adrenomedullin and Ureteral-Obstruction

To test the hypothesis that the gene expression of endothelin-1 and adrenomedullin may be altered in stenotic tissues of patients with congenital hydronephrosis caused by pelvi-ureteric junction (PUJ) obstruction.. Using real-time reverse transcription-polymerase chain reaction, mRNA of smooth muscle-constricting endothelin-1 and of smooth muscle-relaxing adrenomedullin was quantified in tissue specimens of 20 patients with PUJ obstruction (mean age 5.1 years, SD 7.0) and of 21 controls with normal PUJs (mean age 23.5 years, SD 24.2).. The amount of endothelin-1 mRNA in stenotic specimens was higher than in the controls, indicated by significantly lower threshold cycles (Ct values) in real-time PCR for the target gene in the obstructive tissue, with mean (SD) values of 24.9 (1.6) and 26.0 (2.1) (P < 0.05), respectively. The endothelin-1/CD31 ratio was significantly higher in the patients (P < 0.05) than in controls. In addition, adrenomedullin gene expression in the obstructed junctions was significantly lower than in normal junctions, with higher Ct values for the patient group of 26.7 (1.6) vs 25.2 (1.8) (P < 0.05) and lower adrenomedullin mRNA when standardized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (P < 0.05), CD31 (P < 0.01) and smooth muscle alpha-actin mRNA (P < 0.01). The two groups showed no significant differences for GAPDH and CD31 mRNA content, whereas there was about twice as much alpha-actin mRNA in stenotic tissues than in unaffected PUJs, shown by the lower Ct values for the patient group of 16.9 (2.0) vs 17.9 (2.6) (P < 0.05). Furthermore, endothelin-1, adrenomedullin and alpha-actin mRNA amounts were independent of age.. Taken together these results provide evidence that the production of autocrine/paracrine acting endothelin-1 and adrenomedullin is altered in tissues of patients with genuine PUJ obstruction, and may be involved in the pathogenesis of congenital hydronephrosis.

Topics: Adolescent; Adrenomedullin; Adult; Child; Child, Preschool; Endothelin-1; Gene Expression; Humans; Infant; Infant, Newborn; Peptides; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Ureteral Obstruction

Renal fibrosis promotes the progression of chronic renal disease to end-stage renal disease. Microvascular damage and loss play an important role in renal fibrosis. Intermedin (IMD) is expressed mainly in the heart and kidney. IMD has been shown to increase renal blood flow and reduce the loss of glomerular and surrounding renal tubules, but its role in mediating microvascular damage in renal fibrosis remains to be elucidated. Here, we investigated the effects of IMD on microvascular damage in a renal fibrosis model.. We created a rat model of unilateral ureteral obstruction (UUO) to clarify the effect of microvascular damage on renal fibrosis and the effect of intermedin on reversing renal vascular injury and promoting angiogenesis. Rats were divided randomly into three groups: sham, UUO, and UUO + IMD. The sham group underwent free ureteral ligation but not occlusion. Rats in the latter two groups underwent UUO, and rats in the IMD group were additionally administered intermedin (100 ng/kg/h) daily. On the 7th, 14th, 21st, and 28th days after surgery, abdominal aortic blood and the obstructed kidneys were harvested from the rats (n = 6) for analysis.. IMD was found to protect against renal vascular injury and to increase microvessel density. Molecularly, IMD upregulated vascular endothelial growth factor-vascular endothelial growth factor receptor (VEGF-VEGFR2) pathway activity. The VEGF-VEGFR2 pathway might be the underlying mechanism mediating the protective activities of IMD in promoting angiogenesis, delaying renal fibrosis, and improving renal function.. IMD could be a potential candidate treatment for renal fibrosis.

Topics: Adrenomedullin; Animals; Disease Models, Animal; Fibrosis; Kidney; Kidney Diseases; Kidney Tubules; Male; Neovascularization, Pathologic; Neuropeptides; Rats; Rats, Wistar; Up-Regulation; Ureteral Obstruction; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-2

