouse; UACR= urine albumin-to-creatinine ratio; UUO = unilateral ureteral obstruction

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