miR-21 is a key therapeutic target for renal injury in a mouse model of type 2 diabetes
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As microRNA-21 (miR-21) plays a pathological role in fibrosis, we hypothesised that it may be a therapeutic target for diabetic nephropathy.
Abundance of miR-21 was examined in diabetic kidneys from db/db mice. The therapeutic potential of miR-21 in diabetic kidney injury was examined in db/db mice by an ultrasound-microbubble-mediated miR-21 small hairpin RNA transfer. In addition, the role and mechanisms of miR-21 in diabetic renal injury were examined in vitro under diabetic conditions in rat mesangial and tubular epithelial cell lines by overexpressing or downregulating miR-21.
In db/db mice, a mouse model of type 2 diabetes, renal miR-21 at age 20 weeks was increased twofold compared with db/m + mice at the same age, and this increase was associated with the development of microalbuminuria and renal fibrosis and inflammation. More importantly, gene transfer of miR-21 knockdown plasmids into the diabetic kidneys of db/db mice at age 10 weeks significantly ameliorated microalbuminuria and renal fibrosis and inflammation at age 20 weeks, revealing a therapeutic potential for diabetic nephropathy by targeting miR-21. Overexpression of miR-21 in kidney cells enhanced, but knockdown of miR-21 suppressed, high-glucose-induced production of fibrotic and inflammatory markers. Targeting Smad7 may be a mechanism by which miR-21 regulates renal injury because knockdown of renal miR-21 restored Smad7 levels and suppressed activation of the TGF-β and NF-κB signalling pathways.
Inhibition of miR-21 might be an effective therapy for diabetic nephropathy.
KeywordsDiabetic nephropathy MicroRNA-21 TGF-β signalling Smad7
Monocyte chemotactic protein
- miR-21 KD
Nuclear factor κB
Small hairpin RNA
Small interfering RNA
TGF-β receptor II
Tubular epithelial cell
Diabetes is a chronic metabolic syndrome due to insulin deficiency, and its vascular complications are responsible for morbidity and mortality in diabetic patients. Diabetic nephropathy (DN) is a major complication of diabetes that leads to end stage renal disease. Among numerous mediators of DN [1, 2], TGF-β1 is the key one . In vitro studies have shown that induction of extracellular matrix production by high glucose and AGE is TGF-β-dependent . Results from animal models of both type 1 and type 2 diabetes confirm that TGF-β is an essential mediator of diabetic kidney injury . Targeting TGF-β signalling should have therapeutic potential against DN. However, blockade of TGF-β signalling may induce inflammation, as Tgfb1 knockout mice die from massive inflammation . Alternative approaches to inhibiting TGF-β action during DN should be developed in an attempt to protect against renal injury.
Recently, numerous studies have demonstrated that TGF-β regulates specific microRNAs, influencing renal fibrosis in kidney diseases [5, 6, 7, 8, 9]. MicroRNA-21 (miR-21) is the most significant microRNA involved in fibrotic disorders, and its levels are upregulated in human kidney diseases [5, 10, 11]. In a previous study, we demonstrated that TGF-β1 increases miR-21 content during renal fibrosis via a Smad3-dependent mechanism . Suppression of miR-21 abundance in a mouse model of obstructive kidney disease ameliorates renal fibrosis and halts the progression of renal fibrosis in an established obstructive nephropathy [7, 12], revealing a therapeutic potential for miR-21 knockdown in kidney diseases.
Thus, in this study, we hypothesised that targeting miR-21 may have therapeutic potential for DN. First, we determined whether knockdown of miR-21 in vivo would halt the progression of renal injury in db/db mice. We determined the role of miR-21 in high-glucose-induced renal fibrosis and inflammation by altering miR-21 levels in kidney cells. Finally, we determined whether Smad7 was a target of miR-21 during DN.
