Mechanisms of miR-29a-5p involvement in osteogenic phenotype transformation and cellular regulation of vascular smooth muscle and thus influencing calcification in VSMCs in chronic kidney disease.

Vascular calcification is one of the major complications of chronic kidney disease (CKD), which could be further accelerated by the osteogenic transition and apoptosis of smooth muscle cells, thereby advancing the progression of renal diseases and increasing the mortality rate of cardiovascular events. MicroRNA is a kind of key regulator in the phenotypic transition of vascular smooth muscle cells (VSMCs), but its role remains unclear in VSMCs. In this study, VSMCs were stimulated by platelet-derived growth factors - BB (PDGF-BB) in varying concentrations to establish the VSMC dysfunction models. The relative expression of miR-29a-5p was quantified via the quantitative real-time polymerase chain reaction (qRT-PCR). The proliferation of VSMCs was determined via the BrdU method, analysis of cell cycle via flow cytometry, and the migration of VSMCs via Transwell assay. Expression of γ-secretase activating protein (GSAP) and markers of VSMC differentiation, including α-SMA, SM-22α, SMMHC and Calponin, was quantified via the Western blot. The targeting relationship between the 3'-UTR of miR-29a-5p and GSAP was validated through the dual-luciferase reporter gene assay. As a result, we found that PDGF-BB could trigger a decrease of miR-29a-5p in a time- and dose-dependent manner (P < 0.05). Overexpression of miR-29a-5p could curb the effect of PDGF-BB on the proliferation and migration of VSMCs while upregulating the expression of markers of differentiation (P < 0.05). In addition, the expression of GSAP was also affected by the negative regulation of miR-29a-5p, while the restoration of GSAP eliminated the effect of miR-29a-5p on the VSMCs partially (P < 0.05). Moreover, vascular calcification models were also established in the CKD rats, suggesting that the inhibition of GSAP could prevent PTH-induced vascular calcification in CKD rats. In conclusion, miR-29a-5p could inhibit the PDGF-BB-induced proliferation, migration and phenotypic transition of VSMCs via targeting GSAP. Thus, miR-29a-5p/GSAP might be a potential target for the treatment of vascular calcification.

Radix Rehmannia Glutinosa inhibits the development of renal fibrosis by regulating miR-122-5p/PKM axis.

OBJECTIVE: It is acknowledged that Radix Rehmanniae Praeparata (RR) can regulate hormone metabolism, reduce blood glucose, resist aging, help to sedate patients and promote diuresis. The study aims to investigate the mechanism of how RR influences the development of renal fibrosis by regulating the miR-122-5p/PKM axis. METHODS: Unilateral ureteral obstruction (UUO) was applied to induce renal fibrosis in mice in vivo, and human tubular epithelial HK2 cells treated by transforming growth factor-ß (TGF-ß1) were used to induce renal fibrosis in vitro. Interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α) in mouse serum were detected by Enzyme-linked immunosorbent assay (ELISA); fibronectin (FN) and type I collagen (Col-I) in renal tissue were detected by Western blotting; serum creatinine (Cr) and blood urea nitrogen (BUN) were analyzed by kits. Hematoxylin-eosin (HE) staining and Masson staining were utilized to assess the degree of pathological damage and fibrosis. Cell viability and apoptosis in the in vitro model were detected by MTT and Flow cytometry. Dual-luciferase reporter assay was performed to determine intermolecular targeting relationships. RESULTS: RR could inhibit IL-6 and TNF-α levels, decrease the levels of FN and Col-I and improve the renal function indexes (serum Cr and BUN) in UUO mice (all P<0.05). In addition, RR was able to promote the up-regulation of miR-122-5p expression in UUO mice in vivo (P<0.05). MiR-122-5p expression was down-regulated and PKM expression was up-regulated in HK2 cells treated with TGF-ß1 (all P<0.05). RR inhibited renal fibrosis progression by regulating the miR-122-5p/PKM axis. Inhibition of miR-122-5p or overexpression of PKM could promote apoptosis of TGF-ß1-treated HK2 cells, inhibit their viability, aggravate fibrosis, and attenuate the protective effect of RR on the cells. The protective effect of RR promoted by overexpression of miR-122-5p was partially counteracted by PKM. CONCLUSION: RR can inhibit renal fibrosis progression by regulating the miR-122-5p/PKM axis.

