Aims/hypothesis As microRNA-21 (miR-21) plays a pathological role in fibrosis, we hypothesised that it may be a therapeutic target for diabetic nephropathy. Methods 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.

The TGF-β/Smad3 pathway plays a major role in tissue fibrosis, but the precise mechanisms are not fully understood. Here we identified microRNA miR-433 as an important component of TGF-β/Smad3-driven renal fibrosis. The miR-433 was upregulated following unilateral ureteral obstruction, a model of aggressive renal fibrosis. In vitro, overexpression of miR-433 enhanced TGF-β1-induced fibrosis, whereas knockdown of miR-433 suppressed this response. Furthermore, Smad3, but not Smad2, bound to the miR-433 promoter to induce its expression. Delivery of an miR-433 knockdown plasmid to the kidney by ultrasound microbubble-mediated gene transfer suppressed the induction and progression of fibrosis in the obstruction model. The antizyme inhibitor Azin1, an important regulator of polyamine synthesis, was identified as a target of miR-433. Overexpression of miR-433 suppressed Azin1 expression, while, in turn, Azin1 overexpression suppressed TGF-β signaling and the fibrotic response. Thus, miR-433 is an important component of TGF-β/Smad3-induced renal fibrosis through the induction of a positive feedback loop to amplify TGF-β/Smad3 signaling, and may be a potential therapeutic target in tissue fibrosis.

Blockade of transforming growth factor-β (TGF-β) signaling by Smad7 gene therapy is known to prevent experimental renal fibrosis. This study investigated whether Smad7 suppresses renal fibrosis via altering the renal expression of fibrosis-related microRNAs. Application of gene therapy into diseased kidneys of obstructive nephropathy and kidney cells by overexpressing Smad7 restored miR-29b but inhibited the expression of miR-192 and miR-21, resulting in blockade of renal fibrosis. Furthermore, Smad7 overexpression also suppressed advanced glycated end products- and angiotensin II-regulated expression of these microRNAs. In contrast, disruption of Smad7 gene in mice demonstrated opposite results by enhancing the loss of miR-29b and upregulation of miR-192 and miR-21, resulting in promotion of renal fibrosis in ligated kidneys of a model of obstructive nephropathy. More importantly, treatment with anti-miR-29b, miR-21 and miR-192 mimics in Smad7 overexpressing tubular epithelial cells abrogated the suppressive function of Smad7 on renal fibrosis, suggesting that these microRNAs act downstream of Smad7 to override the Smad7 function. In conclusion, Smad7 protects kidneys from fibrosis by regulating TGF-β/Smad3-mediated renal expression of miR-21, miR-192, and miR-29b. Restored renal miR-29b but suppressed miR-192 and miR-21 may be a mechanism by which gene therapy with Smad7 inhibits renal fibrosis.

Abstract-Evidence exists that NO plays a role in the vasodilation that occurs during pregnancy. The purpose of the present study was to determine whether the role of NO is associated with an increase in the activity and protein expression of NO synthase (NOS) in the human uterine artery. Uterine arteries were obtained from pregnant patients (P arteries) and nonpregnant patients (NP arteries). NOS activity was estimated with the L-[ 3 H]-arginine-to-L-[ 3 H]-citrulline conversion method and on the basis of changes in tissue levels of cGMP. Western immunoblotting and immunohistochemistry were used to assess NOS protein expression. Ca 2ϩ -dependent NOS activity was 8 times greater (PϽ0.01) in P than in NP arteries. Although most of this pregnancy-induced increase in NOS activity was Ca 2ϩ dependent (64%), a considerable portion was Ca 2ϩ independent. Expressions of endothelial NOS (eNOS) and neuronal NOS, but not inducible NOS, were demonstrated in P and NP arteries. The eNOS was located in the endothelium and stained with a qualitative order of P arteriesϾNP arteries (follicular)ϾNP arteries (luteal). The neuronal NOS was located in the adventitia of P and NP arteries. Basal NO-dependent and bradykinin-stimulated levels of cGMP were higher (PϽ0.05) in P than in NP arteries. These results indicate that an upregulation of eNOS protein expression could account for the increased NO synthesis/release in the human uterine artery during pregnancy. (Circ Res. 2000;87:406-411.)ormal pregnancy is associated with an increase in uterine blood flow and a decrease in uterine vascular resistance. [1][2][3][4] The low resistance is attributed to a loss of smooth muscle in myometrial resistance vessels (spiral arteries and terminations of radial arteries) as well as to dilation of the larger uterine arteries. 5 The dilation of the uterine arteries could be due to an increased role of endogenous vasodilators.Considerable evidence indicates that NO plays a role in pregnancy-induced uterine vasodilation. We have previously reported that acetylcholine is more potent and efficacious in producing dilation of isolated uterine arteries from pregnant than from nonpregnant patients. 6,7 The acetylcholine-induced relaxation was blocked by NO synthase (NOS) inhibitors and thus is apparently mediated by NO. 6,7 Furthermore, pregnancy-induced increases in basal NO production have been found in the uterine vasculature of rats, 8,9 guinea pigs, 10,11 and sheep. 3,12,13 NO is produced by NOS, of which 3 isoforms have been identified: endothelial NOS (eNOS), neuronal NOS (nNOS), and inducible NOS (iNOS). 14 The NOS isoforms share a common overall catalytic scheme for the oxidation of L-arginine to NO and L-citrulline but can be divided into 2 functional classes based on the dependence of Ca 2ϩ for activity. 14 The constitutive forms, eNOS and nNOS, require Ca 2ϩ for activity, but the inducible isoform, iNOS, has a Ca 2ϩ -independent activity. Ca 2ϩ -independent activity for eNOS also has been reported. [15][16][17][18] In the present study, we te...