Cell-Free Synthesis of Human Endothelin Receptors and Its Application to Ribosome Display.

Engineering G-protein-coupled receptors (GPCRs) for improved stability or altered function is of great interest, as GPCRs consist of the largest protein family, are involved in many important signaling pathways, and thus, are one of the major drug targets. Here, we report the development of a high-throughput screening method for GPCRs using a reconstituted in vitro transcription-translation (IVTT) system. Human endothelin receptor type-B (ETBR), a class A GPCR that binds endothelin-1 (ET-1), a 21-residue peptide hormone, was synthesized in the presence of nanodisc (ND) composed of a phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG). The ET-1 binding of ETBR was significantly reduced or was undetectable when other phospholipids were used for ND preparation. However, when functional ETBR purified from Sf9 cells was reconstituted into NDs, ET-1 binding was observed with two different phospholipids tested, including POPG. These results suggest that POPG likely supports the folding of ETBR into its functional form in the IVTT system. Using the same conditions as ETBR, whose three-dimensional structure has been solved, human endothelin receptor type-A (ETAR), whose three-dimensional structure remains unsolved, was also synthesized in its functional form. By adding POPG-ND to the IVTT system, both ETAR and ETBR were successfully subjected to ribosome display, a method of in vitro directed evolution that facilitates the screening of up to 1012 mutants. Finally, using a mock library, we showed that ribosome display can be applied for gene screening of ETBR, suggesting that high-throughput screening and directed evolution of GPCRs is possible in vitro.

Transfer and Vascular Effect of Endothelin Receptor Antagonists in the Human Placenta.

Increasing evidence suggests a role for the ET (endothelin) system in preeclampsia. Hence, blocking this system with endothelin receptor antagonists (ERAs) could be a therapeutic strategy. Yet, clinical studies are lacking due to possible teratogenic effects of ERAs. In this study, we investigated the placental transfer of ERAs and their effect on ET-1-mediated vasoconstriction. Term placentas were dually perfused with the selective ETAR (ET type A receptor) antagonists sitaxentan and ambrisentan or the nonselective ETAR/ETBR antagonist macitentan and subsequently exposed to ET-1 in the fetal circulation. ET-1 concentration-response curves after incubation with sitaxentan, ambrisentan, macitentan, or the selective ETBR antagonist BQ-788 were also constructed in isolated chorionic plate arteries using wire-myography, and gene expression of the ET-system was quantified in healthy and early onset preeclamptic placentas. At steady state, the mean fetal-to-maternal transfer ratios were 0.32±0.05 for sitaxentan, 0.21±0.02 for ambrisentan, and 0.05±0.01 for macitentan. Except for BQ-788, all ERAs lowered the response to ET-1, both in the perfused cotyledon and isolated chorionic plate arteries. Placental gene expression of ECE-1, ETAR, and ETBR were comparable in healthy and preeclamptic placentas, while ET-1 expression was higher in preeclampsia. Our study is the first to show direct transfer of ERAs across the term human placenta. Furthermore, ETAR exclusively mediates ET-1-induced constriction in the fetoplacental vasculature. Given its limited transfer, macitentan could be considered as potential preeclampsia therapy. Extending knowledge on placental transfer to placentas of preeclamptic pregnancies is required to determine whether ERAs might be applied safely in preeclampsia.

Endothelin A Receptors Expressed in Renal Blood Vessels of Renal Transplant Patients Are Connected With Acute Tubular Necrosis or Antibody-Mediated Rejection.