The normal rat mesangial cell (MC) line, 1099, and tubular epithelial cell (TEC) line, NRK52E (ATCC, Manassas, VA ,USA) were maintained in DMEM/low glucose (5.5 mmol/l; Life Technologies, Grand Island, NY, USA) containing 5% FBS and 0.1% antibiotic/antimycotic solution (Life Technologies), in six-well plastic plates at 37°C in an incubator with 5% CO2. Cells were stimulated with d-glucose at normal (5.5 mmol/l) or high (25 mmol/l) concentration for periods of 0, 24, 48 and 72 h in serum-free medium, in accordance with our own published procedures . d-Mannitol (25 mmol/l; Life Technologies) was used as a control for osmolality.
To impair TGF-β signalling, a stable dominant negative form of the TGF-β receptor II (TβRII)-producing NRK-52E cell line was used as previously described .
Construction of inducible cell lines of doxycycline (Dox)-regulated miR-21 overexpression and knockdown was performed as reported previously . At 48 h before the treatment, Dox (0.5 μg/ml) was added to induce the levels of miR-21 or miR-21 small hairpin RNA (shRNA). The cells were then stimulated with DMEM/high glucose (25 mmol/l; Life Technologies) for 48 h in serum-free medium, in accordance with our own published procedures .
MCs and TECs were transfected with 30 nmol/l Silencer-select Smad7 small interfering RNA (siRNA) or a negative control of siRNA (Life Technologies) in six-well plates using siPort Neo-FX (Life Technologies) according to the manufacturer’s instructions . After transient transfection, we synchronised the cells by culturing them in DMEM/low glucose medium without serum for 24 h. The cells were then stimulated with DMEM/high glucose medium for 48 h following our protocols .
Dox-regulated miR-21-overexpressing TECs were transfected with 1, 2 and 4 μg/well of pcDNA3.1-Smad7 expression plasmid or empty pcDNA 3.1 plasmid (Life Technologies) in six-well plates using Lipofectamine LTX (Life Technologies) according to the manufacturer’s instruction. At 48 h before the treatment, Dox (0.5 μg/ml) was added to increase the abundance of miR-21. After transient transfection, we synchronised the cells by culturing them in DMEM/low glucose medium without serum for 24 h. The cells were then stimulated with DMEM/high glucose medium for 48 h following our protocols .
Adult, male BKS.Cg-Dock7 m +/+ Lepr db /J db/db mice with a C57BLKS/J background (Jackson Laboratory, Bar Harbor, ME, USA), at 10 weeks of age, denoting the overt phases of the type 2 diabetes syndrome , were used in these studies. Details of animal models are provided in electronic supplementary material (ESM) Methods. All animal studies were approved by the Chinese University of Hong Kong’s Animal Experimental Ethics Committee.
Ultrasound-mediated gene transfer of inducible miR-21 knockdown (miR-21 KD) plasmids into the kidneys of db/db mice
The mixture of miR-21 KD and pDNA6/TR tet-on plasmids was combined with Sonovue (Bracco, Milan, Italy) at a ratio of 1:1 (vol./vol.) as described previously [7, 9, 15, 16, 17, 18]. The mixed solution (400 μl) was then injected via the tail vein of eight db/db mice (20–22 g body weight, 6 weeks old). The ultrasound transducer (Therasonic, Electro-Medical Supplies, Wantage, England) was then applied to the kidneys using ultrasound medium with a continuous wave output of 1 MHz at 1 W power output for a total of 5 min for each side. The same procedure was applied to eight control animals with a mixture containing the same amount of empty control plasmids (pSuperior/pcDNA6). To induce transgene expression at the optimal dosage, a dose of Dox solution (200 μg/ml in 200 μl volume) was first injected into the peritoneal cavity after the gene transfer. The mice were then given drinking water containing Dox (200 μg/ml) until they were killed . To maintain the transgene expression levels, gene therapy was given at age 10, 13 and 16 weeks. A group of eight age-matched db/m mice was used as normal controls. All mice were killed at age 20 weeks, and their kidneys were collected for analysis. The experimental procedures were approved by the Chinese University of Hong Kong’s Animal Experimental Ethics Committee.
