RESUMO
Shenkangling plays a role of Yishenhuoxue effect for the treatment of children with nephrotic syndrome. The aim of this study was to investigate the effects of Shenkangling intervention on the mitogen-activated protein kinase (MAPK) pathway in rats with Adriamycin-induced nephropathy (AN) and its underlying mechanism of action. Nephrosis was induced in healthy Sprague-Dawley rats by doxorubicin and the rats were untreated or treated with prednisone, simvastatin, Shenkangling, or a combination thereof. Using real-time PCR, the mRNA expression levels of Chemokine (C-X-C motif) ligand 16 (CXCL16), A Disintegrin and metalloproteinase domain-containing protein 10 (ADAM10), and ADAM17 in the renal tissues of these rats were found to be decreased by the various treatments compared to those in the untreated doxorubicin-induced nephrosis rats. To quantify the activation of the MAPK pathway, western blotting was used to detect the phosphorylation levels of MAPK pathway-associated proteins (p38, ERK1/2, SAPK/JNK) and nuclear factor (NF)-κB p65, which were reduced by the various treatments compared to those in the untreated doxorubicin-induced rats. Serum levels of transforming growth factor (TGF)-ß1, tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6, quantified by ELISA, were decreased by the various treatments compared to the levels in the untreated doxorubicin-induced nephrosis rats. The rats treated with prednisone, simvastatin, and Shenkangling showed the best outcome. The Chinese medicine Shenkangling that is known for nourishing the kidney and promoting blood circulation reduced urinary protein levels, increased serum albumin levels, and reduced cholesterol levels by reducing the release of CXCL16, ADAM10, ADAM17, TGF-ß1, TNF-α, IL-1ß, IL- 6, and other inflammatory mediators and inhibiting the activation of the MAPK signaling pathway, thereby effectively improving the state of nephropathy in AN rats. These results indicate that Shenkangling can be used clinically to treat nephropathy.
Assuntos
Medicamentos de Ervas Chinesas/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Síndrome Nefrótica/tratamento farmacológico , Proteína ADAM10/genética , Proteína ADAM17/genética , Animais , Quimiocina CXCL6/genética , Doxorrubicina/toxicidade , Interleucina-1beta/sangue , Interleucina-6/sangue , Masculino , NF-kappa B/metabolismo , Síndrome Nefrótica/sangue , Síndrome Nefrótica/induzido quimicamente , Síndrome Nefrótica/enzimologia , Proteinúria/tratamento farmacológico , Proteinúria/enzimologia , Proteinúria/metabolismo , Ratos , Ratos Sprague-Dawley , Fator de Crescimento Transformador beta1/sangue , Fator de Necrose Tumoral alfa/sangueRESUMO
The nephrotic syndrome is a renal disease characterized by proteinuria, hypoproteinemia, edema and hyperlipidemia. It has been reported that female nephrotic rats are characterized by loss of the oestrus cycle, follicle atresia, low gonadotropin and steroid concentrations; particularly, undetectable estradiol levels. Therefore, to determine the mechanisms involved in the ovarian steroidogenesis impairment, in this present study we evaluated the ovarian expression of the essential steroidogenesis components: cytochrome P450 side cholesterol chain cleavage enzyme (P450scc) and steroidogenic acute regulatory protein (StAR). The experiments were conducted in the rat experimental model of nephrosis induced by puromycin aminonucleoside (PAN) and in control groups. The evaluation of the expression of P450scc and StAR mRNA were performed during the acute phase of nephrosis as well as after the exogenous administration of 1 or 4 doses of human chorionic gonadotrophin (hCG), or a daily dose of FSH or FSH+hCG for 10 days. In addition, serum hormone concentrations, intra-ovarian steroid content, and the reproductive capacity were determined. The results revealed a decreased expression of mRNA of P450scc enzyme and StAR during nephrosis, and eventhough they increased after gonadotropins treatment, they did not conduce to a normal cycling rat period or fertility recovery. This study demonstrates that the mechanism by which ovarian steroid biosynthesis is altered during acute nephrosis involves damage at the P450scc and StAR mRNA synthesis and processing.