BACKGROUND: The role of non-HLA antibodies named antiendothelin A receptor antibodies is potentially significant but not established. The significance of the endothelin A receptor (ETAR) and its expression in renal biopsy has not been defined. We decided to evaluate the presence and relevance of ETARs in renal transplant biopsy for cause. The aim of our study was to evaluate the immunoreactivity of the ETAR and its significance in patients who had a renal transplant biopsy due to deterioration of transplant function (biopsy for cause) with detailed characterization of staining in small and intermediate arteries of renal transplant biopsies. METHODS: Immunohistochemical expression of ETARs was analyzed in 162 renal transplant biopsies. Microscopic evaluation of ETAR expression (polyclonal antibody) was performed on paraffin sections. ETAR expression was analyzed in renal blood vessels (small and intermediate arteries) based on three-step scale. RESULTS: We analyzed 154 patients who had renal allograft biopsy between 6 days and 24 years (median 597 days) after transplantation. Positive staining of ETAR in small and intermediate arteries was noticed in 9 patients. Among these patients, 4 had early biopsies (<3 months after transplantation), all developed acute tubular necrosis, and 1 developed additionally acute humoral rejection. Further, 4 patients had late biopsy (1-8 years after transplantation) and all developed characteristics of antibody mediated rejection. Lastly, 1 patient had no characteristic changes in the biopsy 4 months after transplantation. Graft loss 1 year after biopsy was higher in patients who were ETAR-positive but statistical significance was not achieved. CONCLUSIONS: The expression of endothelin receptors in renal blood vessels (small and intermediate arteries) seems to be important in diagnosis of damage during acute tubular necrosis and antibody-mediated rejection.

Nephron prorenin receptor deficiency alters renal medullary endothelin-1 and endothelin receptor expression.

The endothelin (ET) and prorenin/renin/prorenin receptor (PRR) systems have opposing physiological effects on collecting duct (CD) salt and water reabsorption. It is unknown if the CD ET and renin/PRR systems interact, hence we examined the effects of deleting CD renin or nephron PRR on CD ET system components. PRR knockout (KO) mice were polyuric and had markedly increased urinary ET-1 and inner medullary CD (IMCD) ET-1 mRNA. PRR KO mice had greatly increased IMCD ETA receptor mRNA and protein, while ETB mRNA and protein were decreased. Water loaded wild-type mice with similar polyuria as PRR KO mice had modestly increased urinary ET-1 excretion and inner medullary ET-1 mRNA, while inner medullary ETA and ETB mRNA or protein expression were unaffected. In contrast to PRR KO, CD prorenin/renin KO did not alter ET system components. Taken together, these results suggest that the nephron PRR is involved in regulating CD ET system expression, but this effect may be independent of CD-derived renin.

Differential somatostatin, CXCR4 chemokine and endothelin A receptor expression in WHO grade I-IV astrocytic brain tumors.

PURPOSE: Glioblastomas represent the most common primary malignant tumor of the nervous system and the most frequent type of astrocytic tumors. Despite improved therapeutic options, prognosis has remained exceptionally poor over the last two decades. Therefore, new treatment approaches are urgently needed. An overexpression of somatostatin (SST) as well as chemokine CXCR4 and endothelin A (ETA) receptors has been shown for many types of cancer. Respective expression data for astrocytic brain tumors, however, are scarce and contradictory. METHODS: SST subtype, CXCR4 and ETA expression was comparatively evaluated in a total of 57 grade I-IV astrocytic tumor samples by immunohistochemistry using well-characterized monoclonal antibodies. RESULTS: Overall, receptor expression on the tumor cells was only very low. SST5 was the most prominently expressed receptor, followed by SST3, ETA, SST2 and CXCR4. In contrast, tumor capillaries displayed strong SST2, SST3, SST5, CXCR4 and ETA expression. Presence of SST5, CXCR4 and ETA on tumor cells and of SST3, CXCR4 and ETA on microvessels gradually increased from grade II to grade IV tumors. Ki-67 values correlated significantly with CXCR4 expression on tumor cells and with vascular SST3, CXCR4 or ETA positivity. SST5 or CXCR4 positivity of tumor cells and vascular SST3 or CXCR4 expression negatively correlated with patient outcome. CONCLUSIONS: Though having some prognostic value, SST, CXCR4 or ETA expression on astrocytic tumor cells is clearly of no therapeutic relevance. Indirect targeting of these highly vascularized tumors via SST3, SST5, CXCR4 or ETA on the microvessels, in contrast, may represent a promising additional therapeutic strategy.

The effects of MEK1/2 inhibition on cigarette smoke exposure-induced ET receptor upregulation in rat cerebral arteries.