In situ hybridisation
A specific 5′-FITC-labelled antisense-locked nucleic acid oligonucleotide for mmu-miR-21 and a scramble probe as a negative control were purchased from Exiqon (Vedbaek, Denmark). The detailed procedure for in situ hybridisation was carried out according to the manufacturer’s protocol . In brief, 10–14 μm slides were prepared from optimal cutting temperature compound- embedded kidney tissues. The slides were then fixed in 4% paraformaldehyde for 10 min, followed by prehybridisation with 1× hybridisation buffer (Exiqon) without the probe. The hybridisation was carried out overnight in 1× hybridisation buffer (30–70 μl) with FITC/anti-sense microRNA probe at 45°C overnight. After being washed, the slides were blocked and incubated with Smad7 antibody (Abcam, Cambridge, MA, USA), and subsequently with Alexa Fluor 555-conjugated secondary antibody (Life Technologies, Carlsbad, CA, USA). Signals were visualised and detected under a fluorescence microscope (Axioplan2 Imaging; Carl Zeiss, Oberkoche, Germany).
RNA extraction and quantitative RT-PCR analyses, western blot analysis, urine albumin excretion measurement, histology, immunohistochemistry and luciferase reporter assay
Details of these methods are provided in ESM Methods.
Construction of Smad7 overexpression plasmid and plasmids for luciferase reporter assay
Details of the cloning procedure are provided in ESM Methods.
Data from real-time PCR and immunohistochemical and western blot analysis were expressed as mean ± SEM and compared using ANOVA with the Newman–Keuls comparison program from GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA).
Knockdown of miR-21 in db/db mice reduces miR-21 levels, improves renal function, and inhibits renal fibrosis and inflammation during kidney damage caused by type 2 diabetes
We then investigated whether knockdown of miR-21 prevented renal injury in db/db mice by delivering miR-21 KD plasmids into kidneys with ultrasound-microbubble-mediated gene transfer at age 10, 13 and 16 weeks (Fig. 1b). As described previously , this knockdown plasmid targets the stem loop sequence of pre-miR-21 and suppresses renal miR-21 abundance for at least 14 days . The dose of miR-21 shRNA was carefully increased by Dox to avoid inducing dramatic cell death in diabetic kidneys .
At age 20 weeks, db/db mice developed similar levels of hyperglycaemia regardless of the treatment (blood glucose concentration 30.15 ± 0.78 mmol/l with control plasmids and 30.97 ± 1.15 mmol/l with miR-21 KD plasmids), indicating that delivery of miR-21 KD plasmids did not affect blood glucose levels. In addition, db/db mice treated with miR-21 KD plasmids developed less severe microalbuminuria than normal db/db mice over the 20-week disease course (Fig. 1c). Pathologically, an increase in mesangial matrix and thickness of the glomerular basement membrane was apparent in db/db mice at week 20 after diabetes onset, which was reduced in diabetic mice receiving miR-21 shRNA treatment (Fig. 1d, e). These results suggest that in vivo inhibition of miR-21 improved renal function in type 2 diabetic mice. Compared with db/m + littermates, the renal content of TGF-β1 and miR-21 in db/db mice was elevated from the age of 10 weeks (Fig. 1f, g). However, delivery of miR-21 KD plasmids into the diabetic kidneys of db/db mice suppressed the renal content of TGF-β1 and miR-21 at age 20 weeks to the levels of 10-week-old db/db mice, suggesting that progression of diabetic kidney injury was suppressed (Fig. 1f, g). No effect was observed when control plasmids with scrambled RNA sequence  were used (Fig. 1f, g).
