Adiponectin attenuates Ang â ¡-induced TGFß1 production in human mesangial cells via an AMPK-dependent pathway.

Glomerulosclerosis is a key element in end-stage renal failure. Angiotensin II (Ang II) plays an important role in modulating cell growth and extracellular matrix (ECM) synthesis and degradation. Adiponectin, a protein derived from adipocytes, is primarily involved in regulating glucose levels and fatty acid break down. It has recently been shown to have antiatherosclerotic and anti-inflammatory properties. However, the role of adiponectin as a renoprotective agent has not been fully explored. We herein examine the effect of adiponectin on Ang II-induced TGFß1 and ECM production in human renal mesangial cells (HRMCs) and explore the signaling pathway involved. In this study, we found that both adiponectin receptor 1 and adiponectin receptor 2 are expressed in HRMCs. Adiponectin (10 µg/mL) attenuated the stimulatory effect of Ang II on TGF-ß1 and fibronectin. Furthermore, adiponectin activated the AMP-activated protein kinase (AMPK), and the AMPK-specific inhibitor (compound C) blocked AMPK activation. We also determined that compound C blocked the inhibitory effect of adiponectin on Ang II-stimulated TGFß1 and fibronectin production. In summary, these results demonstrate that adiponectin suppresses Ang II-induced synthesis of ECM in mesangial cells via activation of the AMPK pathway. Based on our data, we suggest that this mechanism could delay the progression of kidney disease.

[Expression and regulation of adiponectin in human renal glomerular endothelial cells].

OBJECTIVE: To investigate the expression and regulation of adiponectin in human renal glomerular endothelial cells (HRGEC). METHOD: The adiponectin expression in human renal tissue was examined with immunohistochemistry assay, and the adiponectin mRNA and protein expression of HRGEC was confirmed by reverse transcription polymerase chain reaction (RT-PCR) and Western blot, respectively. PCR product was sequenced. To investigate the regulation action of TNF-alpha, the adiponectin produced by HRGEC was semi-quantitatively determined with real-time PCR in mRNA level and quantitatively measured with dissociation enhanced lanthanide fluorescence immunoassay (DELFIA) in protein level, respectively. RESULTS: (1) Positive adiponectin staining was observed on human glomerular capillary wall with immunohistochemistry assay. (2) Adiponectin mRNA expression in HRGEC was detected by RT-PCR, and the DNA sequence of this PCR product is consistent with adiponectin DNA sequence in Gene Bank. Adiponectin protein was also found in the supernatant of cultured HRGEC by Western blot. (3) Compared with control groups, the adiponectin mRNA expression in HRGEC determined by real-time PCR was significantly up-regulated after 250 IU/ml and 500 IU/ml TNF-alpha stimulation for 24 h (P < 0.05 and P < 0.01), and the adiponectin protein levels in culture supernatant measured by DELFIA were also significantly increased in these two groups (P < 0.05 and P < 0.01). (4) Compared with control groups, the adiponectin mRNA expression in HRGEC was significantly up-regulated after 500 IU/ml TNF-alpha stimulation for 6 h, 12 h and 24 h (P < 0.05, P < 0.05 and P < 0.01), and the adiponectin protein levels in culture supernatant were also significantly increased after stimulation for 24 h and 48 h (P < 0.01). CONCLUSIONS: HRGEC is able to synthesize adiponectin, and TNF-alpha can up-regulate its mRNA and protein expression.

Addition of nitric oxide through nitric oxide-paracetamol enhances healing rat achilles tendon.

Nitric oxide is an important messenger molecule in many physiological processes. The addition of NO via NO-flurbiprofen enhances the material properties of healing tendon, however, flurbiprofen has a detrimental effect on healing. We asked if NO delivered by a cyclooxygenase 3 inhibitor (paracetamol/acetaminophen) would enhance healing in a rat Achilles tendon healing model. Rats were injected subcutaneously daily with NO-paracetamol, paracetamol or vehicle from two days before surgery to the day of tissue harvesting. Paracetamol had no effect on tendon healing compared with vehicle alone. NO-paracetamol did not change the failure load, but did decrease the water content, enhance the collagen content, reduce the cross-sectional area and improve the ultimate stress of healing tendon compared with paracetamol and vehicle. The collagen organization of the healing tendon in the NO-paracetamol group, as determined by polarized light microscopy, was enhanced. Our data suggests NO-paracetamol increases the total collagen content and enhances organization while decreasing the cross-sectional area of healing rat Achilles tendon and is consistent with human clinical trials where NO has improved the symptoms and signs of tendinopathy.

[Expression of alpha 1 anti-trypsin in proximal tubular epithelial cell line].

OBJECTIVE: To investigate the expression and regulation of alpha 1 anti-trypsin (AAT) in tubular epithelial cells. METHODS: The expression of AAT in tubular epithelial cell line HKC was detected with indirect immunofluorescence assay and confirmed by reverse transcription polymerase chain reaction (PCR) in transcription level respectively, and PCR product was sequenced. In order to observe the response of HCK to LPS, the different concentrations of LPS (0, 0.5, 1.0, 2.0 microg/ml) were used in the research and the regulation of AAT in HKC was semi-quantitatively detected with Western Blot and Real-Time PCR respectively. RESULTS: Indirect immunofluorescence staining showed that AAT was positive in HKC, but negative in renal interstitial fibroblast. By PCR, a significant messenger RNA band of AAT was found in HKC, but not in renal interstitial fibroblast. DNA sequencing indicated that the sequence of PCR product is consistent with AAT mRNA sequence in Gene Bank. Real-time PCR showed that the expression of AAT mRNA was up-regulated (fluorescence intensity ratio is 3.43 +/- 0.88 versus 1.22 +/- 0.20; P < 0.01) in HKC stimulated with 2.0 microg/ml LPS for 4 h, and Western Blot showed that the synthesis of AAT significantly increased (band density ratio is 0.88 +/- 0.12 versus 0.59 +/- 0.05; P < 0.01) in HKC stimulated with 2.0 microg/ml LPS for 8 h, as compared with unstimulated HKC. 0.5, 1.0 g/ml of LPS has no effect on the expression of AAT mRNA and AAT protein in HKC. CONCLUSIONS: HKC express AAT and the expression can be up-regulated by LPS.

