Rac1 deficiency impairs postnatal development of the renal papilla.

Development of the renal medulla continues after birth to form mature renal papilla and obtain urine-concentrating ability. Here, we found that a small GTPase, Rac1, plays a critical role in the postnatal development of renal papilla. Mice with distal tubule-specific deletion of Rac1 reached adulthood but showed polydipsia and polyuria with an impaired ability to concentrate urine. The elongation of renal papilla that occurs in the first weeks after birth was impaired in the Rac1-deficient infants, resulting in shortening and damage of the renal papilla. Moreover, the osmoprotective signaling mediated by nuclear factor of activated T cells 5, which is a key molecule of osmotic response to osmotic stress in renal medulla, was significantly impaired in the kidneys of the Rac1-deficient infants. These results demonstrate that Rac1 plays an important role in the development of renal papilla in the postnatal period, and suggested a potential link between Rac1 and osmotic response.

Activation of Rac1-Mineralocorticoid Receptor Pathway Contributes to Renal Injury in Salt-Loaded db/db Mice.

[Figure: see text].

Histone methylation and demethylation are implicated in the transient and sustained activation of the interleukin-1ß gene in murine macrophages.

Macrophages play a crucial role in the development of atherosclerosis. To explore the mechanism by which macrophages attain a proinflammatory phenotype for a sustained period, we stimulated macrophages with lipopolysaccharide (LPS) and interferon-γ (IFN-γ) and measured the interleukin-1ß (IL-1ß) expression. The IL-1ß expression increased transiently, and its expression lasted for, at least, 1 week after the cessation of LPS and IFN-γ stimulation. At the promoter region of the IL-1ß gene, the demethylation of histone H3 lysine 27 (H3K27) was significantly induced for 1 week after transient stimulation with LPS and IFN-γ. The expression of H3K27 demethylases ubiquitously transcribed tetratricopeptide repeat, X chromosome (UTX) and jumonji domain-containing 3 (JMJD3) increased significantly for 1 week after transient stimulation with LPS and IFN-γ. When the UTX expression was inhibited by using small interfering RNA (siRNA) for UTX, the IL-1ß expression was significantly suppressed in both transient and sustained phases, whereas siRNA for JMJD3 significantly inhibited only the sustained phase of the IL-1ß expression. These results suggested that H3K27 demethylation was implicated in the transient and sustained increase in the IL-1ß expression after LPS and IFN-γ stimulation.

Salt causes aging-associated hypertension via vascular Wnt5a under Klotho deficiency.

Aging is associated with a high prevalence of hypertension due to elevated susceptibility of BP to dietary salt, but its mechanism is unknown. Serum levels of Klotho, an anti-aging factor, decline with age. We found that high salt (HS) increased BP in aged mice and young heterozygous Klotho-knockout mice and was associated with increased vascular expression of Wnt5a and p-MYPT1, which indicate RhoA activity. Not only the Wnt inhibitor LGK974 and the Wnt5a antagonist Box5 but Klotho supplementation inhibits HS-induced BP elevation, similarly to the Rho kinase inhibitor fasudil, associated with reduced p-MYPT1 expression in both groups of mice. In cultured vascular smooth muscle cells, Wnt5a and angiotensin II (Ang II) increased p-MYPT1 expression but knockdown of Wnt5a with siRNA abolished Ang II-induced upregulation of p-MYPT1, indicating that Wnt5a is indispensable for Ang II-induced Rho/ROCK activation. Notably, Klotho inhibited Wnt5a- and Ang II-induced upregulation of p-MYPT1. Consistently, Klotho supplementation ameliorated HS-induced augmentation of reduced renal blood flow (RBF) response to intra-arterial infusion of Ang II and the thromboxane A2 analog U46619, which activated RhoA in both groups of mice and were associated with the inhibition of BP elevation, suggesting that abnormal response of RBF to Ang II contributes to HS-induced BP elevation. Thus, Klotho deficiency underlies aging-associated salt-sensitive hypertension through vascular non-canonical Wnt5a/RhoA activation.