MiR-21 is an important mediator of high-glucose-induced renal fibrosis and inflammation in vitro
To confirm the role of miR-21 in kidneys under diabetic conditions, we first determined whether a high-glucose concentration upregulated miR-21 abundance in both MCs and TECs. High-glucose concentration, but not the controls, induced miR-21 abundance in a time-dependent manner in both MCs and TECs (ESM Fig. 2a). This upregulation was blocked by the presence of the dominant negative form of TβRII (ESM Fig. 2b), implying that TGF-β signalling was necessary for the high-glucose-induced miR-21 level.
Smad7 is a target of miR-21 during renal fibrosis and inflammation
As reported in lung fibrosis, Smad7 is a target of miR-21 , and overexpression of Smad7 can suppress the TGF-β and NF-κB pathways [16, 23]. We therefore examined whether renal Smad7 abundance was also altered after miR-21 inhibition. Consistent with the type 1 diabetic mouse model , Smad7 (both RNA and protein levels) was significantly reduced during diabetic kidney injury in db/db mice aged 20 weeks (Fig. 7a and ESM Fig. 3a, b). Suppression of miR-21 partially restored the Smad7 content of 20-week-old db/db mice to that at age 10 weeks. Results from in situ hybridisation with FITC-miR-21 showed that miR-21 was induced in both glomeruli and renal tubules during DN (Fig. 7b). More significantly, results from a combination of immunohistochemical staining with Smad7 antibody and in situ hybridisation with FITC-miR-21 showed that the levels of renal Smad7 correlated negatively with miR-21 abundance during renal fibrosis (Fig. 7b). This negative correlation was confirmed by the results from in vitro studies. Overexpression of miR-21 further decreased the Smad7 mRNA content in both MCs and TECs, while knockdown of miR-21 restored Smad7 levels (ESM Fig. 3c–f).
To determine whether Smad7 acted downstream of miR-21 function, we overexpressed Smad7 in Dox-induced miR-21-overexpressing TECs by transiently transfecting different dosages of Smad7 overexpression plasmid without the 3′ untranslated region (UTR) into Dox-induced miR-21-expressing TECs. Results of real-time PCR and western blot assays demonstrated that miR-21 overexpression increased the high-glucose-induced mRNA content of collagen I, fibronectin, Tnf-α and Il-1β (Fig. 8g, h and ESM Fig. 4a–e). Overexpression of Smad7 resulted in >1.5-fold increase in abundance of Smad7, thereby largely reversing the stimulating effect of miR-21 on high-glucose-induced expression of collagen I, fibronectin, Tnf-α and Il-1β in a dose-dependent manner (Fig. 8g, h and ESM Fig. 4a–e). Thus, overexpression of Smad7 in miR-21-overexpressing renal cells reduced the abundance of inflammatory and fibrosis markers, suggesting that Smad7 is downstream of miR-21.
To determine whether miR-21 directly regulates Smad7 content, we assessed the complementarity of miR-21 to the Smad7 3′-UTR using TargetScan (http://www.targetscan.org/; accessed 7 February 2012). The computational algorithm from TargetScan predicted miR-21-binding sites in the Smad7 3′-UTR that are highly conserved between humans and rodents (ESM Fig. 4f), suggesting that Smad7 may be a miR-21 target. Therefore we subcloned various fragments of mouse Smad7 3′-UTR and sequences with native and mutated miR-21-binding sites downstream of the luciferase gene. Overexpression of miR-21 significantly downregulated luciferase activity in the reporter constructs containing the entire Smad7 3′-UTR or the 3′-UTR with a miR-21-binding site compared with normal TECs (Fig. 8i). Furthermore, miR-21 overexpression significantly reduced luciferase activity in the reporter containing the sequences with a native, but not mutant, miR-21-binding site. Collectively, these results suggest that miR-21 binds to the Smad7 3′-UTR and downregulates Smad7 abundance to derepress TGF-β and NF-κB signalling pathways, which are enhanced in renal fibrosis and inflammation during DN.