MicroRNA-363-3p Inhibits the Expression of Renal Fibrosis Markers in TGF-ß1-Treated HK-2 Cells by Targeting TGF-ß2.

This study aimed to explore the role of miR-363-3p in renal fibrosis (RF) in vitro. HK-2 cells were treated with transforming growth factor (TGF)-ß1 for 72 h to establish an in vitro model of RF. Subsequently, western blot analysis and reverse transcription-quantitative PCR were used to detect the protein and mRNA expression levels of RF markers in TGF-ß1-treated HK-2 cells, respectively. The results showed that the protein and mRNA expression levels of TGF-ß2, α-smooth muscle actin (SMA), fibronectin, vimentin, collagen II and N-cadherin were increased, while the protein and mRNA expression levels of E-cadherin were decreased in TGF-ß1-treated HK-2 cells. The level of miR-363-3p was significantly decreased in TGF-ß1-treated HK-2 cells. TargetScan indicated that TGF-ß2 was a direct target gene for miR-363-3p, which was further verified using dual luciferase reporter gene assays. Further analyses revealed that the increased protein and mRNA expression levels of TGF-ß2, α-SMA, fibronectin, vimentin, collagen II, N-cadherin, increased phosphorylated-Smad3 protein level, and decreased E-cadherin protein and mRNA expression in TGF-ß1-treated HK-2 cells were significantly reversed by miR-363-3p mimics. However, all the effects were suppressed by a TGF-ß2-plasmid. The results suggested that miR-363-3p plays a protective role in RF by regulating the TGF-ß2/Smad3 signaling pathway.

[Study on quality control and fingerprint of Chinese traditional medicine Yinhuang oral liquid].

The chromatographic fingerprint of Chinese traditional medicine, Yinhuang Oral Liquid together with its original materials Radix Scutellariae and Flos lonicera was established by high performance liquid chromatography (HPLC). At the same time, baicalin and chlorogenic acid in the Yinhuang Oral Liquid were also quantitatively determined. The analysis was performed on a Lichrospher C18 column (250 mm x 4.6 mm i. d. , 5 microm) and with gradient elution. The optimum conditions were explored. The mobile phase A was methanol and the mobile phase B was 0.1% H3PO4 aqueous solution. The wavelength of 254 nm was selected for detection. Finally, the data of fingerprints of 6 samples of Yinhuang Oral Liquid were processed with two different mathematic methods. This proposed method demonstrated good stability for the 6 samples of Yinhuang Oral Liquid. In the comparison of the common peaks in chromatogram of Yinhuang Oral Liquid with those of crude herbs, most of them could be matched well.

[Elevated plasma concentration of IP-10 in patients with type 2 diabetes mellitus].