Mineralocorticoid receptor blockade suppresses dietary salt-induced ACEI/ARB-resistant albuminuria in non-diabetic hypertension: a sub-analysis of evaluate study.

Excessive dietary salt intake can counteract the renoprotective effects of renin-angiotensin system (RAS) blockade in hypertensive patients with chronic kidney disease (CKD). In rodents, salt loading induces hypertension and renal damage by activating the mineralocorticoid receptor (MR) independently of plasma aldosterone levels. Thus, high salt-induced resistance to RAS blockade may be mediated by MR activation. To test this, a post hoc analysis of the Eplerenone Combination Versus Conventional Agents to Lower Blood Pressure on Urinary Antialbuminuric Treatment Effect (EVALUATE) trial was conducted. Thus, 304 non-diabetic hypertensive patients on RAS-blocking therapy were divided into tertiles according to salt intake (estimated 24-h urinary sodium excretion at baseline) and compared in terms of percent reduction in urinary albumin-to-creatinine ratio (UACR) at 52 weeks relative to baseline. The eplerenone-treated patients in the highest sodium excretion tertile exhibited significantly greater reduction in UACR than the placebo subjects in the same tertile (-22.5% vs. +21.8%, p = 0.02). This disparity was not observed in the lowest (-10.2% vs. -0.84%, p = 0.65) or middle (-19.5% vs. +9.5%, p = 0.22) tertiles. Similar systolic blood pressure changes were observed. In the whole cohort, reduction in UACR correlated positively with reduction in systolic blood pressure (r2 = 0.04, p = 0.02). These results support the hypothesis that excessive salt intake can enhance resistance to RAS blockade by activating MR. They also suggest that eplerenone plus RAS blockade may be effective for CKD in hypertensive patients, especially those with excessive salt intake.

Aberrant DNA methylation of hypothalamic angiotensin receptor in prenatal programmed hypertension.

Maternal malnutrition, which causes prenatal exposure to excessive glucocorticoid, induces adverse metabolic programming, leading to hypertension in offspring. In offspring of pregnant rats receiving a low-protein diet or dexamethasone, a synthetic glucocorticoid, mRNA expression of angiotensin receptor type 1a (Agtr1a) in the paraventricular nucleus (PVN) of the hypothalamus was upregulated, concurrent with reduced expression of DNA methyltransferase 3a (Dnmt3a), reduced binding of DNMT3a to the Agtr1a gene, and DNA demethylation. Salt loading increased BP in both types of offspring, suggesting that elevated hypothalamic Agtr1a expression is epigenetically modulated by excessive glucocorticoid and leads to adult-onset salt-sensitive hypertension. Consistent with this, dexamethasone treatment of PVN cells upregulated Agtr1a, while downregulating Dnmt3a, and decreased DNMT3a binding and DNA demethylation at the Agtr1a locus. In addition, Dnmt3a knockdown upregulated Agtr1a independently of dexamethasone. Hypothalamic neuron-specific Dnmt3a-deficient mice exhibited upregulation of Agtr1a in the PVN and salt-induced BP elevation without dexamethasone treatment. By contrast, dexamethasone-treated Agtr1a-deficient mice failed to show salt-induced BP elevation, despite reduced expression of Dnmt3a. Thus, epigenetic modulation of hypothalamic angiotensin signaling contributes to salt-sensitive hypertension induced by prenatal glucocorticoid excess in offspring of mothers that are malnourished during pregnancy.

Aberrant DNA methylation of Tgfb1 in diabetic kidney mesangial cells.