Our findings identify miR-21 as an important signature microRNA in DN because renal miR-21 was shown to be markedly upregulated in diabetic kidneys of db/db mice, and this is associated with the development of microalbuminuria and renal fibrosis and inflammation. More importantly, our study shows that in vivo inhibition of miR-21 ameliorated progression of DN in an established experimental model of type 2 diabetes. Suppressing Smad7 levels to promote activation of the TGF-β and NF-κB signalling pathways may be the mechanism whereby miR-21 promotes renal fibrosis and inflammation in diabetic kidney injury. Targeting miR-21 may be a good approach to preventing diabetic kidney injury.
We have previously shown that, during renal fibrosis, TGF-β1 upregulates the miR-21 level via a Smad3-dependent mechanism . Results from this study confirm that TGF-β signalling is necessary for high-glucose-induced miR-21 production. Upregulation of renal miR-21 content during DN in mouse models of type 1  and type 2 diabetes shows a positive correlation between miR-21 and progression of renal injury in DN. This study further shows that, under diabetic conditions, overexpression of miR-21 increases renal fibrosis, but suppression of miR-21 reduces renal fibrosis. These results are consistent with the pathological role of miR-21 in cardiac, lung and renal fibrosis [7, 22, 26]. Furthermore, we demonstrate for the first time that overexpression of miR-21 in kidney cells promotes, and knockdown of miR-21 reduces, renal inflammation under diabetic conditions. Thus, inhibiting both renal fibrosis and inflammation by in vivo suppression of miR-21 may be the mechanism by which miR-21 ameliorates progression of DN in type 2 diabetes.
In this study, we identified Smad7 as a target of miR-21 during renal injury and demonstrated that overexpression of Smad7 was able to reverse the pathological effects of miR-21 on renal fibrosis and inflammation. These findings are significant because, during DN, renal abundance of Smad7 is downregulated and gene therapy with Smad7 ameliorates proteinuria and suppresses both renal fibrosis and inflammation . Overexpression of Smad7 can suppress the TGF-β and NF-κB pathways by blocking the activation of Smad3 and stimulating IκBα production, respectively [16, 23]. Now, we demonstrate that knockdown of miR-21 in diabetic kidneys restores Smad7 levels and reduces phosphorylation of Smad3 and NF-κB/p65. Results from in vitro studies confirm the ability of miR-21 to reduce Smad7 content by binding to the Smad7 3′-UTR. As Smad7 knockout mice develop more severe renal fibrosis in both obstructive nephropathy and diabetic kidney disease as renal TGF-β/Smad3 signalling is increased [24, 27], we also demonstrated that knockdown of Smad7 in kidney cells increases renal fibrosis and inflammation, as observed when miR-21 was overexpressed. More importantly, we showed that the renal fibrosis and inflammation present under diabetic conditions associated with miR-21 overexpression were reversed by restoration of target protein, Smad7, levels when transfection of miR-21-expressing cells with the Smad7 expression construct rendered miR-21 insensitive by deletion of its 3′-UTR. Thus the possible mechanism by which miR-21 regulates high-glucose-induced renal fibrosis and inflammation has been deduced to be via targeting of Smad7 so as to induce the TGF-β and NF-κB signalling pathways. MiR-21 may function in a feed-forward loop, which amplifies TGF-β signalling during renal injury.
However, the precise mechanism by which miR-21 affects fibrosis and inflammation may also be related to other putative target genes of miR-21. Studies of cardiac fibrosis indicate that SPRY and PTEN are potential targets of miR-21 [26, 28]. Recent studies of miR-21 knockout mice show that miR-21 promotes renal fibrosis by silencing metabolic pathways via suppression of peroxisome proliferator-activated receptor-α . Another study on diabetic kidney injury has shown that PRAS40, a negative regulator of TORC1, is a target of miR-21 . For renal inflammation, we and others have shown that blocking miR-21 reduces macrophage infiltration in diseased kidneys . However, other studies demonstrate anti-inflammatory properties of miR-21 in macrophages by targeting the proinflammatory programmed cell death 4 gene (PDCD4) [29, 30]. Negative correlation between miR-21 and PDCD4 has been reported in TECs, with induction of ischaemia . Further studies should be performed to clarify whether miR-21 regulates inflammation in a cell-type-dependent fashion.
