Cell-type specific interaction of endothelin and the nitric oxide system: pattern of prepro-ET1 expression in kidneys of L-NAME treated prepro-ET1 promoter-lacZ-transgenic mice.

  • Nitric oxide (NO) and endothelin-1 (ET-1) are known to play a major role in renal and vascular pathophysiology and exhibit a close interaction with ET-1, stimulating NO production; NO in turn inhibits ET-1 expression. Our objectives were (1) to establish a novel transgenic mouse model facilitating ET-1 expression assessment in vivo, (2) to validate this model by assessing prepro-ET-1 promoter activity in mice embryos by means of our novel model and comparing expression sites to well-established data on ET-1 in fetal development and (3) to investigate renal ET-NO interaction by assessing prepro-ET-1 promoter activity in different structures of the renal cortex in the setting of blocked NO synthases via L-NAME administration. We established transgenic mice carrying a lacZ reporter gene under control of the human prepro-ET-1 gene promoter sequence (8 kb of 5′ sequences).
  • Bluo-Gal staining of tissue sections revealed intracellular blue particles as indicators of prepro-ET-1 promoter activity. In mouse embryos, we detected high prepro-ET-1 promoter activity in the craniofacial region, as well as in bone and cartilage consistent with the literature. In order to investigate the interaction of ET-1 and NO in the kidney in vivo, transgenic mice at the age of 3-4 months were treated with a single dose of the NO synthase inhibitor L-NAME (25 mg (kg bw)(-1) i.p.) 12 h before kidney removal. Bluo-Gal staining of kidney sections revealed intracellular blue particles as indicators of prepro-ET-1 promoter activity in tubular and vascular endothelium and glomerular cells.
  • Particle count was closely correlated to kidney tissue ET-1 content (R=0.918, P<0.001). Comparison of counts revealed an increase by 135+/-53% in L-NAME treated (n=12) compared to non-treated mice (n=10, P=0.001). Cell-type specific evaluation revealed an increase of 136+/-51% in tubular (P=0.001) and 105+/-41% in glomerular cells (P=0.046), but no significant increase in vascular endothelium. In conclusion, our study revealed a close interaction of renal endothelin and the NO system in a cell-type specific manner. Our new transgenic model provides a unique opportunity to analyse regulation of the ET system on a cellular level in vivo.

<em>Endothelin</em>-<em>1</em> promotes cell survival in renal cell carcinoma through the <em>ET</em>(A) receptor.

Endothelin-1 (ET-1) is a potent vasoconstrictor that has been shown to significantly impact many benign and malignant tissues by signaling through its two cognate receptors: ET(A) and ET(B). As ET-1 has a role in both normal and diseased kidney, we initiated studies to investigate endothelin axis expression and function in renal cell carcinoma (RCC). In this study, relatively high levels of ET-1 were detected in all six human RCC cell lines investigated. RT-PCR and Southern analyses revealed that all six RCC cell lines expressed ET(A) receptor mRNA, while 3/6 cell lines also expressed ET(B) mRNA.
High affinity ET-1 binding occurred in all but one RCC cell line and quantitative RT-PCR demonstrated ET(A) mRNA expression in all six cell lines. Methylation of the ET(B) promoter (EDNRB) in 4/6 RCC cell lines was observed, suggesting a mechanism for repressed ET(B) expression. Moreover, methylation occurred in 32/48 of renal tumors and in 27/55 of histologically normal adjacent tissue samples studied, while no methylation was evident in any normal tissue isolated from nephrectomy or at autopsy. Functionally, ET-1 significantly inhibited paclitaxel-induced apoptosis in RCC cells through binding ET(A) with the ET-1 signaling mediated via the PI3-kinase/Akt pathway. Collectively, these data support the therapeutic targeting of the ET(A) receptor as a novel treatment strategy for RCC.

<em>Endothelin</em>-<em>1</em> and its receptors <em>ET</em>(A) and <em>ET</em>(B) in drug-induced gingival overgrowth.