Cigarette smoking, a major stroke risk factor, upregulates endothelin receptors in cerebral arteries. The present study examined the effects of MEK1/2 pathway inhibition on cigarette smoke exposure-induced ET receptor upregulation. Rats were exposed to the secondhand smoke (SHS) for 8weeks followed by intraperitoneal injection of MEK1/2 inhibitor, U0126 for another 4weeks. The urine cotinine levels were assessed with high-performance liquid chromatography. Contractile responses of isolated cerebral arteries were recorded by a sensitive wire myograph. The mRNA and protein expression levels of receptor and MEK/ERK1/2 pathway molecules were examined by real-time PCR and Western blotting, respectively. Cerebral artery receptor localization was determined with immunohistochemistry. The results showed the urine cotinine levels from SHS exposure group were significantly higher than those from the fresh group. In addition, the MEK1/2 inhibitor, U0126 significantly reduced SHS exposure-increased ETA receptor mRNA and protein levels as well as contractile responses mediated by ETA receptors. The immunoreactivity of increased ETA receptor expression was primarily cytoplasmic in smooth muscle cells. In contrast, ETB receptor was noted in endothelial cells. However, the SHS-induced decrease in endothelium-dependent relaxation was unchanged after U0126 treatment. Furthermore, SHS increased the phosphorylation of MEK1/2 and ERK1/2 protein in cerebral arteries. By using U0126 could inhibit the phosphorylated ERK1/2 protein but not MEK1/2. Taken together, our data show that treatment with MEK1/2 pathway inhibitor offsets SHS exposure-induced ETA receptor upregulation in rat cerebral arteries.

Immunohistochemical assessment of endothelin-1 axis in psoriasis, basal cell carcinoma and squamous cell carcinoma.

AIM: Endothelin-1 is an autocrine growth factor for keratinocytes, an effect controlled by its A and B receptors, with no previous comparison of endothelin axis expression in inflammatory and neoplastic skin diseases showing keratinocyte proliferation. The aim of the study was to investigate endothelin-1 axis expression in skin lesions of psoriasis, basal cell carcinoma (BCC), and squamous cell carcinoma (SCC). METHODS: This study included 40 subjects (8 patients with SCC, 12 patients with BCC, 10 patients with psoriasis, and 10 healthy controls). Biopsies from lesional skin of patients and normal skin of controls were examined immunohistochemically for endothelin-1 and its receptors A and B frequency and grade of expression. RESULTS: Endothelin-1 and receptor A were detected in all patients with SCC and psoriasis, with a higher frequency and grade of expression than controls and BCC. The frequency of receptor B expression was significantly lower while higher staining grade was found in BCC (8.3%) rather than other studied groups. CONCLUSION: A comparable higher frequency and grade of expression of endothelin-1 and its receptor A are documented in psoriasis and SCC than in BCC and controls denoting their involvement in keratinocyte proliferation in both diseases. Receptor A is the predominately expressed receptor in psoriasis and SCC.

Sex differences in ET-1 receptor expression and Ca2+ signaling in the IMCD.

The inner medullary collecting duct (IMCD) is the nephron segment with the highest production of endothelin-1 (ET-1) and the greatest expression of ET-1 receptors that function to adjust Na(+) and water balance. We have reported that male rats have reduced natriuresis in response to direct intramedullary infusion of ET-1 compared with female rats. Our aim was to determine whether alterations of ET-1 receptor expression and downstream intracellular Ca(2+) signaling within the IMCD could account for these sex differences. IMCDs from male and female rats were isolated for radioligand binding or microdissected for intracellular Ca(2+) ([Ca(2+)]i) measurement by fluorescence imaging of fura-2 AM. IMCD from male and female rats had similar ETB expression (655 ± 201 vs. 567 ± 39 fmol/mg protein, respectively), whereas male rats had significantly higher ETA expression (436 ± 162 vs. 47 ± 29 fmol/mg protein, respectively; P < 0.05). The [Ca(2+)]i response to ET-1 was significantly greater in IMCDs from male compared with female rats (288 ± 52 vs. 118 ± 32 AUC, nM × 3 min, respectively; P < 0.05). In IMCDs from male rats, the [Ca(2+)]i response to ET-1 was significantly blunted by the ETA antagonist BQ-123 but not by the ETB antagonist BQ-788 (control: 137 ± 27; BQ-123: 53 ± 11; BQ-788: 84 ± 25 AUC, nM × 3 min; P < 0.05), consistent with greater ETA receptor function in male rats. These data demonstrate a sex difference in ETA receptor expression that results in differences in ET-1 Ca(2+) signaling in IMCD. Since activation of ETA receptors is thought to oppose ETB receptor activation, enhanced ETA function in male rats could limit the natriuretic effects of ETB receptor activation.