Epigenetic modulation may underlie the progression of diabetic nephropathy (DN). Involvement of TGFB1 in mesangial fibrosis of DN led us to hypothesize that Tgfb1 DNA demethylation contributes to progression of DN. In primary mesangial cells from diabetic (db/db) mouse kidneys, demethylation of Tgfb1 DNA and upregulation of Tgfb1 mRNA progressed simultaneously. USF1 binding site in Tgfb1 promoter region were demethylated, and binding of USF1 increased, with decreased binding of DNMT1 in db/db compared with control. Given downregulation of Tgfb1 expression by folic acid, antioxidant Tempol reversed DNA demethylation, with increased and decreased recruitment of DNMT1 and USF1 to the promoter, resulting in decreased Tgfb1 expression in db/db mice. Addition of H2O2 to mesangial cells induced DNA demethylation and upregulated Tgfb1 expression. Finally, Tempol attenuated mesangial fibrosis in db/db mice. We conclude that aberrant DNA methylation of Tgfb1 due to ROS overproduction play a key to mesangial fibrosis during DN progression.

Endogenous Interleukin-1ß Is Implicated in Intraplaque Hemorrhage in Apolipoprotein E Gene Null Mice.

BACKGROUND: Intraplaque hemorrhage (IPH) has been implicated in plaque instability and rupture in atherosclerotic lesions, although the mechanisms by which IPH progresses remain largely unknown. In this study, apolipoprotein E-deficient mice with carotid artery ligation and cuff placement around the artery were used, and pro-inflammatory cytokines that are implicated in IPH were analyzed.Methods and Results:The expression of interleukin-1ß (IL-1ß) increased significantly following cuff placement compared with mice with carotid artery ligation alone. IPH occurred in the cuff-placed carotid artery following treatment with the negative control (NC) small interfering RNA (siRNA). However, the occurrence was significantly reduced in the cuff-placed carotid artery following treatment with an IL-1ß siRNA. Neovessel formation was significantly reduced in the carotid artery treated with the NC siRNA compared with that treated with IL-1ß siRNA. IL-1ß significantly inhibited the tube formation and wound healing capacities of vascular endothelial cells in vitro. Furthermore, immunostaining of matrix metalloproteinase-9 (MMP-9) significantly increased in the carotid artery treated with the NC siRNA compared with that treated with IL-1ß siRNA. CONCLUSIONS: These results suggest that endogenous IL-1ß is implicated in the progression of IPH via the inhibition of physiological angiogenesis in the atherosclerotic plaque, leading to the formation of leaky neovessels. Furthermore, the stimulation of MMP-9 expression may also contribute to the formation of leaky neovessels.

Therapeutic Effects of Mesenchymal Stem Cell-Derived Exosomes in Cardiovascular Disease.

Mesenchymal stem cells (MSCs) are multipotent stem cells that reside in various organs. They have the capacity to differentiate into various cell types, including cardiomyocytes, vascular endothelial cells, and vascular smooth muscle cells. Among the various MSCs, bone marrow-derived MSCs (BMMSCs) have been widely used for treating acute myocardial infarction (AMI) and ischemic heart failure (IHF) in preclinical and clinical studies. Although the beneficial effects of BMMSCs in treating AMI and IHF were originally attributed to their capacity to differentiate into cardiac cell types, recent evidence suggests that the differentiation capacity of BMMSCs appears to be minimal and that BMMSCs exert cardioprotective effects by secreting paracrine factors. In this context, MSC-derived exosomes have recently gained much attention. In this chapter, we introduce preclinical studies in which MSC-derived exosomes are used for treating cardiovascular diseases (CVDs) such as AMI, stroke, pulmonary hypertension, and septic cardiomyopathy. Future clinical studies are required to confirm the efficacy of exosome administration in treating CVDs.

Stem cell-derived exosomes as a therapeutic tool for cardiovascular disease.

