C66 ameliorates diabetic nephropathy in mice by both upregulating NRF2 function via increase in miR-200a and inhibiting miR-21
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Diabetic nephropathy is the leading cause of end-stage renal disease. Previously we reported that C66, a novel analogue of curcumin with a very high bioavailability, ameliorated diabetic nephropathy in mice, with little known about the mechanism. The present study aimed to define the mechanism by which C66 ameliorates diabetic nephropathy.
Our aim was to discover whether C66 acts through the activation of nuclear factor (erythroid-derived 2)-like 2 (NFE2L2 or NRF2), which governs the antioxidant response. Streptozotocin-induced Nrf2 (also known as Nfe2l2)-knockout and wild-type (WT) diabetic mice were treated with C66. To determine whether the actions of C66 on NRF2 are mediated by microRNA (miR)-200a, WT diabetic mice were treated with C66 in the presence or absence of an in vivo miR-200a inhibitor (locked nucleic acid-modified anti-miR-200a [LNA-200a]) for 6 months. To determine whether miR-21 downregulation provided an NRF2-independent basis for C66 protection, Nrf2-knockout diabetic mice were treated with either C66 or an inhibitor of miR-21 (locked nucleic acid-modified anti-miR-21 [LNA-21]).
Deletion of Nrf2 partially abolished diabetic nephropathy protection by C66, confirming the requirement of NRF2 for this protection. Diabetic mice, but not C66-treated diabetic mice, developed significant albuminuria, renal oxidative damage and fibrosis. C66 upregulated renal miR-200a, inhibited kelch-like ECH-associated protein 1 and induced NRF2 function, effects that were prevented by LNA-200a. However, LNA-200a only partially reduced the protection afforded by C66, suggesting the existence of miR-200a/NRF2-independent mechanisms for C66 protection. C66 was also found to inhibit diabetes induction of miR-21. Both C66 and LNA-21 produced similar reductions in miR-21, albuminuria and renal fibrosis.
The present study indicates that in addition to upregulating NRF2 by increasing miR-200a, C66 also protects against diabetic nephropathy by inhibiting miR-21.
KeywordsAlbuminuria Curcumin Diabetic nephropathy Fibrosis KEAP1 miR-200 miR-21 NRF2
c-Jun N-terminal kinase
Kelch-like ECH-associated protein 1
Locked nucleic acid-modified anti-miR-200a
Locked nucleic acid-modified anti-miR-21
- NFE2L2, NRF2
Nuclear factor (erythroid-derived 2)-like 2
Programmed cell death protein 4
Urinary albumin-to-creatinine ratio
Diabetic nephropathy is the leading cause of end-stage renal disease , resulting in high mortality. As a long-term complication of diabetes, diabetic nephropathy still cannot be effectively treated and so there is an urgent need to develop more effective medicines to prevent or slow down its progression .
Oxidative stress contributes to the pathogenesis of diabetic nephropathy . Nuclear factor (erythroid-derived 2)-like 2 (NFE2L2 or NRF2) controls cellular defence mechanisms against oxidative stress  by turning on transcription of antioxidant genes, such as Ho1 and Nqo1 . NRF2 has been demonstrated to play a key role in protection from diabetic nephropathy [2, 3]. Curcumin is a natural compound that induces NRF2  and prevents diabetes-induced renal injury in animal models [7, 8]. Although curcumin has beneficial effects in animal models of many diseases, poor bioavailability limits its clinical application. The doses of curcumin used in animal models of diabetic nephropathy range from 50 mg/kg to 200 mg/kg daily [7, 9, 10, 11]. C66 is a novel curcumin analogue with a much lower effective dose of 5 mg/kg administered every other day, and has been shown to establish protection from diabetic nephropathy and diabetic cardiomyopathy [12, 13]. We have also demonstrated that C66 activated NRF2 in diabetic aorta . However, it is not certain that activation of NRF2 is necessary for C66 to provide protection against diabetic nephropathy. Therefore our first question to address is whether renal protection by C66 in diabetes is mediated by NRF2.
