C66 ameliorates diabetic nephropathy in mice by both upregulating NRF2 function via increase in miR-200a and inhibiting miR-21.
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.
Department of Nephrology, the Second Hospital of Jilin University, 218 Ziqiang St, Changchun, Jilin, 130041, People's Republic of China.|Kosair Children's Hospital Research Institute at the Department of Pediatrics, Wendy L. Novak Diabetes Care Center, University of Louisville, 570 S Preston St, Baxter I., Louisville, KY, 40202, USA.
Hao
H
Wu
Department of Nephrology, the First Hospital of Jilin University, Changchun, Jilin, People's Republic of China.|Kosair Children's Hospital Research Institute at the Department of Pediatrics, Wendy L. Novak Diabetes Care Center, University of Louisville, 570 S Preston St, Baxter I., Louisville, KY, 40202, USA.
Lili
L
Kong
Kosair Children's Hospital Research Institute at the Department of Pediatrics, Wendy L. Novak Diabetes Care Center, University of Louisville, 570 S Preston St, Baxter I., Louisville, KY, 40202, USA.|Chinese-American Research Institute for Diabetic Complications, School of Pharmaceutical Science, Wenzhou Medical University, Wenzhou, Zhejiang, People's Republic of China.
Yi
Y
Tan
Kosair Children's Hospital Research Institute at the Department of Pediatrics, Wendy L. Novak Diabetes Care Center, University of Louisville, 570 S Preston St, Baxter I., Louisville, KY, 40202, USA.
Paul N
PN
Epstein
Kosair Children's Hospital Research Institute at the Department of Pediatrics, Wendy L. Novak Diabetes Care Center, University of Louisville, 570 S Preston St, Baxter I., Louisville, KY, 40202, USA.
Jun
J
Zeng
Kosair Children's Hospital Research Institute at the Department of Pediatrics, Wendy L. Novak Diabetes Care Center, University of Louisville, 570 S Preston St, Baxter I., Louisville, KY, 40202, USA.
Junlian
J
Gu
Chemical Biology Research Center, School of Pharmaceutical Science, Wenzhou Medical University, Wenzhou, Zhejiang, People's Republic of China.
Guang
G
Liang
Department of Bioinformatics and Biostatistics, SPHIS, University of Louisville, Louisville, KY, USA.
Maiying
M
Kong
Department of Nephrology, Chinese PLA General Hospital, Beijing, People's Republic of China.
Xiangmei
X
Chen
Department of Nephrology, the Second Hospital of Jilin University, 218 Ziqiang St, Changchun, Jilin, 130041, People's Republic of China. miaolining55@163.com.
Lining
L
Miao
Kosair Children's Hospital Research Institute at the Department of Pediatrics, Wendy L. Novak Diabetes Care Center, University of Louisville, 570 S Preston St, Baxter I., Louisville, KY, 40202, USA. l0cai001@louisville.edu.|Chinese-American Research Institute for Diabetic Complications, School of Pharmaceutical Science, Wenzhou Medical University, Wenzhou, Zhejiang, People's Republic of China. l0cai001@louisville.edu.
Lu
L
Cai
D000818:Animals; D001794:Blood Pressure / Q000235:genetics / Q000502:physiology; D015153:Blotting, Western; D003474:Curcumin / Q000627:therapeutic use*; D003921:Diabetes Mellitus, Experimental / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D003928:Diabetic Nephropathies / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D006579:Heterozygote; D000072019:Kelch-Like ECH-Associated Protein 1 / Q000235:genetics / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D051267:NF-E2-Related Factor 2 / Q000235:genetics / Q000378:metabolism*
D000072019:Kelch-Like ECH-Associated Protein 1; C531950:MIRN21 microRNA, mouse; D035683:MicroRNAs; C543719:Mirn200 microRNA, mouse; D051267:NF-E2-Related Factor 2; C495636:Nfe2l2 protein, mouse; D003474:Curcumin
Albuminuria; Curcumin; Diabetic nephropathy; Fibrosis; KEAP1; NRF2; miR-200; miR-21