Intermedin [IMD, adrenomedullin-2 (ADM-2)] attenuates renal fibrosis by inhibition of oxidative stress. However, the precise mechanisms remain unknown. Heme oxygenase-1 (HO-1), an antioxidant agent, is associated with antifibrogenic effects. ADM is known to induce HO-1. Whether IMD has any effect on HO-1 is unclear. Herein, we determined whether the antifibrotic properties of IMD are mediated by induction of HO-1.. Renal fibrosis was induced by unilateral ureteral obstruction (UUO) performed on male Wistar rats. Rat proximal tubular epithelial cell line (NRK-52E) was exposed to rhTGF-β1 (10 ng/ml) to establish an in vitro model of epithelial-mesenchymal transition (EMT). IMD was over-expressed in vivo and in vitro using the vector pcDNA3.1-IMD. Zinc protoporphyrin (ZnPP) was used to block HO-1 enzymatic activity. IMD effects on HO-1 expression in the obstructed kidney of UUO rat and in TGF-β1-stimulated NRK-52E were analyzed by real-time RT-PCR, Western blotting or immunohistochemistry. HO activity in the obstructed kidney, contralateral kidney of UUO rat and NRK-52E was examined by measuring bilirubin production. Renal fibrosis was determined by Masson trichrome staining and collagen I expression. Macrophage infiltration and IL-6 expression were evaluated using immunohistochemical analysis. In vivo and in vitro EMT was assessed by measuring α-smooth muscle actin (α-SMA) and E-cadherin expression using Western blotting or immunofluorescence, respectively.. HO-1 expression and HO activity were increased in IMD-treated UUO kidneys or NRK-52E. The obstructed kidneys of UUO rats demonstrated significant interstitial fibrosis on day 7 after operation. In contrast, kidneys that were treated with IMD gene transfer exhibited minimal interstitial fibrosis. The obstructed kidneys of UUO rats also had greater macrophage infiltration and IL-6 expression. IMD restrained infiltration of macrophages and expression of IL-6 in UUO kidneys. The degree of EMT was extensive in obstructed kidneys of UUO rats as indicated by decreased expression of E-cadherin and increased expression of α-SMA. In vitro studies using NRK-52E confirmed these observations. EMT was suppressed by IMD gene delivery. However, all of the above beneficial effects of IMD were eliminated by ZnPP, an inhibitor of HO enzyme activity.. This study demonstrates that IMD attenuates renal fibrosis by induction of HO-1.

Topics: Adrenomedullin; Animals; Cells, Cultured; Enzyme Induction; Fibrosis; Gene Transfer Techniques; Heme Oxygenase (Decyclizing); Kidney Diseases; Male; Neuropeptides; Random Allocation; Rats; Rats, Wistar; Ureteral Obstruction

Transforming growth factor-β1 (TGF-β1) plays a pivotal role in the progression of renal fibrosis. Reactive oxygen species mediate profibrotic action of TGF-β1. Intermedin (IMD) has been shown to inhibit oxidative stress, but its role in renal fibrosis remains unclear. Here, we investigated the effects of IMD on renal fibrosis in a rat model of unilateral ureteral obstruction (UUO).. The expression of IMD and its receptors, calcitonin receptor-like receptor (CRLR) and receptor activity-modifying proteins (RAMP1/2/3), in the obstructed kidney was detected by real-time polymerase chain reaction (PCR), western blotting and immunohistochemistry. To evaluate the effects of IMD on renal fibrosis, we locally overexpressed exogenous IMD in the obstructed kidney using an ultrasound-microbubble-mediated delivery system. Renal fibrosis was determined by Masson trichrome staining. The expression of TGF-β1, connective tissue growth factor (CTGF), α-smooth muscle actin (α-SMA) and fibronectin was measured. Smad2/3 activation and macrophage infiltration were evaluated. We also studied oxidative stress by measuring superoxide dismutase (SOD) activity and malondialdehyde (MDA) content.. mRNA and protein expression of IMD increased after UUO. CRLR, RAMP1, RAMP2 and RAMP3 were also induced by ureteral obstruction. IMD overexpression remarkably attenuated UUO-induced tubular injury and blunted fibrotic response as shown by decreased interstitial collagen deposition and downregulation of fibronectin. Macrophage infiltration, α-SMA and CTGF upregulation caused by UUO were all relieved by IMD, whereas TGF-β1 upregulation and Smad2/3 activation were not affected. Meanwhile, we noted increased oxidative stress in obstruction, which was also attenuated by IMD gene delivery.. Our results indicate that IMD is upregulated after UUO. IMD plays a protective role in renal fibrosis via its antioxidant effects.

Topics: Adrenomedullin; Animals; Calcitonin Receptor-Like Protein; Collagen; Disease Models, Animal; Fibronectins; Fibrosis; Genetic Therapy; Kidney; Kidney Diseases; Male; Microbubbles; Neuropeptides; Oxidative Stress; Rats, Wistar; Receptor Activity-Modifying Protein 1; Receptor Activity-Modifying Protein 2; Receptor Activity-Modifying Protein 3; RNA, Messenger; Signal Transduction; Smad2 Protein; Smad3 Protein; Time Factors; Transfection; Transforming Growth Factor beta1; Ultrasonics; Up-Regulation; Ureteral Obstruction