Kelch-like ECH-associated protein 1 (KEAP1) sequesters NRF2 in the cytoplasm, promoting its proteasomal degradation . Thus, one strategy for increasing the activity of NRF2 is to reduce cytoplasmic KEAP1. MicroRNAs (miRs) are short non-coding RNAs that modulate gene expressions . miR-200a ameliorates diabetic nephropathy and promotes degradation of Keap1 mRNA . In diabetic mice, suppression of renal miR-200a increases KEAP1, decreases NRF2 and worsens renal fibrosis and albuminuria . Interestingly, a difluorinated analogue of curcumin upregulates expression of the miR-200 family [18, 19]. Curcumin analogue C66 may also upregulate miR-200a which could mediate the ability of C66 to upregulate NRF2 and prevent diabetic nephropathy. miR-21 also has a key role in the pathogenesis of diabetic nephropathy [20, 21, 22] but unlike miR-200a, it exacerbates diabetic nephropathy. Curcumin downregulates miR-21 [18, 23] and this likely contributes to the beneficial effect of curcumin against diabetic nephropathy. C66 has not been tested for its ability to modulate renal levels of miR-200a or miR-21; this could be an essential step in the pathway by which C66 provides protection from diabetic nephropathy.
The present study was performed to determine the mechanism(s) by which C66 provides protection against diabetic nephropathy by answering the following questions: (1) does prevention of diabetic nephropathy by C66 require NRF2?; (2) does C66 upregulate miR-200a or downregulate miR-21?; (3) is C66 upregulation of NRF2 mediated by miR-200a? and (4) is C66 modulation of either miR-21 or miR-200a sufficient to protect against diabetic nephropathy? To these ends, Nrf2 (also known as Nfe2l2)-knockout (Nrf2-null) mice and their wild-type (WT) counterparts were used with and without C66 treatment, and in combination with specific in vivo inhibitors of miR-200a or miR-21.
Nrf2-null (Nrf2 −/−) male mice with C57BL/6J background (WT) were obtained through breeding of homozygote (Nrf2 −/−) with heterozygote (Nrf2 +/−) following the mating system suggested by Jackson Laboratory (Bar Harbor, ME, USA). C57BL/6J male mice (Nrf2 +/+) were also purchased from the Jackson Laboratory. All mice were housed in University of Louisville Research Resources Center at 22°C, on a 12 h light–dark cycle, with free access to standard rodent feed and tap water. The Institutional Animal Care and Use Committee at University of Louisville approved all experimental procedures for these animals, and all procedures complied with the Guide for the Care and Use of Laboratory Animals by the US National Institutes of Health (2011, eighth edition). To test the role of NRF2 in C66 protection against diabetic nephropathy, 8-week-old male WT mice were randomised into the following four groups (n = 7): control (Ctrl), control treated with C66 (Ctrl/C66), diabetes (DM) and diabetes treated with C66 (DM/C66). To further explore whether miR-200a mediates NRF2 activation by C66, 8-week-old male WT mice were randomised into six groups (n = 7): Ctrl, Ctrl/C66, DM, DM/C66, C66/locked nucleic acid-modified anti-miR-200a (LNA-200a) negative control co-treated DM (DM/C66/LNA-200a-NC) and C66/LNA-200a co-treated DM (DM/C66/LNA-200a). To explore whether the inhibition of miR-21 by C66 provides additional protection against diabetic nephropathy in the absence of NRF2, 8-week-old male Nrf2-null mice were randomised into six groups (n = 7): Ctrl, Ctrl/C66, DM, DM/C66, locked nucleic acid-modified anti-miR-21 (LNA-21) negative control treated DM (DM/LNA-21-NC) and LNA-21 treated DM (DM/LNA-21). Mice received an intraperitoneal injection of either sodium citrate or streptozotocin (STZ) (Sigma-Aldrich, St Louis, MO, USA; 50 mg/kg, dissolved in 0.1 mol/l sodium citrate, pH 4.5) on five consecutive days. One week after the last injection of STZ, fasting glucose levels (4 h fast) were measured and mice with a fasting glucose level above 13.89 mmol/l were considered diabetic. Diabetic mice and age-matched controls were then treated by gavage with C66 (5 mg/kg, dissolved in 1% carboxymethyl cellulose-Na solution) or vehicle every other day for 24 weeks. LNA-200a, LNA-21 and their respective mismatched controls (Exiqon, Woburn, MA, USA; 2 mg/kg, dissolved in normal saline [154 mmol/l NaCl]) were delivered subcutaneously at the same time as C66, twice weekly for 24 weeks. Blood glucose levels were recorded on days 28, 56, 84, 112, 140 and 168 after diabetes onset. Mice were then killed and their kidneys taken for analysis. Experimenters of this study were blind to group assignment and outcome assessment.
