RESUMO
Nephrotoxicity is a major constraint of cisplatin application in many solid tumors. Since the lack of preventive strategies, the necessity exists to identify critical molecular targets involved in cisplatin nephrotoxicity. The Purinergic ligand-gcotedion channel 7 receptor (P2X7R) is a ligand-gated ion channel that is predominantly implicated in inflammation and cell death. Our aim is to investigate the role P2X7R in cisplatin-induced acute and chronic kidney injury, as well as the underlying mechanism. In this study, we found that cisplatin can cause an increase in the expression of P2X7R in mouse kidney tissue, and P2X7R knockout can alleviate acute renal function damage caused by cisplatin, as well as the expression of kidney injury molecule 1 (KIM-1) and interleukin-18 (IL-18). Cisplatin can cause an increase in the expression of nucleotide-binding domain-like receptor protein 3 (NLRP3) inflammasome in mouse kidney tissue. Compared with wild-type mice, P2X7R -/- mice showed decreased expression of NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), cleaved Caspase-1, and cleaved IL-1ß in kidney tissue after cisplatin administration, and the apoptosis of renal tubular epithelial cells were also decreased. In addition, we also found that NLRP3 knockout can improve cisplatin induced degeneration, detachment, and necrosis of renal tubular epithelial cells. Furthermore, P2X7R -/- mice also showed reduced renal fibrosis and better long-term renal prognosis. In conclusion, our study identified that P2X7R knockout can improve cisplatin induced acute renal injury and chronic renal fibrosis by inhibiting the activation of NLRP3 inflammasome.
Assuntos
Injúria Renal Aguda , Cisplatino , Inflamassomos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR , Receptores Purinérgicos P2X7 , Animais , Cisplatino/toxicidade , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/deficiência , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Receptores Purinérgicos P2X7/metabolismo , Receptores Purinérgicos P2X7/genética , Receptores Purinérgicos P2X7/deficiência , Camundongos , Inflamassomos/metabolismo , Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/prevenção & controle , Injúria Renal Aguda/patologia , Masculino , Antineoplásicos/toxicidadeRESUMO
BACKGROUND: Renin-angiotensin system inhibitors (RASi) treatment is the basic therapy for IgA nephropathy (IgAN) patients. However, there is few of biomarker that can predict the efficacy of RASi. This study aimed to find urinary exosomal mRNAs related to the therapeutic effect of RASi in the treatment of proteinuria in IgAN patients. METHODS: We divided IgAN patients in screening cohort into A1 (proteinuria increase at 3 months), B1 (proteinuria decrease less than 50 % at 3 months), C1 (proteinuria decrease more than 50 % at 3 months) groups according to changes of proteinuria after treatment. The urinary exosomes were collected before biopsy, RNAs were extracted and analyzed with the microarray assay. The candidate genes were screened by differentially expressed genes (DEGs) analysis and then validated by quantitative real-time polymerase chain reaction (qPCR) in a validation cohort. A receiver operating characteristic (ROC) curve was used to evaluate gene performance in predicting therapeutic effect on RASi reducing proteinuria in IgAN patients. RESULTS: ECE1 and PDE1A mRNAs were significantly different among the three groups, and were gradually decreased among A1, B1 and C1 groups. In the validation cohort, the level of urinary exosomal ECE1 and PDE1A mRNAs were also significantly lower in A2 group compared with C2 group(ECE1, P < 0.001;PDE1A, P < 0.01). Besides, the level of ECE1 mRNA was also lower in B2 group compared with C2 group (P < 0.01). The ROC curve verified that urinary exosomal ECE1 and PDE1A gene level predicted RASi efficacy in IgAN patients with area under curve (AUC) 0.68 and 0.63 respectively. CONCLUSION: Urinary exosomal ECE1 and PDE1A mRNAs expression can serve as potential biomarkers for predicting the RASi efficacy to reduce proteinuria in IgAN patients.