The endothelin A receptor mediates fibrocyte differentiation in chronic obstructive asthma. The involvement of connective tissue growth factor.

RATIONALE: Fibrocytes possess increased differentiability into α-smooth muscle actin (α-SMA)(+) myofibroblasts in chronic obstructive asthma (COA) and contribute to pulmonary fibrosis. Endothelin-1 (ET-1) induces matrix-associated gene expression through the ETA receptor (ETAR) and promotes fibroblast differentiation. However, the mechanism of fibrocyte differentiation remains unclear. OBJECTIVES: To define the roles of the ETAR and connective tissue growth factor (CTGF) expression in fibrocytes in the development of fibrosis in COA. METHODS: Blood nonadherent non-T (NANT) cells were isolated, and fibrocytes expressing CD45, collagen I, CTGF, ETAR, or α-SMA were identified by flow cytometry. MEASUREMENTS AND MAIN RESULTS: We showed the accumulation of fibrocytes in bronchial walls and overexpression of CTGF in fibrocytes from patients with COA. After being cultured, CTGF was increased in fibrocytes from patients with COA, but not from those of normal participants or patients with asthma without obstruction. Serum levels of ET-1 and the expression of the ETAR in fibrocytes were significantly higher in patients with COA compared with normal participants and patients with asthma without obstruction. Treatment with the ETAR antagonist (BQ123), but not ETBR antagonist (BQ788), reduced the expression of CTGF and α-SMA in fibrocytes and fibrocyte differentiation in patients with COA. Furthermore, treatment with BQ123 or an anti-CTGF antibody attenuated α-SMA expression induced by ET-1 in fibrocytes from normal participants. CONCLUSIONS: Our findings demonstrate for the first time that the ETAR pathway is vital for CTGF expression, which results in fibrocyte differentiation in COA, and suggests that an ETAR antagonist may be a potential antifibrotic agent in preventing the development of fibrosis in patients with COA.

Ethanol consumption increases endothelin-1 expression and reactivity in the rat cavernosal smooth muscle.

AIMS: We investigated the effects of chronic ethanol consumption on the cavernosal smooth muscle (CSM) reactivity to endothelin-1 (ET-1) and the expression of ET system components in this tissue. METHODS: Male Wistar rats were treated with heavy dose of ethanol (20% v/v) for 6 weeks. Reactivity experiments were performed in the isolated rat CSM. Plasma and CSM nitrate generation and also superoxide anion generation in rat CSM were measured by chemiluminescence. Protein and mRNA levels of pre-pro-ET-1, endothelin-converting enzyme-1 (ECE-1), ETA and ETB receptors, eNOS, nNOS and iNOS were assessed by western immunoblotting and quantitative real-time polymerase chain reaction, respectively. RESULTS: Chronic ethanol consumption increased plasma ET-1 levels and the contractile response induced by this peptide in the isolated CSM. The relaxation induced by acetylcholine, but not IRL1620, a selective ETB receptor agonist, was reduced in CSM from ethanol-treated rats. BQ123, a selective ETA receptor antagonist, produced a rightward displacement of the ET-1 concentration-response curves in CSM from control, but not ethanol-treated rats. Reduced levels of nitrate were found in the plasma and CSM from ethanol-treated rats. Ethanol consumption increased superoxide anion generation in the rat CSM. The mRNA levels of pre-pro-ET-1, ECE-1, ETA and ETB receptors, eNOS, nNOS and iNOS were not altered by ethanol consumption. Protein levels of ET-1, ETA receptor and iNOS were higher in the CSM from rats chronically treated with ethanol. CONCLUSION: The major findings of the present study are that heavy ethanol consumption increases plasma ET-1 levels and the contraction induced by the peptide in the CSM. Increased CSM reactivity to ET-1 and altered protein levels of ET-1 and ETA receptors could play a role in the pathogenesis of erectile dysfunction associated with chronic ethanol consumption.