Analysis of kidney function
Urine albumin and creatinine were measured on a spot urine sample by a mouse albumin ELISA kit (Bethyl Laboratories, Montgomery, TX, USA) and a QuantiChrom Creatinine Assay Kit (BioAssay Systems, Hayward, CA, USA). The urinary albumin-to-creatinine ratio (UACR) was calculated.
Measurement of blood pressure
Systolic BP was recorded on day 168 after the onset of diabetes with a non-invasive tail cuff Plethysmograph system (CODA6TM; Kent Scientific, Torrington, CT, USA) as previously described .
Western blot analysis
Western blot was performed using kidney cortex as described in our previous study . The primary antibodies were anti-KEAP1 (Santa Cruz Biotechnology, Dallas, TX, USA; 1:1,000), anti-NRF2 (Santa Cruz Biotechnology; 1:1,000), anti-Histone H3 (Santa Cruz Biotechnology; 1:500), anti-4-HNE (Alpha Diagnostic, San Antonio, TX, USA; 1:3,000), anti-3-NT (Millipore, Temecula, CA, USA; 1:1,000), anti-TGF-β1 (Cell Signaling, Beverly, MA, USA; 1:500), anti-COL4 (Abcam, Cambridge, MA, USA, 1:500), anti-FN (Santa Cruz Biotechnology; 1:500), anti-Smad7 (Santa Cruz Biotechnology; 1:1,000), anti-Smad3 (Santa Cruz Biotechnology; 1:1,000), anti-p-Smad3 (Cell Signaling; 1:500), anti-p-JNK (Cell Signaling; 1:500), anti-PDCD4 (Santa Cruz Biotechnology; 1:1,000), anti-t-JNK (Cell Signaling; 1:1,000), anti-Actin (Santa Cruz Biotechnology; 1:2,000) and anti-GAPDH (Santa Cruz Biotechnology; 1:3,000). These antibodies were routinely validated when they arrived from suppliers with previous positive tissues that had been defined either based on the knockout or overexpression of the target protein.
Quantitative reverse transcription PCR
Quantitative RT-PCR (qRT-PCR) was performed as described in our previous studies [26, 27]. qRT-PCR for miRNAs was performed with the TaqMan MicroRNA Reverse Transcription Kit (Life Technologies, Grand Island, NY, USA). Primers for Keap1, NAD(P)H quinone oxidoreductase (Nqo1), haem oxygenase-1 (Ho1), primary-miR-21 (pri-miR-21), miR-21, Smad7, programmed cell death protein 4 (Pdcd4), actin and U6 were all purchased from Life Technologies.
Histological and immunohistochemical staining
Kidney tissues were fixed immediately in 10% buffered formalin solution after harvesting and were embedded in paraffin and sectioned into 5 μm thick sections onto glass slides. The sections were processed for periodic acid–Schiff (PAS), Masson’s trichrome and immunohistochemical staining using antibody against NRF2 (Santa Cruz Biotechnology; 1:100 dilution).
Isolation of nuclei
The renal nuclei were isolated using a nuclei isolation kit (Sigma-Aldrich). Kidney tissue (30 mg) from each mouse was homogenised for 45 s in 150 μl of cold lysis buffer containing 0.5 μl of dithiothreitol (DTT) and 0.1% Triton X-100. After that, 300 μl of cold 1.8 mol/l cushion solution (Sucrose cushion solution:sucrose cushion buffer:DTT 900:100:1) was added to the lysis solution. The mixture was transferred to a new tube preloaded with 150 μl of 1.8 mol/l sucrose cushion solution followed by centrifugation at 30,000 g for 45 min. The supernatant fraction containing cytosolic components was aspirated and the nuclei were visible as a thin pellet at the bottom of the tube.
Morphometric analyses were performed using Image-Pro Plus 6.0 software (Media Cybernetics, Bethesda, MD, USA). Areas to be photographed were selected by people blind to the identity of the samples, as was scoring.