Mesenchymal stem cells (MSCs) have been used to treat patients suffering from acute myocardial infarction (AMI) and subsequent heart failure. Although it was originally assumed that MSCs differentiated into heart cells such as cardiomyocytes, recent evidence suggests that the differentiation capacity of MSCs is minimal and that injected MSCs restore cardiac function via the secretion of paracrine factors. MSCs secrete paracrine factors in not only naked forms but also membrane vesicles including exosomes containing bioactive substances such as proteins, messenger RNAs, and microRNAs. Although the details remain unclear, these bioactive molecules are selectively sorted in exosomes that are then released from donor cells in a regulated manner. Furthermore, exosomes are specifically internalized by recipient cells via ligand-receptor interactions. Thus, exosomes are promising natural vehicles that stably and specifically transport bioactive molecules to recipient cells. Indeed, stem cell-derived exosomes have been successfully used to treat cardiovascular disease (CVD), such as AMI, stroke, and pulmonary hypertension, in animal models, and their efficacy has been demonstrated. Therefore, exosome administration may be a promising strategy for the treatment of CVD. Furthermore, modifications of exosomal contents may enhance their therapeutic effects. Future clinical studies are required to confirm the efficacy of exosome treatment for CVD.

miR-133 regulates Evi1 expression in AML cells as a potential therapeutic target.

The Ecotropic viral integration site 1 (Evi1) is a zinc finger transcription factor, which is located on chromosome 3q26, over-expression in some acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Elevated Evi1 expression in AML is associated with unfavorable prognosis. Therefore, Evi1 is one of the strong candidate in molecular target therapy for the leukemia. MicroRNAs (miRNAs) are small non-coding RNAs, vital to many cell functions that negatively regulate gene expression by translation or inducing sequence-specific degradation of target mRNAs. As a novel biologics, miRNAs is a promising therapeutic target due to its low toxicity and low cost. We screened miRNAs which down-regulate Evi1. miR-133 was identified to directly bind to Evi1 to regulate it. miR-133 increases drug sensitivity specifically in Evi1 expressing leukemic cells, but not in Evi1-non-expressing cells The results suggest that miR-133 can be promising therapeutic target for the Evi1 dysregulated poor prognostic leukemia.

Adult stem cells as a tool for kidney regeneration.

Kidney regeneration is a challenging but promising strategy aimed at reducing the progression to end-stage renal disease (ESRD) and improving the quality of life of patients with ESRD. Adult stem cells are multipotent stem cells that reside in various tissues, such as bone marrow and adipose tissue. Although intensive studies to isolate kidney stem/progenitor cells from the adult kidney have been performed, it remains controversial whether stem/progenitor cells actually exist in the mammalian adult kidney. The efficacy of mesenchymal stem cells (MSCs) in the recovery of kidney function has been demonstrated in animal nephropathy models, such as acute tubular injury, glomerulonephritis, renal artery stenosis, and remnant kidney. However, their beneficial effects seem to be mediated largely via their paracrine effects rather than their direct differentiation into renal parenchymal cells. MSCs not only secrete bioactive molecules directly into the circulation, but they also release various molecules, such as proteins, mRNA, and microRNA, in membrane-covered vesicles. A detailed analysis of these molecules and an exploration of the optimal combination of these molecules will enable the treatment of patients with kidney disease without using stem cells. Another option for the treatment of patients with kidney disease using adult somatic cells is a direct/indirect reprogramming of adult somatic cells into kidney stem/progenitor cells. Although many hurdles still need to be overcome, this strategy will enable bona fide kidney regeneration rather than kidney repair using remnant renal parenchymal cells.

Adipose tissue-derived stem cells as a therapeutic tool for cardiovascular disease.