Functional properties of cell-free expressed human endothelin A and endothelin B receptors in artificial membrane environments.

The human endothelin receptors are members of the rhodopsin class A of G-protein coupled receptors and key modulators of blood pressure regulation. Their functional in vitro characterization has widely been limited by the availability of high quality samples. We have optimized cell-free expression protocols for the human endothelin A and endothelin B receptors by implementing co-translational association approaches of the synthesized proteins with supplied liposomes or nanodiscs. Efficiency of membrane association and ligand binding properties of the receptors have systematically been studied in correlation to different membrane environments and lipid types. Ligand binding was analyzed by a number of complementary assays including radioassays, surface plasmon resonance and fluorescence measurements. High affinity binding of the peptide ligand ET-1 to both endothelin receptors could be obtained with several conditions and the highest Bmax values were measured in association with nanodiscs. We could further obtain the characteristic differential binding pattern of the two endothelin receptors with a panel of selected agonists and antagonists. Two intrinsic properties of the functionally folded endothelin B receptor, the proteolytic processing based on conformational recognition as well as the formation of SDS-resistant complexes with the peptide ligand ET-1, were observed with samples obtained from several cell-free expression conditions. High affinity and specific binding of ligands could furthermore be obtained with non-purified receptor samples in crude cell-free reaction mixtures, thus providing new perspectives for fast in vitro screening applications.

Endothelin axis is upregulated in human and rat right ventricular hypertrophy.

RATIONALE: Right ventricular (RV) function is the most important determinant of morbidity and mortality in pulmonary arterial hypertension (PAH). Endothelin (ET)-1 receptor antagonists (ERAs) are approved therapies for PAH. It is not known whether ERAs have effects on the RV, in addition to their vasodilating/antiproliferative effects in pulmonary arteries. OBJECTIVE: We hypothesized that the ET axis is upregulated in RV hypertrophy (RVH) and that ERAs have direct effects on the RV myocardium. METHODS AND RESULTS: RV myocardial samples from 34 patients with RVH were compared with 16 nonhypertrophied RV samples, and from rats with normal RV versus RVH attributable to PAH. Confocal immunohistochemistry showed that RVH myocardial ET type A (but not type B) receptor and ET-1 protein levels were increased compared with the nonhypertrophied RVs and positively correlated with the degree of RVH (RV thickness/body surface area; r(2)=0.838 and r(2)=0.818, respectively; P<0.01). These results were recapitulated in the rat model. In modified Langendorff perfusions, ERAs (BQ-123 and bosentan 10(-7,-6,-5) mol/L) decreased contractility in the hypertrophied, but not normal RV, in a dose-dependent manner (P<0.01). CONCLUSIONS: Patients and rats with PAH have an upregulation of the myocardial ET axis in RVH. This might be a compensatory mechanism to preserve RV contractility, as the afterload increases. ERAs use might potentially worsen RV function, and this could explain some of the peripheral edema noted clinically with these agents. Further studies are required to evaluate the effects of ERAs on the RV in patients with RVH and PAH.

Altered expression and signal transduction of endothelin-1 receptors in heritable and idiopathic pulmonary arterial hypertension.