Recent studies have shown the important role of proinflammatory cytokines and chemokines in the pathogenesis of atherosclerosis and diabetes mellitus(DM). Interferon-inducible protein of 10 kD (IP-10/ CXCL10), a member of the C-X-C chemokine superfamily, is a potent chemoattractant for activated T lymphocytes and is reported to be involved in various disease states including atheroma plaque formation, inhibition of tumor angiogenesis and maintenance of podocyte function. However, the involvement of IP-10 in type 2 DM, especially in its vascular and renal complications, is largely unknown. To elucidate the etiopathological role of IP-10 in type 2 DM, we measured the concentrations of IP-10 together with IFN-gamma, TNF-alpha, IL-18, IL-6 and MCP-1 in plasma samples from 103 type 2 DM patients with various degrees of nephropathy. A significant difference in the plasma level of IP-10 was observed between the patients and the control subjects (183.3+/-12.5 pg/m/ vs 65.6+/-9.3 pg/ml, p<0.05). IP-10 correlated IL-18, IL-6, TNF-alpha and MCP-1. The IFN-gamma level was below the detectable range. IP-10 levels became higher with the progression of nephropathy : IP-10 levels were 148.9+/-14.5, 174.2+/-17.2 and 231.9+/-31.3 pg/m/ in patients with an urinary albumin creatinine ratio of <30, 30 to 300 and >300 microg/mg Cr, respectively. Similarly, IL-18, IL-6, MCP-1 and TNF-alpha levels in patients with overt albuminuria were significantly higher as compared with those without albuminuria (IL-18, 367.3 45.6 vs 203.5+/-17.6 pg/ml; IL-6, 1.61+/-0.26 vs 0.87+/-0.13 pg/ml; TNF-alpha, 1.83+/-0.48 vs 0.61+/-0.07 pg/ml; p<0.05, respectively) in consistent with previous reports. These results suggested that IP-10 may have an etiopathogenic role in type2 DM and diabetic nephropathy as one of the downstream effectors of proinflammatory cytokines.

Endothelium-derived nitric oxide modulates vascular action of aldosterone in renal arteriole.

We have recently demonstrated that aldosterone causes nongenomic vasoconstriction by activating phospholipase C (PLC) in the preglomerular afferent arteriole (Af-Art). In the present study, we tested the hypothesis that endothelium modulates this vasoconstrictor action by releasing nitric oxide (NO). In addition, to study the post-PLC mechanism, we examined possible contributions of phosphoinositol hydrolysis products. Rabbit Af-Arts were microperfused at 60 mm Hg in vitro, and increasing doses of aldosterone (10(-10) to 10(-8) mol/L) were added to the bath and lumen. Aldosterone caused dose-dependent vasoconstriction (within 10 minutes); significant (P<0.01) constriction was observed from 5x10(-9) mol/L, and at 10(-8) mol/L, intraluminal diameter decreased by 29%+/-3% (n=9). Disrupting the endothelium augmented vasoconstriction; significant constriction was observed from 10(-10) mol/L, and at 10(-8) mol/L, the diameter decreased by 38%+/-2% (n=6). NO synthesis inhibition reproduced this augmentation (n=7). Pretreatment with chelerythrine (10(-6) mol/L), a protein kinase C (PKC) inhibitor, slightly attenuated the constriction; aldosterone at 10(-8) mol/L now decreased the diameter by 18%+/-3% (n=7). However, in Af-Arts treated with thapsigargin (10(-6) mol/L) or dantrolene (3x10(-5) mol/L), which blocks inositol 1,4,5-triphosphate (IP3)-induced intracellular calcium release, aldosterone at 10(-8) mol/L decreased the diameter by only 9%+/-1% (n=6) or 9%+/-2% (n=5), respectively. These results demonstrate that in the Af-Art endothelium-derived NO modulates vasoconstrictor actions of aldosterone that are mediated by the activation of both IP3 and PKC pathways. Such vasoconstrictor actions of aldosterone may contribute to the development or aggravation of hypertension by elevating renal vascular resistance in cardiovascular diseases associated with endothelium dysfunction.

Nongenomic vascular action of aldosterone in the glomerular microcirculation.