Assuntos
Biomarcadores , Exossomos , Glomerulonefrite por IGA , RNA Mensageiro , Sistema Renina-Angiotensina , Humanos , Glomerulonefrite por IGA/tratamento farmacológico , Glomerulonefrite por IGA/urina , Glomerulonefrite por IGA/genética , Masculino , RNA Mensageiro/genética , RNA Mensageiro/urina , Adulto , Feminino , Exossomos/genética , Exossomos/metabolismo , Biomarcadores/urina , Sistema Renina-Angiotensina/efeitos dos fármacos , Sistema Renina-Angiotensina/genética , Pessoa de Meia-Idade , Inibidores da Enzima Conversora de Angiotensina/uso terapêuticoRESUMO
BACKGROUND: Uremia-associated immunodeficiency, mainly characterized by T cell dysfunction, exists in patients on maintenance hemodialysis (MHD) and promotes systemic inflammation. However, T cell senescence, one of the causes of T cell dysfunction, has not been clearly revealed yet. In this cross-sectional research, we aimed to study the manifestation of T cell premature senescence in MHD patients and further investigate the associated clinical factors. METHODS: 76 MHD patients including 33 patients with cardiovascular diseases (CVD) and 28 patients with arteriovenous fistula (AVF) event history were enrolled in this study. Complementarity determining region 3 (CDR3) of T cell receptor (TCR) was analyzed by immune repertoire sequencing (IR-Seq). CD28- T cell subsets and expression of senescence marker p16 and p21 genes were detected by multicolor flow cytometry and RT-qPCR, respectively. RESULTS: MHD patients had significantly decreased TCR diversity (P < 0.001), increased CDR3 clone proliferation (P = 0.001) and a left-skewed CDR3 length distribution. The proportion of CD4 + CD28- T cells increased in MHD patients (P = 0.014) and showed a negative correlation with TCR diversity (P = 0.001). p16 but not p21 expression in T cells was up-regulated in MHD patients (P = 0.039). Patients with CVD exhibited increased expression of p16 and p21 genes (P = 0.010 and 0.004, respectively), and patients with AVF events showed further TCR diversity and evenness reduction (P = 0.002 and 0.017, respectively) compared to patients without the comorbidities. Moreover, age, average convection volume, total cholesterol, high-density lipoprotein cholesterol and transferrin saturation were associated with TCR diversity or CD4 + CD28- T cell proportion (P < 0.05). CONCLUSIONS: MHD patients undergo T cell premature senescence characterized by significant TCR diversity reduction and repertoire skew, as well as accumulation of the CD4 + CD28- subset and up-regulation of p16 gene. Patients with CVD or AVF events show higher level of immunosenescence. Furthermore, T cell senescence in MHD patients is associated with blood cholesterol and uremic toxin retention, suggesting potential intervention strategies in the future.
Assuntos
Senescência Celular , Receptores de Antígenos de Linfócitos T , Diálise Renal , Humanos , Feminino , Masculino , Pessoa de Meia-Idade , Idoso , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Estudos Transversais , Inibidor de Quinase Dependente de Ciclina p21/genética , Linfócitos T/imunologia , Inibidor p16 de Quinase Dependente de Ciclina/genética , Antígenos CD28 , Uremia/imunologia , Regiões Determinantes de Complementaridade/genética , Adulto , Doenças Cardiovasculares/imunologia , Linfócitos T CD4-Positivos/imunologiaRESUMO
End-stage renal disease is a worldwide health burden, but the pathogenesis of uremia-associated cognitive impairment (CI) is poorly recognized. We hypothesized that uremia brings about deficiency of thiamin and folic acid and causes CI by inducing oxidative stress. Therefore, 24 Sprague-Dawley rats were randomly divided into two groups: a 5/6 nephrectomy group (n = 12) and a sham-operated group (n = 12). The Morris water maze was used to assess the cognitive function eight weeks post-surgery, and serum levels of thiamin, folic acid and homocysteine were detected subsequently. Brain and kidney tissues were collected for pathological examination and 8-Hydroxy-2'-deoxyguanosine (8-OHdG) immunochemistry staining. Results showed that the escape latency on training days 1-2 was longer, and the time in quadrant IV on experimental day 6 was significantly shorter in 5/6 nephrectomy group. Meanwhile, the uremic rats showed decreased thiamin, folic acid and increased homocysteine. We also found the time in quadrant IV was positively correlated with thiamin and folic acid level, while negatively correlated with the blood urea nitrogen and 8-OHdG positive cell proportion. Furthermore, in 5/6 nephrectomy group, the hippocampal neuron count was significantly reduced, and a greater proportion of 8-OHdG positive cells were detected. Pretreating LPS-stimulated rat microglial cells with thiamin or folic acid in vitro alleviated the inflammatory impairment in terms of cell viability and oxidative stress. In summary, we applied a uremic rat model and proved that uremia causes serum thiamin and folic acid deficiency, homocysteine elevation, along with neuron reduction and severe oxidative stress in hippocampus, finally leading to CI.