Adipose tissue-derived stem cells (ADSCs) are adult stem cells that can be easily harvested from subcutaneous adipose tissue. Many studies have demonstrated that ADSCs differentiate into vascular endothelial cells (VECs), vascular smooth muscle cells (VSMCs), and cardiomyocytes in vitro and in vivo. However, ADSCs may fuse with tissue-resident cells and obtain the corresponding characteristics of those cells. If fusion occurs, ADSCs may express markers of VECs, VSMCs, and cardiomyocytes without direct differentiation into these cell types. ADSCs also produce a variety of paracrine factors such as vascular endothelial growth factor, hepatocyte growth factor, and insulin-like growth factor-1 that have proangiogenic and/or antiapoptotic activities. Thus, ADSCs have the potential to regenerate the cardiovascular system via direct differentiation into VECs, VSMCs, and cardiomyocytes, fusion with tissue-resident cells, and the production of paracrine factors. Numerous animal studies have demonstrated the efficacy of ADSC implantation in the treatment of acute myocardial infarction (AMI), ischemic cardiomyopathy (ICM), dilated cardiomyopathy, hindlimb ischemia, and stroke. Clinical studies regarding the use of autologous ADSCs for treating patients with AMI and ICM have recently been initiated. ADSC implantation has been reported as safe and effective so far. Therefore, ADSCs appear to be useful for the treatment of cardiovascular disease. However, the tumorigenic potential of ADSCs requires careful evaluation before their safe clinical application.

Chronic kidney disease and erectile dysfunction.

Erectile dysfunction (ED) is a common condition among male chronic kidney disease (CKD) patients. Its prevalence is estimated to be approximately 80% among these patients. It has been well established that the production of nitric oxide from the cavernous nerve and vascular endothelium and the subsequent production of cyclic GMP are critically important in initiating and maintaining erection. Factors affecting these pathways can induce ED. The etiology of ED in CKD patients is multifactorial. Factors including abnormalities in gonadal-pituitary system, disturbance in autonomic nervous system, endothelial dysfunction, anemia (and erythropoietin deficiency), secondary hyperparathyroidism, drugs, zinc deficiency, and psychological problems are implicated in the occurrence of ED. An improvement of general conditions is the first step of treatment. Sufficient dialysis and adequate nutritional intake are necessary. In addition, control of anemia and secondary hyperparathyroidism is required. Changes of drugs that potentially affect erectile function may be necessary. Further, zinc supplementation may be necessary when zinc deficiency is suspected. Phosphodiesterase type 5 inhibitors (PDE5Is) are commonly used for treating ED in CKD patients, and their efficacy was confirmed by many studies. Testosterone replacement therapy in addition to PDE5Is may be useful, particularly for CKD patients with hypogonadism. Renal transplantation may restore erectile function. ED is an early marker of cardiovascular disease (CVD), which it frequently precedes; therefore, it is crucial to examine the presence of ED in CKD patients not only for the improvement of the quality of life but also for the prevention of CVD attack.

Adipose-derived stem cells stimulate reendothelialization in stented rat abdominal aorta.

BACKGROUND: Although drug-eluting stents (DES) have been widely used for the treatment of coronary artery disease, they potentially increase the risk of late thrombosis. It is, therefore, desirable to establish a strategy to stimulate reendothelialization. Endothelial injury models have been widely used to analyze the mechanisms of coronary restenosis. However, animal models deployed with coronary stents in the blood vessels are necessary to accurately analyze the mechanisms of coronary restenosis and late thrombosis because persistent inflammation occurs around the coronary stents. METHODS AND RESULTS: Coronary stents were implanted into rat abdominal aorta and adipose tissue-derived stem cells (ASC) were administered from the adventitial side. Reendothelialization was then visualized by Evans blue staining, and neointimal formation was analyzed histologically. ASC significantly stimulated reendothelialization and inhibited neointimal formation in bare metal stents (BMS)-implanted aorta. In addition, ASC promoted reendothelialization in DES-implanted aorta; however, the effects were weaker than in BMS-implanted aorta. Among the cytokines that ASC produce, adrenomedullin (AM) significantly stimulated reendothelialization and inhibited neointimal formation in BMS-implanted aorta, when an adenovirus expressing AM was administered from the adventitial side. CONCLUSIONS: These results suggest that ASC produce several cytokines that stimulate reendothelialization and inhibit neointimal formation in stent-deployed vessels, and that AM could mediate these effects.

A useful method of identifying of miRNAs which can down-regulate Zeb-2.