Assuntos
Enzima de Clivagem da Cadeia Lateral do Colesterol/biossíntese , Síndrome Nefrótica/enzimologia , Ovário/metabolismo , Fosfoproteínas/biossíntese , Esteroides/biossíntese , Animais , Northern Blotting , Enzima de Clivagem da Cadeia Lateral do Colesterol/genética , Gonadotropina Coriônica/farmacologia , Ciclo Estral , Feminino , Hormônio Foliculoestimulante/farmacologia , Indicadores e Reagentes , Síndrome Nefrótica/genética , Ovário/enzimologia , Fosfoproteínas/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Ratos WistarRESUMO
Alterations at the pituitary-testicular axis has been already characterized in nephrotic syndrome. Particularly low concentrations of progesterone, testosterone and undetectable levels of estradiol have been associated to the nephrotic condition. Accordingly, to determine if the hormonal withdrawal is the result of steroidogenic failure, we evaluated the expression of testicular cytochrome P450 side-chain-cleavage (P450scc) and steroidogenic acute regulatory (StAR) protein in PAN-induced nephrotic rats. The mRNAs were evaluated on day 10 before and after hCG administration to control and nephrotic rats. The concentrations of progesterone, testosterone, estradiol, and corticosterone were measured by radioimmunoassay. The testicular and adrenal expression of P450scc and StAR were evaluated by Northern hybridization. On day 10, gonadal steroids decreased in nephrotic rats, while after hCG administration the hormonal responses from nephrotic groups were similar to the controls, except estradiol which remained undetectable. Interestingly, in nephrotic testis P450scc and StAR mRNAs were not detected neither on day 10 nor after hCG stimulation, except by the increased expression of StAR after one dose of hCG. In conclusion, this study demonstrates that the expression of P450scc and StAR protein are highly impaired during nephrotic syndrome.
Assuntos
Enzima de Clivagem da Cadeia Lateral do Colesterol/metabolismo , Síndrome Nefrótica/metabolismo , Fosfoproteínas/metabolismo , Testículo/metabolismo , Animais , Enzima de Clivagem da Cadeia Lateral do Colesterol/genética , Expressão Gênica , Masculino , Síndrome Nefrótica/enzimologia , Síndrome Nefrótica/genética , Fosfoproteínas/genética , RNA Mensageiro/genética , Ratos , Ratos Wistar , Testículo/enzimologiaRESUMO
1. The concentration of renin and angiotensinogen (Ao) and the activity of angiotensin I-converting enzyme (ACE) was measured in the ascites fluid of nephrotic rats obtained 8 days after puromycin aminonucleoside (PAN) injection. 2. Ascites fluid, serum and urine proteins of these rats were analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). 3. Renin, Ao and ACE were found in the ascites fluid and the percentage of the ratio ascites fluid/(plasma or serum) ranged from 5.9 to 9.9%. The electrophoretic analysis revealed that the ascites fluid contained low (Mr < 66 kDa) and high (Mr < 66 kDa) molecular weight proteins. Albumin and six proteins higher than 66 kDa were present both in the ascites fluid and in serum from nephrotic rats. 4. Data from the study suggest that some proteins in the ascites fluid, including renin, Ao and ACE, come from the plasma. It is possible that the loss of renin, Ao and ACE to the ascites fluid may be playing a role in the metabolic alterations of these three proteins in PAN-nephrotic rats.