Aldosterone (Aldo) accelerates hypertension, proteinuria, and glomerulosclerosis in animal models of malignant hypertension or chronic renal failure. Aldo may exert these deleterious renal effects by elevating renal vascular resistance and glomerular capillary pressure. To test this possibility, directly examined were the action of Aldo on the afferent (Af) and efferent (Ef) arterioles (Arts). Examined were the effect of Aldo added to both the bath and lumen on the intraluminal diameter (measured at the most responsive point) of rabbits. Aldo caused dose-dependent constriction in both arterioles with a higher sensitivity in Ef-Arts. Vasoconstrictor action of Aldo was not affected by a mineralocorticoid receptor antagonist spironolactone and was reproduced by membrane-impermeable albumin-conjugated Aldo, suggesting that the vasoconstrictor actions are nongenomic. Pretreatment with neomycin (a specific inhibitor of phospholipase C) abolished the vasoconstrictor action of Aldo in both arterioles. In addition, the vasoconstrictor action of Aldo on Af-Arts was inhibited by both nifedipine and efonidipine, whereas that on Ef-Arts was inhibited by efonidipine but not nifedipine. The results demonstrate that Aldo causes nongenomic vasoconstriction by activating phospholipase C with a subsequent calcium mobilization thorough L- or T-type voltage-dependent calcium channels in Af- or Ef-Arts, respectively. These vasoconstrictor actions on the glomerular microcirculation may play an important role in the pathophysiology and progression of renal diseases by elevating renal vascular resistance and glomerular capillary pressure.

Effects of exercise training on glomerular structure in fructose-fed spontaneously hypertensive rats.

A high-fructose diet (HFD) has been shown to elevate blood pressure (BP) and to decrease insulin sensitivity in rats. Although running exercise can attenuate these phenomena, its effect on target organ protection is not clear. We investigated whether exercise training has renal protective effects in this model. Nine-week-old spontaneously hypertensive rats were allocated to groups that received HFD or a control diet (control group) for 15 weeks. At the age of 10 weeks, fructose-fed rats were allocated to groups that were given vehicle (FRU group), temocapril, an angiotensin converting enzyme inhibitor (TEM group), exercise training (EX group; treadmill running), or temocapril plus exercise training (TEM+EX group). BP was higher in the FRU group than in the control group. Exercise training tended to decrease BP and temocapril treatment decreased BP significantly. Proteinuria was similar in the five groups. Plasma leptin concentration and epididymal fat weight were lower in the EX and TEM+EX groups than in the FRU group. In the soleus muscle of the FRU group, the composite ratio of type I fiber was decreased and that of type IIa fiber was increased compared with those in the control group. Both temocapril and exercise training restored these ratios. The glomerular sclerosis index (GSI) was higher in the FRU group than in the control group. GSI was decreased equally in the TEM, EX, and TEM+EX groups and was positively correlated with plasma leptin concentration. The results suggest that exercise training ameliorates glomerular sclerosis through mechanisms other than a reduction in BP.

Chronic angiotensin-converting enzyme inhibition and angiotensin II antagonism in rats with chronic renal failure.

The current study was undertaken to compare the organ protective effects of an angiotensin-converting enzyme inhibitor, temocapril, with those of an angiotensin II type 1 receptor antagonist, CS-866 (olmesartan medoxomil), alone or combined, in the remnant kidney model of rats. Eight-week-old spontaneously hypertensive male rats were subjected to five-sixths nephrectomy. At the age of 10 weeks, the rats were randomly allocated to groups that received two doses of CS-866 (CS-L, 3 mg/kg/day; CS-H, 10 mg/kg/day), temocapril (TEM, 10 mg/kg/day), CS-866 (3 mg/kg/day) plus temocapril (10 mg/kg/day), or a vehicle alone (untreated control group). Systolic blood pressure (SBP) and urinary protein excretion (UprotV) were measured every 2 weeks. When the rats were 18 weeks old, biochemical measurement and histologic examination were performed. All the drug treatments significantly reduced SBP, UprotV, glomerular sclerosis index (GSI), relative interstitial volume (RIV), and heart weight. The hypotensive effects were on the order of combination therapy > CS-H = TEM > CS-L. Correlational analysis was based on the values for SBP and UprotV derived from the average of values obtained when the rats were 12 to 18 weeks of age. UprotV, GSI, and RIV were found to be highly correlated with SBP among the individual rats pooled from all the groups, and the correlation was maintained among the group means. A similar correlation was found between heart weight and SBP. The results suggest that the organ protective effects of temocapril, CS-866, and combination therapy are closely related to the magnitude of their antihypertensive effects.