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...

We have previously shown that transforming growth factor-β/Smad3-dependent miRNAs play a critical role in renal inflammation and fibrosis. However, off-target effects of miRNAs limit their therapeutic application. Recently, emerging roles of long noncoding RNAs (lncRNAs) in diseases have been recognized. In this study, we used high-throughput RNA sequencing to identify the Smad3-dependent lncRNAs related to renal inflammation and fibrosis in Smad3 knockout mouse models of unilateral ureteral obstructive nephropathy and immunologically induced anti-glomerular basement membrane glomerulonephritis. Compared with wild-type mice, 151 lncRNAs in the unilateral ureteral obstructive nephropathy kidney and 413 lncRNAs in kidneys with anti-glomerular basement membrane glomerulonephritis were significantly altered in Smad3 knockout mice. Among them, 21 common lncRNAs were up-regulated in wild-type, but down-regulated in Smad3 knockout, kidneys in both disease models in which progressive renal inflammation and fibrosis were abolished when the Smad3 gene was deleted or suppressed. Real-time PCR confirmed these findings and revealed the functional link between Smad3-dependent lncRNAs np_5318/np_17856 and progressive kidney injury. Results demonstrate that the identification and characterization of functional lncRNAs associated with kidney disease may represent a promising research direction into renal disorder and may lead to the development of new lncRNA therapies for kidney diseases.

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In one event, Chinese male individuals accidentally exposed to unknown chemicals and emerged erythema or blisters on contacted organism derma, then hospitalized. To identify the causative agents, blood, urine and exudate samples were collected from the patients during the therapeutic course. Five established liquid chromatography–mass spectrometry (LC–MS) and gas chromatography (GC)–MS methods were employed to analyze the samples. Here, an overall analysis of four types of sulfur mustard biomarkers, including the hydrolysis/oxidation products, β-lyase metabolites, DNA adducts and hemoglobin adducts, was conducted toward the samples from exposed individuals. The results of all the four types of biomarkers in different biomedical matrices showed high relevance, and verified that this exposure is indeed originated from sulfur mustard. The concentrations of the biomarkers in specimens revealed a good correlation with the severity of the patient's symptom. The concentration-time profile demonstrated that most of the biomarkers quickly achieved maximum at the beginning of the course, and then decreased and kept a detectable level until the 7th day after exposure. The DNA adducts in urine samples still appeared on the 30th day, and the N-terminal valine adducts in hemoglobin could be monitored for over 90 days, which was meaningful for the concurrent study of clinical samples. To the best of our knowledge, this work provides the total analysis and profile of four categories of biomarkers in human specimens for the first time, and the good accordance between concentration and level of burns, between time course and biomarkers will be of great importance for early diagnosis and medical treatment monitoring of sulfur mustard exposure.

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