Although our present findings are consistent with previous studies showing that miR-21 plays a pathological role in tumorigenesis [32, 33], immunity [34, 35], inflammation  and fibrosis [7, 12, 22, 26], they are inconsistent with a report on miR-21 during diabetic kidney injury suggesting that it has a protective role in DN . In that report, Zhang et al showed by microarray analysis that renal miR-21 levels were downregulated threefold in 8-week-old db/db mice. In addition, overexpression of miR-21 by intraperitoneal injection in 4-week-old mice improved kidney function and inhibited glomerular expansion at 8 weeks of age. The authors suggest that miR-21 suppresses Pten expression in MCs and diabetic kidneys, resulting in mesangial hypertrophy . Although these discrepancies are as yet unexplained, the differences in disease nature, conditions and timing should be considered. First, we used BKS.Cg-Dock7 m +/+ Lepr db /J with a C57BLKS/J background because it is widely used as a type 2 diabetic mouse model. Zhang et al used C57BL/6JLep mice , and the diabetic phenotype of this strain has been reported to be less severe than that in C57BLKS/J . Second, in the present study, we chose to study db/db mice at the age of 10 weeks because db/db mice rapidly develop hyperglycaemia between 6 and 10 weeks of age and diabetic renal injury occurs after 10 weeks of age [39, 40, 41]. In the study by Zhang et al , miR-21 treatment in mice started at the age of 4 weeks, before the increase in microalbuminuria. Third, we used ultrasound-microbubble-mediated gene transfer to deliver miR-21 KD plasmids specifically into the living kidneys . However, Zhang et al delivered miR-21-overexpression plasmids into diabetic kidneys by systemic intraperitoneal injection. However, the quantitative measurement of miR-21 abundance and functional role of miR-21 in kidneys and cells under diabetic conditions have not been investigated, which prevents further discussion about the discrepancies between these studies. In the present study, the pathological role of miR-21 during diabetic kidney injury is based on two observations. On the one hand, miR-21 levels were further increased in 20-week-old db/db mice compared with 10-week-old mice, when hyperglycaemia and microalbuminuria worsen. On the other hand, suppression of miR-21 ameliorated progression of DN at the age of 20 weeks.
In summary, we propose that miR-21 is an important component of high-glucose-induced signalling, as upregulation of miR-21 reduces Smad7 abundance, increasing high-glucose-induced fibrosis and NF-κB-mediated inflammation. Delivery of miR-21 KD plasmids offers an alternative therapeutic treatment for preventing diabetic kidney injury.
We are grateful to F. A. Pereira of the Huffington Center on Aging, Baylor College of Medicine, Houston, TX, USA and D. J. Wilmshurst of the Research Administration Office, the Chinese University of Hong Kong, for commenting on a draft of this paper.
This work was supported by grants from the Research Grant Council of Hong Kong (RGC GRF 468711, 469110, 768409 and CUHK5/CRF/09 to H.Y. Lan; GRF 464010, 463612, 763908, and 764109 to A.C.K. Chung) and the Focused Investment Scheme B (1902061) and by direct grants from the Chinese University of Hong Kong (2041656 and 2041735 to A.C.K. Chung), 2011 Research Grant from Hong Kong Society of Nephrology (6903213 to A.C.K. Chung), National Natural Science Foundation of China (General Program, 81170681 to A.C.K. Chung), and Major State Basic Research Development Program of China (973 program, No.2012CB517700 to H.Y. Lan).
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
XZ, YD, HC, XMM, RL, WY and ACKC designed the study, performed the research and analysed the data. HYL and FFH analysed data and revised the manuscript. ACKC was responsible for the conception of the study and drafting the article. All authors contributed to writing the paper and approved the final version.
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