BASIC RESEARCH www.jasn.org Variations in MicroRNA-25 Expression Influence the Severity of Diabetic Kidney Disease † † † Yunshuang Liu,* Hongzhi Li,* Jieting Liu,* Pengfei Han, Xuefeng Li, He Bai,* Chunlei Zhang,* Xuelian Sun,* Yanjie Teng,* Yufei Zhang,* Xiaohuan Yuan,* Yanhui Chu,* and Binghai Zhao* *Heilongjiang Key Laboratory of Anti-Fibrosis Biotherapy, Medical Research Center, Heilongjiang, People’s Republic of China; and †Clinical Laboratory of Hong Qi Hospital, Mudanjiang Medical University, Heilongjiang, People’s Republic of China ABSTRACT Diabetic nephropathy is characterized by persistent albuminuria, progressive decline in GFR, and second- ary hypertension. MicroRNAs are dysregulated in diabetic nephropathy, but identification of the specific microRNAs involved remains incomplete. Here, we show that the peripheral blood from patients with diabetes and the kidneys of animals with type 1 or 2 diabetes have low levels of microRNA-25 (miR-25) compared with those of their nondiabetic counterparts. Furthermore, treatment with high glucose decreased the expression of miR-25 in cultured kidney cells. In db/db mice, systemic administration of an miR-25 agomir repressed glomerular fibrosis and reduced high BP. Notably, knockdown of miR-25 in normal mice by systemic administration of an miR-25 antagomir resulted in increased proteinuria, extracellular matrix accumulation, podocyte foot process effacement, and hypertension with renin-angiotensin system activation. However, excessive miR-25 did not cause kidney dysfunction in wild-type mice. RNA sequencing showed the alteration of miR-25 target genes in antagomir-treated mice, including the Ras-related gene CDC42. In vitro,cotrans- fection with the miR-25 antagomir repressed luciferase activity from a reporter construct containing the CDC42 39 untranslated region. In conclusion, these results reveal a role for miR-25 in diabetic nephropathy and indicate a potential novel therapeutic target for this disease. J Am Soc Nephrol 28: ccc–ccc, 2017. doi: https://doi.org/10.1681/ASN.2015091017 Diabetic nephropathy (DN) is a progressive kidney and microRNAs (miRNAs) in the pathogenesis of disease that develops secondary to diabetes, and it is hypertension, which serves as a reminder that the the single most common disorder leading to renal deregulation of miRNA expression plays a key role failure in developing countries.1 The percentage of in hypertension, especially originating from DN.6 individuals with DN in China also exceeds the per- miRNAs are short noncoding RNAs of approxi- centage with CKD related to GN.2 DN is charac- mately 22 nucleotides in length that exert their terized by persistent albuminuria (.300 mg/d), a progressive decline in the GFR, and elevated arte- rial BP.3 BP control and efficient blockade of the Received September 14, 2015. Accepted August 16, 2017. renin-angiotensin system (RAS) to reduce micro- Y.L., H.L., and J.L. contributed equally to this work. albuminuria together with tight glycemic control 4 Published online ahead of print. Publication date available at are important therapeutic strategies for DN. www.jasn.org. However, this strategy is insufficient to prevent progression to renal failure in a substantial propor- Correspondence: Dr. Yanhui Chu or Dr. Binghai Zhao, Heilongjiang Key Laboratory of Anti-Fibrosis Biotherapy, Mudanjiang Medical 5 tion of patients. Thus, novel effective therapeutic University, 3 Tongxiang Street, Mudanjiang, Heilongjiang approaches are critical for the treatment and pre- 157001, People’s Republic of China. Email: yanhui_chu@163. vention of the disease. Intriguingly, recent studies com or [email protected] have shown a link between ACE2/Apelin signaling Copyright © 2017 by the American Society of Nephrology J Am Soc Nephrol 28: ccc–ccc, 2017 ISSN : 1046-6673/2812-ccc 1 BASIC RESEARCH www.jasn.org canonical function by binding the 39 un- translated region (39UTR) of mRNAs and regulate the expression of 50% of genes.7,8 It is well known that one miRNA can target multiple mRNAs, whereas one mRNA can be bound by a combination of numerous miRNAs.Therefore,miRNAsandtheir target mRNAs form an intricate regulatory network that can be expressed as a recip- rocal “one-to-more” relationship, which has been hypothesized as a “fail-safe” mechanism to control gene expression.9 In mice, a podocyte-specificknockoutof Dicer, an enzyme critical to the miRNA biogenesis pathway, leads to progressive re- nal glomerular and tubular damage.10–12 A similar phenotype is also observed when Drosha is inducibly deleted in podocytes.13 Thus, we conclude that excessive deficiency of miRNAs due to deletion of Dicer and Drosha contributes to renal dysfunction and suggest that dysregulation of miRNAs can promote kidney disease. Emerging ev- idence confirms that individual miRNAs, including microRNA-23b (miR-23b),14 miR-192/216a/217,15,16 and miR-377,17 are involved in DN. Recent studies have also shown that miRNAs, including miR- 143/145, are associated with the