BACKGROUND: Although identification of the target mRNAs of micro RNAs (miRNAs) is essential to understanding their function, the low complementarity between miRNAs and their target mRNAs has complicated this process. In this study, we sought to identify miRNAs which reduce the expression of the transcription factor Zeb-2, a transcriptional repressor of E-cadherin which is known to be down regulated by members of the miR-200 family (miR-200a,b,c miR-429, and miR-141). FINDINGS: We first used a computational target predicting system to identify 82 candidate miRNAs which bound the 3'UTR region of the Zeb-2 mRNA. Of these 82 miRNAs, precursors for 51 were available in our miRNA precursor library. Pre-miR™ Precursor Molecules for these 51 miRNAs were co-transfected into NIH3T3 cells with a luciferase reporter vector containing the 3'UTR region of the Zeb-2 mRNA. Seven miRNAs (miR-141, mi-183, miR-200a, miR-200b, miR-200c, miR-429 and miR-666-5p) were shown to down-regulate luciferase activity and Western blotting analysis confirmed that Pre-miR™ Precursor Molecules for these seven miRNAs induced expression of E-cadherin and miScript target protector against miR-183 and miR-666-5p abrogated this effect. Moreover, an Anti-miR™ miRNA Inhibitor targeting miR-183 and miR-666-5p repressed expression of E-cadherin. CONCLUSIONS: We have established a method to identify miRNAs that bind the 3'UTR region of the Zeb-2 mRNA and that induce expression of E-cadherin, possibly by down-regulating the expression of Zeb-2. Our method may be more widely applicable for identifying miRNAs that bind target mRNA 3'UTR regions and down-regulate the expression of proteins encoded by these mRNAs.

Oncogene- and oxidative stress-induced cellular senescence shows distinct expression patterns of proinflammatory cytokines in vascular endothelial cells.

Senescent cells are metabolically active and produce a variety of proinflammatory cytokines. It was previously reported that atherosclerotic plaques contain senescent cells, suggesting that senescence may contribute to the progression of atherosclerosis. In this study, we induced cellular senescence in vascular endothelial cells (VECs) using hydrogen peroxide (H2O2) or an adenovirus that expresses a constitutively active mutant of Ras (AdRas12V) and studied the expression of cytokines. Both H2O2 treatment and AdRas12V infection induced senescence in VECs, as assessed by senescence-associated ß-Gal activity and the expression of proteins such as p53 and p21(CIP1). In addition, both treatments induced the expression of a variety of cytokines, including interleukin-1ß (IL-1ß) and nerve growth factor (NGF). AdRas12V infection induced IL-1ß expression more significantly than H2O2 treatment, whereas both treatments induced comparable mRNA and protein expression levels of NGF. These results suggest that senescent cells express different patterns of proinflammatory cytokines, depending on the trigger that induced senescence. It is therefore possible that senescent cells can differentially induce inflammation in the surrounding tissues, depending on the cause of senescence.

Angiopoietin-1 mediates adipose tissue-derived stem cell-induced inhibition of neointimal formation in rat femoral artery.

BACKGROUND: Adipose tissue-derived stem cells (ASC) produce a variety of cytokines that potentially mediate the proangiogenic and antiapoptotic effects of the ASC. We examined whether ASC produced angiopoietin-1 (Ang1) and whether Ang1 functionally mediated ASC-induced suppression of neointimal formation. METHODS AND RESULTS: Ang1 production was measured by enzyme-linked immunosorbent assay. Production of endogenous Ang1 by ASC was inhibited with small interfering RNA (siRNA) for Ang1. Overproduction of Ang1 was achieved with an adenovirus that expresses Ang1 (AdAng1). ASC expressing Ang1 siRNA, or AdAng1 were administered around the femoral artery after wire injury, and immunohistochemical analysis was performed to examine their effects on neointimal formation. ASC produced Ang1 in a time-dependent manner, especially when cultured in medium containing growth factors for vascular endothelial cells. When ASC were treated with Ang1 siRNA, the inhibitory effect of ASC on neointimal formation was significantly reduced. Knockdown of Ang1 significantly increased macrophage infiltration in the neointima, and significantly decreased endothelial regeneration. In contrast, forced expression of Ang1 using AdAng1 significantly suppressed neointimal formation and macrophage infiltration, and stimulated reendothelialization. CONCLUSIONS: Ang1 was implicated in ASC-induced suppression of neointimal formation. The results also suggested that Ang1 inhibited neointimal formation via stimulation of reendothelialization and suppression of macrophage infiltration in the neointima.