[Renoprotective effects of nonselective endothelin receptor antagonist bosentan on rats with interstitial fibrosis following unilateral ureteral obstruction].

OBJECTIVE: To investigate the effects of nonselective endothelin receptor antagonist bosentan on the progression of renal interstitial fibrosis following unilateral ureteral obstruction (UUO). METHODS: Eighteen female SD rats were randomly divided into 3 groups: intervention group (administered with bosentan for 7 days after left ureter ligation), animal model group (administered with solution of Arabic gum for 7 days left ureter ligation) and sham operation group (administered with Arabic gum for 7 days after sham operation). Seven days after the operation the rats were killed and their left kidneys were harvested. The mRNA expressions of preproendothelin-1 (prepro ET-1), type I collagen (ColI), transforming growth factor beta1 (TGF-beta1), tissue inhibitors of metalloproteinase-1 (TIMP-1) and type 1 plasminogen activator inhibitor (PAI-1) were detected semiquantitatively with reverse transcription-polymerase chain reaction (RT-PCR). The protein expressions of endothelin-1 (ET-1), ColI, TGF-beta1, TIMP-1 and PAI-1were determined semiquantitatively by immunohistochemical staining assay. RESULTS: The mRNA expressions of prepro ET-1, ColI, TGF-beta1, TIMP-1 and PAI-1 were significantly upregulated in obstructed renal tissue compared to those in sham operation group (all P < 0.05). The positive staining areas of ET-1, ColI, TGF-beta1, TIMP-1 and PAI-1 in tubulointerstitium were markedly enhanced in the animal model group in comparison with those in the sham operation group (all P < 0.05). The mRNA expressions of ColI, TGF-beta1 and TIMP-1 in renal tissue subjected to ureter ligation were significantly lower in the bosentan group then in the animal model group (all P < 0.05). The positive staining areas of ColI, TGF-beta1 and TIMP-1 in the tubulointerstitium were significantly smaller in bosentan group than in the animal model group (all P < 0.05). However, there was no significant difference in the mRNA expression of PAI-1 and in the positive staining area between the animal model group and bosentan group (both P > 0.05). CONCLUSION: ET-1 may be involved in the progression of renal interstitial fibrosis following unilateral ureter ligation and blockage of its receptors with bosentan attenuates the fibroticlesion to a certain extent by possibly inhibiting TGF-beta1 and TIMP-1.

[The effects of endothelin-1 and selective endothelin receptor-type A antagonist on human renal interstitial fibroblasts in vitro].

OBJECTIVE: (1) To investigate the effects of endothelin-1 (ET-1) on cell proliferation of cultured human renal interstitial fibroblasts (hRIFs), and the mRNA expression of type I Collagen (Col I), transforming growth factor-beta (TGF-beta), matrix metalloproteinase-1 (MMP-1) and tissue inhibitors of metalloproteinase-1, -2 (TIMP-1, TIMP-2) by hRIFs. (2) To investigate the changes of above effects after the hRIFs were pretreated with selective endothelin receptor-type A antagonist (ETaRA). METHODS: (1) The proliferation of hRIFs was determined by MTT method. (2) The mRNA expression of Col I, TGF-beta, MMP-1, TIMP-1 and TIMP-2 was detected semiquantitatively with reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: (1) The hRIFs proliferation was increased by ET-1 stimulation in dose dependent manner (10(-11) approximately 10(-7) mol/L, 24 h), and the peak growth level was at the concentration of 10(-7) mol/L (P < 0.05). (2) The hRIFs proliferation was significantly increased at 8th hour after ET-1 stimulation (10(-7) mol/L) (P < 0.01), and the peak growth level was attained at 24th hour (P < 0.01). (3) The mRNA expression of Col I, TGF-beta, MMP-1, TIMP-1 and TIMP-2 by hRIFs was upregulated with ET-1 in dose dependent manner (10(-11) approximately 10(-7) mol/L, 16 h), and the peak expression level was at the concentration of 10(-7) mol/L (P < 0.05). (4) When hRIFs were stimulated by ET-1 (10(-7) mol/L), the mRNA expression of Col I and TGF-beta was significantly increased at 8th hour (P < 0.05), and the peak expression levels were at 24th hour and 8th hour respectively; the mRNA expression of TIMP-1 and TIMP-2 was significantly increased at 16th hour (P < 0.05) and reached the peak level at 24th hour; the mRNA expression of MMP-1 was significantly increased and attained the peak level at 16th hour (P < 0.05). (5) The above effects of ET-1 were significantly inhibited by ETaRA (P < 0.05). CONCLUSION: The stimulating effects of ET-1 on hRIFs proliferation and the mRNA expression of Col I, TGF-beta, MMP-1, TIMP-1 and TIMP-2, and the inhibiting effects of ETaRA on the above effects suggest that ET-1 may participate in the process of renal interstitial fibrosis under pathological condition.