pathogen- esis of hypertension.18–20 In this study, we have shown that miR-25 is commonly downregulated in the serum and kidneys of patients with DN, the kidneys of mouse models of type 1 and 2 diabetes, and spe- cific kidney cells exposed to high glucose. We also observed that downregulation of miR-25 in normal mice causes severe renal disease with hypertension, and upregula- tion of miR-25 in db/db mice can reverse DN alterations and reduce high BP. RESULTS Figure 1. Excessive miR-25 reverse albuminuria and fibrosis in diabetic nephropathy. (A) miR- 25 levels in the sera of patients with type 1 diabetes (T1DM), type 2 diabetes (T2DM), or DN Overexpression of miR-25 Reverses were reduced compared with the healthy controls (Con). (B and C) Similarly, miR-25 was re- Albuminuria and Fibrosis in Diabetes duced in the kidneys of type 1/2 diabetic (Dia [n=5] and db/db [n=5]) animals compared with miR-25 levels in the sera of patients with controls (Con; n=5).(D)miR-25expressionlevelsinvarioustissuesofnormalC57BL/6mice.(E) diabetes both with and without nephropa- fi – Quanti cation of 24-hour albumin excretioninmiR-25agomir treated mice. (F) Representative thyweredownregulatedcomparedwith images of H&E (row 1), PAS (row 2), Sirius Red (row 3), a-SMA (row 4), and TEM (row 5) staining those in healthy controls (Figure 1A). A sim- from kidney sections of miR-25 agomir–injected mice. Green arrows indicate areas of fibrosis, an asterisk indicates the GBM, and a red arrow represents a foot process. (G) Quantification of ilar downregulation of miR-25 occurred as the a-SMA–positive area within glomeruli from miR-25 agomir–treated kidney sections. (H) detected by quantitative PCR (qPCR) in the Quantification of GBM thickening after miR-25 agomir injection. (I) Quantification of foot kidney tissues of streptozotocin-induced di- process fusion from miR-25 agomir–treated db/db mice. (J) Measurement of BP from miR-25 abetic and db/db mice (Figure 1, B and C) agomir–injected db/db mice. Data are shown as the mean6SEM. **P,0.01; ***P,0.001. and cultured cells exposed to high glucose 2 Journal of the American Society of Nephrology J Am Soc Nephrol 28: ccc–ccc,2017 www.jasn.org BASIC RESEARCH Figure 2. Overexpression of miR-25 inhibit RAS activation. (A) Representative images of KRI (row 1), angiotensin 1 (AT1; row 2), renin (row 3), and vWf (row 4) staining from miR-25 agomir–treated db/db mouse kidney sections (right panel represents a quantification of the images). (B) Quantification of an ELISA for serum renin, angiotensin I/II, and aldosterone (ALD) from miR-25 agomir– and antagomir– treated mice. Data are shown as the mean6SEM. Con, control. ***P,0.001. (Supplemental Figure 1, A–C). We also found that miR-25 in the miR-25 was highly expressed in mouse heart, muscle, adipose serum of db/db mice was decreased significantly compared with tissue, and kidneys compared with other tissues (Figure 1D). in the wild type (Supplemental Figure 1D). To regulate their To establish the functional consequence of miR-25 upregula- target mRNAs, the miRNA minimum threshold of expression tion in the kidneys,we performed intravenous miR-25 agomir must be reached, and the abundance of miRNAs in the miR- (2.5 mg/kg) injection in db/db mice. Delivery efficiencies NAome of a specific cell or tissue may be more important for showed an approximately fourfold increase in kidney miR-25 their function at the organismal level.21 We examined miR-25 expression (Supplemental Figure 1E). Importantly, miR-25 levels in multiple tissues, including the kidneys, and found that agomir injection in db/db mice decreased their proteinuria J Am Soc Nephrol 28: ccc–ccc, 2017 Inhibition of MicroRNA-25 Causes Kidney Failure 3 BASIC RESEARCH www.jasn.org (Figure 1E, Supplemental Figure 1F) and reversed their glomer- (Supplemental Figure 1I). In keeping with these findings, ular fibrosis as shown by H&E, PAS, Sirius Red, and a-SMA mesangial expansion, GBM thickening, and podocyte foot staining (Figure 1, F and G). Furthermore, miR-25 agomir collapse were observed under electron microscopy in miR-25 treatment reversed the upregulation of a series of fibrosis gene antagomir–treated mice but were not observed in the mice re- markers; however, miRNEG treatment had no such effect ceiving miRNEG injections (Figure 3, D and G). Because accu- (Supplemental Figure 1G). miR-25 overexpression also alle- mulated renal fibrosis and the collapse of the foot processes are viated the amount of foot process fusion and glomerular base- the main characteristics of glomerulosclerosis, we, therefore, ment membrane (GBM) thickening observed in db/db mice measured immunostaining for glomerulosclerosis marker (Figure1,F,H,andI). PODXL and found that its expression was significantly decreased (Figure 3, D and H). A series of genes related to glomeruloscle- Overexpression of miR-25 Alleviates Secondary rosis displayed a similar decrease compared with in control and Hypertension in Diabetes miRNEG-treated mice (Supplemental Figure 2A).