Renoprotective effect of erythropoietin in ischemia/reperfusion injury: possible roles of the Akt/endothelial nitric oxide synthase-dependent pathway.

OBJECTIVES: It has been reported that erythropoietin protects the kidneys from ischemia/reperfusion injury. In the present study, we examined the role of Akt and endothelial nitric oxide synthase in the protective effect of erythropoietin on ischemia/reperfusion injury of the kidney. METHODS: Erythropoietin was injected in the peritoneal space of ICR mice after ischemia/reperfusion injury and its effect was assessed by measuring blood urea nitrogen and creatinine, and by histological analysis. Phosphorylation of Akt and endothelial nitric oxide synthase was examined by western blot analysis. Endothelial nitric oxide synthase gene null mice were also used to examine the role of endothelial nitric oxide synthase in the renoprotective effect of erythropoietin. RESULTS: Erythropoietin administration significantly inhibited the increase in blood urea nitrogen and creatinine after ischemia/reperfusion injury compared with control mice. Accordingly, erythropoietin administration significantly ameliorated the histological damages, including apoptotic cell death. Erythropoietin significantly stimulated phosphorylation of Akt and endothelial nitric oxide synthase in the kidneys. When endothelial nitric oxide synthase gene null mice were subjected to ischemia/reperfusion injury, erythropoietin did not significantly suppress the increase in blood urea nitrogen or creatinine. CONCLUSIONS: Erythropoietin seems to activate the Akt/endothelial nitric oxide synthase-dependent pathway in the kidneys. This pathway might be implicated in the renoprotective effect of erythropoietin in the ischemia/reperfusion injury model.

miR-200b precursor can ameliorate renal tubulointerstitial fibrosis.

Members of the miR-200 family of micro RNAs (miRNAs) have been shown to inhibit epithelial-mesenchymal transition (EMT). EMT of tubular epithelial cells is the mechanism by which renal fibroblasts are generated. Here we show that miR-200 family members inhibit transforming growth factor-beta (TGF-beta)-induced EMT of tubular cells. Unilateral ureter obstruction (UUO) is a common model of EMT of tubular cells and subsequent tubulointerstitial fibrosis. In order to examine the role of miR-200 family members in tubulointerstitial fibrosis, their expression was investigated in the kidneys of UUO mice. The expression of miR-200 family miRNAs was increased in a time-dependent manner, with induction of miR-200b most pronounced. To clarify the effect of miR-200b on tubulointerstitial fibrosis, we injected miR-200b precursor intravenously. A single injection of 0.5 nM miR-200b precursor was sufficient to inhibit the increase of collagen types I, III and fibronectin in obstructed kidneys, and amelioration of fibrosis was confirmed by observation of the kidneys with Azan staining. miR-200 family members have been previously shown to inhibit EMT by reducing the expression of ZEB-1 and ZEB-2 which are known repressors of E-cadherin. We demonstrated that expression of ZEB-1 and ZEB-2 was increased after ureter obstruction and that administration of the miR-200b precursor reversed this effect. In summary, these results indicate that miR-200 family is up-regulated after ureter obstruction, miR-200b being strongly induced, and that miR-200b ameliorates tubulointerstitial fibrosis in obstructed kidneys. We suggest that members of the miR-200 family, and miR-200b specifically, might constitute novel therapeutic targets in kidney disease.