We evaluated the effects of adrenomedullin (Peptide Institute, Minoh-shi, Osaka, Japan) on mediators, including nitric oxide and transforming growth factor-beta, and parameters of renal injury in a murine unilateral ureteral obstruction model.. Three study groups of control, adrenomedullin treated and adrenomedullin plus L-NAME treated BALB/C mice, respectively, underwent left unilateral ureteral obstruction. A 24-hour urine sample was collected to measure urinary NO(2)/NO(3) 1 day before unilateral ureteral obstruction and kidneys were harvested on postoperative day 14. Tubulointerstitial damage markers were evaluated by immunohistochemistry. Tissue transforming growth factor-beta was determined by enzyme-linked immunosorbent assay. Endothelial and inducible nitric oxide synthase immunolocalization was also determined.. Urinary NO(2)/NO(3) was significantly higher in the adrenomedullin group than in controls, confirming increased renal nitric oxide production. Immunohistochemistry showed increased endothelial nitric oxide synthase in vascular endothelial cells in the adrenomedullin group but tissue transforming growth factor-beta did not significantly differ in controls vs the adrenomedullin group. Interstitial collagen deposition and fibroblasts in the obstructed kidney were significantly decreased in the adrenomedullin group. The number of leukocytes and apoptotic cells in the obstructed kidney were significantly decreased by adrenomedullin. Renal injury amelioration resulting from adrenomedullin was blunted by the nitric oxide synthase inhibitor L-NAME.. Adrenomedullin increased renal nitric oxide, and suppressed tubular apoptosis, interstitial fibrosis and inflammatory cell infiltration in mice with unilateral ureteral obstruction. The renoprotective peptide adrenomedullin may be useful for that condition.

Topics: Adrenomedullin; Animals; Mice; Mice, Inbred BALB C; Nitric Oxide; Renal Insufficiency; Transforming Growth Factor alpha; Ureteral Obstruction

Ureteral obstruction is characterized by decreased renal blood flow that is associated with hypoxia within the kidney. Adrenomedullin (AM) is a peptide hormone with tissue-protective capacity that is stimulated through hypoxia. We tested the hypothesis that ureteral obstruction stimulates expression of AM and hypoxia-inducible factor-1 (HIF-1alpha) in kidneys. Rats were exposed to bilateral ureteral obstruction (BUO) for 2, 6, 12, and 24 h or sham operation and compared with unilateral obstruction (UUO). AM mRNA expression was measured by quantitative PCR in cortex and outer medulla (C+OM) and inner medulla (IM). AM and HIF-1alpha protein abundance and localization were determined in rats subjected to 24-h BUO. AM mRNA expression in C+OM increased significantly after 12-h BUO and further increased after 24 h. In IM, AM mRNA expression increased significantly in response to BUO for 6 h and further increased after 24 h. AM peptide abundance was enhanced in C+OM and IM after 24-h BUO. Immunohistochemical labeling of kidneys showed a wider distribution and more intense AM signal in 24-h BUO compared with Sham. In UUO rats, AM mRNA expression increased significantly in IM of the obstructed kidney compared with nonobstructed and Sham kidney whereas AM peptide increased in IM compared with Sham. HIF-1alpha protein abundance increased significantly in IM after 24-h BUO compared with Sham and HIF-1alpha immunoreactive protein colocalized with AM. In summary, AM and HIF-1alpha expression increases in response to ureteral obstruction in agreement with expected oxygen gradients. Hypoxia acting through HIF-1alpha accumulation may be an important pathway for the renal response to ureteral obstruction.

Topics: Adrenomedullin; Animals; Disease Models, Animal; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Immunohistochemistry; Kidney; Male; Oxygen; Rats; Rats, Wistar; RNA, Messenger; Time Factors; Tumor Necrosis Factor-alpha; Up-Regulation; Ureteral Obstruction

To investigate the expression of endothelin-1 (ET-1) and adrenomedullin (ADM) in the renal pelvis, stenotic ureteropelvic junction, and ureter of 20 male Wistar rats with congenital unilateral ureteropelvic junction obstruction; the normal contralateral kidneys served as controls. The molecular pathophysiology of congenital ureteropelvic junction obstruction is still unclear. The implication of altered peptidergic innervation is under discussion. Our study group has recently been able to demonstrate a significant increase in ET-1 and a significant decrease in ADM in prestenotic and stenotic tissue, but not in the remainder of the ureter, compared with controls.. Twenty animals were killed, and samples of the renal pelvis, ureteropelvic junction, upper ureter, middle part of the ureter, and lower ureter were immediately snap-frozen and stored in liquid nitrogen. Total RNA was extracted, and subsequently 1 microg of RNA was reversely transcribed. mRNA expression of ET-1 and ADM was determined semiquantitatively using on-line polymerase chain reaction. The expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was determined to relate the specific mRNA expression to the expression of a housekeeping gene.. We found a significant increase in the expression of ET-1 in the obstructed junctions related to GAPDH (P <0.001). The expression of ADM, however, revealed no statistically significant differences. No differences at all could be detected in the tissue samples from the rest of the ureter.. Alterations in the local production of peptidergic neurotransmitters, especially ET-1, may contribute to the molecular pathogenesis of ureteropelvic junction obstruction. Results previously obtained in the stenotic tissue from children were confirmed in the stenotic tissue from the rat model. We hypothesize that the alterations are disease-, but not age-specific.

Topics: Abnormalities, Multiple; Adrenomedullin; Animals; Atrophy; Computer Systems; Constriction, Pathologic; Disease Models, Animal; Endothelin-1; Gene Expression Profiling; Hydronephrosis; Kidney Pelvis; Male; Peptides; Polymerase Chain Reaction; Rats; Rats, Mutant Strains; Rats, Wistar; RNA, Messenger; Ureter; Ureteral Obstruction