C66 retains partial renal protection against diabetes-induced albuminuria despite deletion of the Nrf2 gene
Diabetic WT and Nrf2-null mice had higher blood glucose levels compared with their respective controls (Fig. 1a, b). Blood glucose levels were not altered by C66 (Fig. 1a, b). Nrf2-null mice had lower systolic BP compared with WT mice (Fig. 1c), which was in line with findings reported in a previous publication . Diabetes mildly increased BP (Fig. 1c) and decreased body weight (Fig. 1d) in both strains 24 weeks post diabetes. C66 had no impact on either BP or body weight (Fig. 1c, d). Diabetes increased UACR 2.57-fold in WT mice and 3.70-fold in Nrf2-null mice (Fig. 1e), suggesting the importance of NRF2 in preventing diabetic nephropathy. Protein levels of TGF-β1 (Fig. 1f) and FN (Fig. 1g) were increased by diabetes in both strains but these elevated levels were decreased by C66. C66 decreased UACR, TGF-β1 and FN protein by 44.72%, 50.80% and 48.94% in WT diabetic mice and by 21.77%, 21.68% and 24.83% in Nrf2-null diabetic mice; these effects were all significantly lower in Nrf-null diabetic mice than in WT diabetic mice. This confirmed the requirement of NRF2 in C66 protection. On the other hand, the fact that C66 still provided protection (Fig. 1e–g) suggested the existence of an NRF2-independent protective effect of C66 against diabetic nephropathy.
C66 upregulated renal NRF2 expression via miR-200a targeting Keap1
LNA-200a blocks renal C66 protection against diabetic oxidative damage more effectively than C66 protection against diabetic renal fibrosis, remodelling or albuminuria
Diabetes-induced renal structural remodelling was accompanied by a slight increase in kidney size, shown by increased ratio of kidney weight to tibia length (Fig. 4e). Diabetic renal dysfunction was reflected by increased UACR at 12 and 24 weeks post diabetes and this effect was significantly reduced by C66 treatment (Fig. 4f). The protective effect of C66 on UACR was partially abolished by LNA-200a after 24 weeks of treatment (Fig. 4f).
In summary, LNA-200a completely abolished the effect of C66 on KEAP1–NRF2 signalling (Fig. 2) and oxidative damage (Fig 3a, b) but only partially abolished the protective effect of C66 against renal fibrosis (Fig. 3c–e), remodelling (Fig. 4a–d) and albuminuria (Fig. 4f). These results suggest that the C66-stimulated miR-200a–KEAP1–NRF2 pathway is not the full explanation for C66 protection against diabetic nephropathy and this conclusion is consistent with the finding that C66 retains partial efficacy against albuminuria in diabetic Nrf2-null mice (Fig. 1e).
C66 decreased miR-21 in normal and diabetic WT mice accompanied by downregulation of p-Smad3
Both C66 and LNA-21 downregulate miR-21 in Nrf2-null diabetic mice, resulting in partial amelioration of diabetes-induced renal fibrosis
Oxidative stress contributes to the pathogenesis of diabetic nephropathy  and the KEAP1–NRF2 system even governs antioxidant redox signalling to prevent the initiation of diabetes mellitus [34, 35]. We and others have demonstrated the beneficial role of NRF2 induction in the prevention of diabetic nephropathy [2, 36]. High BP accelerates diabetic nephropathy. In the present study, the systolic BP of Nrf2-null mice was lower than that of WT mice. This result is in accordance with the finding by Erkens et al , which showed that elevated endothelial nitric-oxide synthase in the heart and aorta of Nrf2-null mice was the reason for the low BP. Given that high BP accelerates diabetic nephropathy whereas the Nrf2-null mice had lower BP along with severer diabetic renal injury, we may further conclude that NRF2 does play an important role in the prevention of diabetic nephropathy.
Curcumin is reported to be an activator of NRF2 [6, 37], although little is known about how this occurs. Curcumin disrupts the binding between KEAP1 and NRF2, leading to the release of NRF2 . However, the fact that curcumin downregulates KEAP1 expression  challenges the notion that curcumin only modifies the KEAP1–NRF2 complex. Our results also demonstrate that the curcumin analogue C66 decreases the expression of Keap1 mRNA and KEAP1 protein. Therefore, in addition to modification of the KEAP1–NRF2 complex, curcumin-decreased expression of Keap1 may also account for NRF2 induction. The present study identified miR-200a as the mediator between C66 and Keap1 mRNA. miR-200a degrades Keap1 mRNA . Moreover, LNA-200a exacerbates diabetic cortical and glomerular fibrogenesis and increases urinary albumin excretion . Using LNA-200a, we provide the first evidence that C66 stimulates miR-200a to downregulate Keap1 expression, resulting in the activation of NRF2.