Human pulmonary arterial smooth muscle cells (PASMC) were isolated from elastic pulmonary arteries dissected from lungs of individuals with and without pulmonary arterial hypertension (PAH). Reflecting increased smooth muscle constriction in cells from PAH subject, Ca(2+) influx in response to endothelin-1 (ET-1) increased in all the PAH PASMC populations relative to the normal donor control cells. The ETA receptor mRNA levels remained unchanged, whereas the ETB receptor mRNA levels decreased in both heritable and idiopathic PAH-derived PASMC. All the PASMC populations expressed considerably higher ETA compared to ETB receptor number. Both ETA and ETB receptor numbers were reduced in bone morphogenetic protein receptor type II (BMPR2) mutation PAH. ETB receptors showed a particular reduction in number. Phospho-antibody array analysis of normal and BMPR2 deletion PASMC illustrated ERK and Akt activation to be the most prominent and to be taking place principally through ETB receptors in normal PASMC, but primarily through ETA receptors in PASMC from BMPR2 PAH subjects. Additionally in the PAH cells the total relative ET-1 signal response was markedly reduced. Western analysis from the BMPR2 PASMC duplicated the array results, whereas PASMC from iPAH subjects showed variability with most samples continuing to signal through ETB. In sum, these results indicate that generally both receptors are reduced in PAH particularly ETB, and that ETB signaling through protein kinases becomes markedly reduced in BMPR2 PASMC, while it continues in IPAH. Importantly, the data suggest that caution must be taken when applying ET-1 receptor antagonist therapy to PAH patients.

Repeated administration of centhaquin to pregnant rats did not affect postnatal development and expression of endothelin receptors in the brain, heart or kidney of pups.

The effect of repeated administration of centhaquin to pregnant rats on postnatal development, and expression of ETA and ETB receptors was determined. Pregnant rats were treated daily with either saline or centhaquin for 2 weeks. Male rat pups were sacrificed on day 1, 7, 14 and 28 of birth. Brain, kidney and heart were removed to study the expression of ETA and ETB receptor protein levels. Body weight of pregnant rats increased steadily in both vehicle and centhaquin groups. Expression of ETA receptors in the heart and kidney was similar in vehicle and centhaquin treated postpartum rats, but was significantly increased in the brain of centhaquin treated postpartum rats. No change in expression of ETB receptors was observed. In postnatal rats, mean body weight and weights of the brain, kidney and heart increased proportionally with advancing age and were similar in vehicle and centhaquin groups. The expression of ETA receptors in the brain, heart and kidneys was similar in vehicle and centhaquin groups. ETB receptor expression significantly (p<0.001) decreased by 72% and 70% on day 28 compared to rats of age 1, 7 and 14 days in control and centhaquin groups, respectively. Centhaquin treated rats showed similar expression of ETA and ETB receptors compared to vehicle treatment. This study suggests that repeated administration of centhaquin was well tolerated by pregnant rats that gave birth to normal pups. Centhaquin did not affect postnatal development of rats and had similar expression of ETA and ETB receptors compared to control pups.

ET-1 deletion from endothelial cells protects the kidney during the extension phase of ischemia/reperfusion injury.

BACKGROUND: The prognosis of patients after acute kidney injury (AKI) is poor and treatment is limited. AKI is mainly caused by renal ischemia/reperfusion injury (IRI). During the extension phase of IRI, endothelial damage may participate in ischemia and inflammation. Endothelin-1 (ET-1) which is mostly secreted by endothelial cells is an important actor of IRI, particularly through its strong vasoconstrictive properties. We aimed to analyze the specific role of ET-1 from the endothelial cells in AKI. METHODS: We used mice lacking ET-1 in the vascular endothelial cells (VEETKO). We induced IRI in VEETKO mice and wild type controls by clamping both kidneys for 30min. Sham operated mice were used as controls. Mice were sacrificed one day after IRI in order to investigate the extension phase of IRI. Kidney function was assessed based on serum creatinine concentration. Levels of expression of ET-1, its receptor ET(A), protein kinase C, eNOS, E-Cadherin and inflammation markers were evaluated by real time PCR or western blot. Tubular injury was scored on periodic acid Schiff stained kidney preparations. Lumen and wall area of small intrarenal arteries were measured on kidney slices stained for alpha smooth muscle cell actin. Oxidative stress, macrophage infiltration and cell proliferation was evaluated on slices stained for 8-hydroxy-2'-deoxyguanosine, F4/80 and PCNA, respectively. RESULTS: IRI induced kidney failure and increased ET-1 and ET(A) receptor expression. This was accompanied by tubular injury, wall thickening and reduction of lumen area/wall area ratio of small renal arteries, increased oxidative stress and inflammation. These parameters were attenuated in VEETKO mice. CONCLUSION: Our results suggest that suppression of ET-1 from the endothelial cells attenuates IRI kidney injury. Blocking ET-1 effects may represent a therapeutic strategy in the management of AKI.