BACKGROUND
The purpose of this study was to study the expression of endothelin-1 (ET-1) and its receptors ETA and ETB in normal human gingiva and cyclosporin-induced gingival fibroblasts.
METHODS
Gingival samples were collected from eight normal healthy individuals, eight patients with periodontitis, and eight patients with cyclosporin A (CsA)-induced gingival overgrowth. Total RNA was extracted from tissue samples, and reverse transcriptase-polymerase chain reaction was performed for ET-1, ETA, and ETB. ET-1 protein was estimated from the tissues by enzyme-linked immunosorbent assay. The expression of ET-1 and its receptors was also examined in gingival fibroblast cells treated with CsA.
RESULTS
ET-1 mRNA expression was significantly higher in patients with CsA-induced gingival overgrowth (P <0.001) than in patients with periodontitis and the controls. ETA mRNA was expressed more than the ETB in all examined samples. In human gingival fibroblasts, ET-1 expression was increased with CsA incorporation compared to controls (P <0.001).
CONCLUSIONS
These results suggest that CsA can modulate the expression of ET-1 in gingival fibroblasts and CsA-induced gingival overgrowth.

Regulation and expression of <em>endothelin</em>-<em>1</em> (<em>ET</em>-<em>1</em>) and <em>ET</em>-receptors in rat epithelial cells of renal and intestinal origin.

  • The hormone endothelin-1 (ET-1) is involved in many functions of the kidney and intestine. In addition to its vasoactive and proliferative effects, ET-1 is involved in the maintenance of water and salt balance, and in drug excretion by influencing the activity of different transporters in the epithelial cells of these two organs. To study ET-1 function and its role in pathophysiological processes in epithelial cells in vitro, we investigated ET-1 and ET-receptor expression and inducibility of ET-1 excretion by cytokines in three rat cell lines of intestinal (IEC-6) and renal (NRK-52E and GERP) origin.
  • Immunocytochemistry showed that all three cell lines express ET-1 and the ET-A and ET-B receptor. ET-1 was expressed intracellularly, and also the ET-A receptor showed a punctate intracellular staining pattern. The ET-B receptor was localized in the membrane, which was confirmed by Western blot analysis. Real-time RT-PCR and ELISA showed that exposure of IEC-6 cells to the cytokines, interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNFalpha), induced ET-1 mRNA expression and excretion, while IL-2 was ineffective.
  • In NRK-52E cells, IL-1beta and TNFalpha induced ET-1 excretion as well. In GERP cells, adequate measurement of cytokine effects on ET-1 excretion was not possible, since ET-1 excretion under non-stimulated conditions was around the lowest level of detection. In conclusion, we showed ET-1 and ET-receptor expression, and inducibility of ET-1 by cytokines in IEC-6, NRK-52E, and GERP cells.
  • These rat intestinal and renal cell lines appear to be suitable for further characterisation of ET-1 function and its role in pathophysiological processes in epithelial cells.

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[Relationship of <em>endothelin</em>-<em>1</em> (<em>ET</em>-<em>1</em>) TaqI and tumor necrosis factor (TNF) a gene polymorphism with portal hypertension in liver cirrhosis].

OBJECTIVE
To study whether liver cirrhosis and portal hypertension are associated with ET-1 TaqI polymorphism and TNFa promoter-308G to A polymorphism.
METHODS
A case control study of 106 patients with liver cirrhosis following HBV C infection was performed in comparison with 108 controls by PCR-RFLP.
RESULTS
The frequency of C allele and CC+TC genotype in TaqI polymorphism of ET-1 gene in the portal hypertension group (LC+) was significantly higher than that in the healthy controls, and the frequency of TNF2/1 genotype in TNFa promoter -308 G to A polymorphism in LC+ group was significantly higher than that in the control group. The results by stratification analysis showed that TCF2 genotype frequency was higher in the LC+ group than in the control group. ET-1 TaqI polymorphism and TNFa polymorphism were risk factors for the occurrence of portal hypertension by Logistic regression analysis.
CONCLUSIONS
ET-1 TaqI polymorphism and TNFa polymorphism are associated with portal hypertension, and are new risk factors for the occurrence of portal hypertension. TCF2 genotype may be a susceptible gene of portal hypertension.

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