Assuntos
Líquido Ascítico/química , Síndrome Nefrótica/metabolismo , Proteínas/metabolismo , Sistema Renina-Angiotensina/fisiologia , Angiotensinogênio/metabolismo , Animais , Antibióticos Antineoplásicos , Líquido Ascítico/enzimologia , Eletroforese em Gel de Poliacrilamida , Indicadores e Reagentes , Masculino , Peso Molecular , Síndrome Nefrótica/enzimologia , Síndrome Nefrótica/patologia , Peptidil Dipeptidase A/metabolismo , Proteínas/química , Puromicina Aminonucleosídeo , Radioimunoensaio , Ratos , Ratos WistarRESUMO
An oxidant stress has been shown to prevail in experimental and clinical nephrotic syndrome. Such oxidant stress may be induced by a reduced activity of antioxidant systems. We examined the altered expression of manganese-superoxide dismutase (Mn-SOD), an antioxidant enzyme, in patients with idiopathic nephrotic syndrome, in whom an increased oxidant stress had been demonstrated. The Mn-SOD activities in peripheral blood mononuclear cells obtained from 12 patients with active nephrotic syndrome (6.0 +/- 1.1 years of age, mean +/- SE) and hypoproteinemia were 42% lower (p < 0.05) than in 12 control subjects (5.5 +/- 0.5 years of age) with normal serum total protein concentrations. Reverse-transcriptase polymerase chain reaction also demonstrated that Mn-SOD messenger RNA expression in the patients with nephrotic syndrome was, on average, 59% lower than in control subjects. Because expressions of some genes are sensitive to serum, the serum dependency of Mn-SOD gene transcription was studied in glomerular endothelial cells transfected with a luciferase reporter gene fused with a rat Mn-SOD DNA fragment of -806 to +22 bp of the transcription initiation site (-806:+22). When these cells were exposed to different concentrations of fetal bovine serum (0.5% to 15%), the transcriptional activities determined by luciferase activities were proportional to serum concentrations. This serum-dependent transcriptional activation was also demonstrated by the fragment (-220:+22) but not by the fragment (-220:-20). When glomerular endothelial cells transfected with the fragment (-220:+22) were treated with 5% serum from patients with active nephrotic syndrome, transcriptional activation was more than 80% less than that by 5% serum from control subjects without nephrosis. These results indicate that Mn-SOD gene transcription is regulated at least in part by serum, and that the serum-dependent transcription of the gene is diminished in patients with idiopathic nephrotic syndrome. The regulatory region of serum-dependent gene transcription resides within its early promoter region. Our findings suggest that down-regulation of antioxidant enzyme transcription may contribute increased oxidant stress in idiopathic nephrotic syndrome.
Assuntos
Regulação Enzimológica da Expressão Gênica , Síndrome Nefrótica/genética , Superóxido Dismutase/genética , Animais , Criança , Regulação para Baixo , Feminino , Genes Reporter , Humanos , Luciferases/metabolismo , Masculino , Síndrome Nefrótica/enzimologia , Reação em Cadeia da Polimerase , Ratos , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismo , Transcrição GênicaRESUMO
Puromycin aminonucleoside (PAN)-nephrotic rats have high serum angiotensin I-converting enzyme (ACE) activity. We studied ACE activity in serum, urine, and tissues from PAN-nephrotic rats on days 2, 6, 11, and 16 after PAN injection. Proteinuria and hypoproteinemia were evident on days 6 and 11. Though significantly decreased, proteinuria was still evident on day 16. Serum ACE activity increased on days 2, 6, and 11. Urinary ACE activity became evident on days 6, 11, and 16 and correlated positively with proteinuria, suggesting that the source of urine ACE is the blood serum. ACE activity increased in testis on days 2 and 6, in lungs and aorta on days 6 and 11, in adrenal glands and small intestine on day 11, and in kidney on days 11 and 16. Heart ACE activity decreased on days 2 and 6, and increased on day 16; brain ACE activity decreased on day 6 and increased on day 11. These data implicate that changes in tissue ACE content may contribute to elevate serum ACE in PAN-nephrotic rats.
Assuntos
Síndrome Nefrótica/enzimologia , Peptidil Dipeptidase A/metabolismo , Puromicina Aminonucleosídeo/toxicidade , Animais , Proteínas Sanguíneas/análise , Hipuratos/análise , Masculino , Síndrome Nefrótica/induzido quimicamente , Peptidil Dipeptidase A/sangue , Peptidil Dipeptidase A/urina , Proteinúria/metabolismo , Ratos , Ratos Endogâmicos , Distribuição TecidualRESUMO
1. Serum angiotensin converting enzyme activity (ACEA) and plasma renin activity (PRA) were determined in rats under different experimental conditions such as: nephrotic syndrome (NS), bilateral nephrectomy (BN), renovascular hypertension (RH), dehydration (DEH), anaesthesia (AN), low sodium diet (LSD) and high sodium diet (HSD), and injection with propranolol (PRO) and isoprenaline (ISO). 2. PRA increased in LSD, AN, NS, RH, DEH and IPT groups, and decreased in HSD, BN, and PRO groups. Serum ACEA did not change in RH, HSD, IPT, DEH, AN, and PRO groups, increased in NS group, and decreased in LSD and BN groups. 3. Serum ACEA changed in the opposite direction to PRA only in the LSD group. This finding suggests that ACE may limit the full expression of the renin-angiotensin system in the LSD group, but not in the other groups.