C66, sulforaphane (SFN) and cinnamic aldehyde (CA) all activate NRF2. The protection from diabetic nephropathy afforded by SFN and CA is abolished in Nrf2-null diabetic mice . The present study showed that C66 still provided significant protection in the absence of NRF2 (Fig. 1e–g). The difference between C66 and the other two NRF2 activators must be due to additional actions of C66. SFN and CA exclusively provide protection through NRF2 and, accordingly, no preventive effect on diabetic nephropathy is observed in Nrf2-null diabetic mice . Unlike SFN and CA, we show that C66 has additional function  other than NRF2 upregulation [6, 37].
Curcumin is a regulator of epigenetic events, including miRNAs , among which miR-21 has been demonstrated to play a key role in the pathogenesis of diabetic nephropathy [20, 21, 22]. Recent findings shed light on the involvement of miR-21 as a key component of TGF-β–Smad and PDCD4–JNK signalling. miR-21 degrades Smad7 or Pdcd4 mRNA to lower their protein translation , leading to impaired function of Smad7 or PDCD4 in the inhibition of Smad3 or JNK phosphorylation [32, 41]. The transcription factors p-Smad3 or p-c-Jun turn on the expression of a cohort of renal fibrotic and inflammatory genes [41, 42, 43, 44, 45]. Therefore, inhibition of miR-21 may exert an effect different from activation of NRF2. This notion is supported by our results that knockdown of NRF2 by LNA-200a in WT mice completely abolished C66 protection against renal oxidative damage (Fig. 3a, b) whereas C66 still protected against renal fibrosis and kidney function (Figs 3c–e and 4d, f). Furthermore, inhibition of miR-21 by either C66 or LNA-21 negatively altered TGF-β–Smad3 signalling, PDCD4–JNK signalling and albuminuria even in the absence of NRF2 (Figs 6 and 7), confirming that inhibition of miR-21 offered an additional role for C66 distinct from that of inducing NRF2 function.
Inhibition of diabetic nephropathy-inducing miRNAs has attracted a great interest in recent years. miRNA inhibitors or mimics have even been studied in clinical trials . The advantage of C66 compared with LNA-21 is that C66 also upregulates NRF2 in addition to knockdown of miR-21. NRF2 activation has already been applied in clinical trials. Initial human studies of bardoxolone methyl were promising but its use was terminated in a phase III study due to heart complications . However, NRF2 remains a viable drug target as demonstrated by the approval of BG-12 (dimethyl fumarate) for use in multiple sclerosis . In addition to initiating treatment at an earlier stage of diabetic nephropathy and diminishing drug–drug interactions, greater attention should have been paid to the dosage of bardoxolone . More recently, Tan et al showed that dh404, a derivative of bardoxolone methyl, prevented diabetic nephropathy , although inflammation was triggered at higher doses. These studies indicate a sweet spot for NRF2 activation . Therefore the dosage of NRF2 activators should be carefully considered. The dose-effectiveness of C66 is very much better than native curcumin (20- to 80-fold), and this confers a unique advantage in future clinical trials.
In summary, the present study demonstrates for the first time that C66 ameliorates diabetic nephropathy by both inhibiting miR-21 and activating NRF2 through the stimulation of miR-200a. Thus, this novel curcumin analogue with dual effects and high bioavailability urgently needs to be further tested in various diabetic nephropathy models and small clinical trials.
Some of the data were presented as an abstract at the American Diabetes Association 75th Scientific Sessions in Boston in 2015.
This work was supported in part by the National Science Foundation of China (81170669 to LM, 81200572 to GL, 2011BAI10B00 to XC), JDRF (1-INO-2014-122-A-N to YT, 1-INO-2014-116-A-N to PE) and the National Institutes of Health (1R01DK 091338-01A1 to LC).
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
HW and LK researched the data. YT, JZ, JG, PNE and XC were involved in the experiments and acquisition of data. MK did essential statistical analysis. HW, LM, LC and GL contributed to the initial discussion and design of the project. GL provided compound C66 used in the study. LC, LM, GL, PNE and XC provided funding for this work. HW, PNE and LC wrote the manuscript. LK, YT, PNE, JZ, JG, GL, MK, XC, LM and LC reviewed and revised the manuscript. YT, PNE and XC were involved in discussion of the manuscript. All the authors approve the version to be published. LC and LM are guarantors of this work and, as such, had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.
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