Endothelin-1 and endothelin a receptor immunoreactivity is increased in patients with diabetic nephropathy.

BACKGROUND: Endothelin-1 (ET-1) is associated with progression of renal disease, acting as a vasoconstrictor and growth factor for mesangial cells. ET-1 and endothelin A receptor (ET-RA) might have a role in the development of diabetic nephropathy (DN). The aims of this study were to determine ET-1 and ET-RA expressions in patients with DN and to correlate these expressions with renal function and proteinuria. MATERIALS AND METHODS: This is a cross-sectional study comprising 13 patients with type 2 diabetes mellitus and DN, 10 patients with proteinuric IgA nephropathy, and 13 samples of normal kidney from tumor nephrectomies. Demographic and selected data were collected from medical charts. The distribution and intensity of ET-1 and ET-RA immunostaining in renal biopsies were determined by immunohistochemistry and these correlated with the estimated glomerular filtration rate (eGFR) and proteinuria. RESULTS: Patients with DN and IgA nephropathy on biopsy had markedly increased staining for ET-1 in endothelial cells of glomerular and peritubular capillaries when compared with controls (p < 0.001). ET-RA staining was also more intense and more diffuse in DN and IgA nephropathy than in controls (p = 0.019) and was restricted to tubular epithelial cells. A positive correlation was observed between ET-1 expression and proteinuria (r = 0.634, p = 0.027), but both ET-1 and ET-RA expressions did not correlate with eGFR. CONCLUSION: In this preliminary report, the higher expressions of ET-1 and ET-RA found in both DN and IgA nephropathy suggest a potential role for the endothelin system in DN as well as in other nondiabetic glomerular diseases.

Hyperinsulinemia increases placenta endothelin-converting enzyme-1 expression in trophoblasts.

BACKGROUND: Exogenous hyperisulinemia causes pregnancy, induced hypertension and intrauterine growth restriction (IUGR) in pregnant rats. Hyperinsulinemia may increase production of endothelin-1 (ET-1), produced by sequential proteolysis of the big endothelin by the endothelin-converting enzyme (ECE)-1, the expression of which is examined here in the placenta, kidney, heart, and liver. METHODS: Rats were rendered hyperinsulinemic by subcutaneous insulin pellet, mated and followed to the twenty-first day of pregnancy. They were then killed, and their fetuses and placentas were examined. RESULTS: Hyperinsulinemic dams (HD) had higher blood pressure (BP) (130 ± 17 mm Hg in HD vs. 115 ± 16 mm Hg in normal pregnant dams (NPD), P < 0.05), lower placenta weight (0.44 ± 0.08 g in HD vs. 0.47 ± 0.08 NPD, P < 0.05) and lower fetus weight: males 4.9 ± 0.4 g in HD vs. 5.5 ± 0.4 g in NPD, P < 0.0001; females 4.7 ± 0.4 g in HD vs. 5.2 ± 0.4 g in NPD (P < 0.0001). ECE-1 expression as determined by western blot was significantly increased in the placenta and its implantation site, i.e., the mesometrial triangle (MT) of HD by 46 and 48%, respectively. In the kidney and heart of HD ECE-1, protein expression was increased by 230 and 220%, respectively, but its level in the liver was similar in both groups. Immunohistochemical staining revealed ECE-1 expression in endothelial cells and trophoblastic cells of the placenta and MT. Endothelin receptor A (ET-A), a mediator of vasoconstriction by ET-1, was also expressed in the endothelium and in trophoblasts of the placenta and MT. The expression of both ECE-1 and ET-A, as measured by automated image analysis, was generally stronger in placentas of HD. CONCLUSIONS: ECE-1 and ET-A are expressed in the trophoblastic cells of the placenta and MT. This may affect local endothelin levels, BP and IUGR.