Assuntos
Insuficiência Renal , Uremia , Ratos , Animais , Ácido Fólico , Tiamina , Ratos Sprague-Dawley , Uremia/complicações , Cognição , HomocisteínaRESUMO
BACKGROUND: The relationship between Bowman's capsule thickening and progression of diabetic kidney disease (DKD) remains uncertain. METHODS: Renal biopsy specimens from 145 DKD patients and 20 control subjects were evaluated for Bowman's capsule thickness. Immunohistochemical staining assessed col4α2, laminin ß1, and albumin expression. In a discovery cohort of 111 DKD patients with eGFR ≥ 30 ml/min/1.73 m2, thickening was classified as fibrotic or exudative. The composite endpoint included CKD stage 5, dialysis initiation, and renal disease-related death. Prognosis was analyzed using Kaplan-Meier and Cox regression analyses. Two validation cohorts were included. RESULTS: Three types of thickening were observed: fibrotic, exudative, and periglomerular fibrosis. Parietal epithelial cell matrix protein accumulation contributed to fibrotic thickening, while albumin was present in exudative thickening. Bowman's capsule was significantly thicker in DKD patients (5.74 ± 2.09 µm) compared to controls (3.38 ± 0.43 µm, P < 0.01). In discovery cohort, the group of exudative thickning had a poorer prognosis(median time 20 months vs 57 months, P = 0.000). Cox multivariate analysis revealed that exudative thickening of Bowman's capsule were associated with a poor prognosis. The validation cohorts confirmed the result. CONCLUSIONS: Various mechanisms contribute to Bowman's capsule thickening in DKD. The proportion of exudative thickening may serve as a valuable prognostic indicator for DKD patients.
Assuntos
Diabetes Mellitus , Nefropatias Diabéticas , Falência Renal Crônica , Humanos , Cápsula Glomerular/metabolismo , Cápsula Glomerular/patologia , Nefropatias Diabéticas/patologia , Falência Renal Crônica/patologia , Diálise Renal , Albuminas , Diabetes Mellitus/patologiaRESUMO
Diabetic nephropathy is one of the leading causes of end-stage renal disease worldwide. In our study we found that Adenosine triphosphate (ATP) content was significantly increased in the urine of diabetic mice. We examined the expression of all purinergic receptors in the renal cortex and found that only purinergic P2X7 receptor (P2X7R) expression was significantly increased in the renal cortex of wild-type diabetic mice and that the P2X7R protein partially co-localized with podocytes. Compared with P2X7R(-/-) non-diabetic mice, P2X7R(-/-) diabetic mice showed stable expression of the podocyte marker protein podocin in the renal cortex. The renal expression of microtubule associated protein light chain 3 (LC-3II) in wild-type diabetic mice was significantly lower than in wild-type controls, whereas the expression of LC-3II in the kidneys of P2X7R(-/-) diabetic mice was not significantly different from that of P2X7R(-/-) non-diabetic mice. In vitro, high glucose induced an increase in p-Akt/Akt, p-mTOR/mTOR and p62 protein expression along with a decrease in LC-3II levels in podocytes, whereas after transfection with P2X7R siRNA, Phosphorylated protein kinase B (p-Akt)/Akt, Phosphorylated mammalian target of rapamycin (p-mTOR)/mTOR, and p62 expression were restored and LC-3II expression was increased. In addition, LC-3II expression was also restored after inhibition of Akt and mTOR signaling with MK2206 and rapamycin, respectively. Our results suggest that P2X7R expression is increased in podocytes in diabetes, and that P2X7R is involved in the inhibition of podocyte autophagy by high glucose, at least in part through the Akt-mTOR pathway, thereby exacerbating podocyte damage and promoting the onset of diabetic nephropathy. Targeting P2X7R may be a potential treatment for diabetic nephropathy.
Assuntos
Nefropatias Diabéticas , Podócitos , Camundongos , Animais , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Nefropatias Diabéticas/genética , Receptores Purinérgicos P2X7/genética , Receptores Purinérgicos P2X7/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Glucose/metabolismo , Autofagia , Mamíferos/metabolismoRESUMO
In intracellular transport, the cargo is usually simultaneously carried by two types of motor proteins that move oppositely, widely described as a "tug-of-war". We show theoretically that apart from the apparent competition, there is also a unintuitive cooperation between motors with opposite directionality. The model reproduces the in vivo experimental data with high accuracy. Under certain conditions, the cooperation can significantly increase the transport distance, rationalizing the choice of bidirectional over unidirectional transport in evolution. We further derive the exact analytical solution for the transport distance. Our results pave the road to understanding the physical nature of intracellular transport by motor proteins.
Assuntos
Dineínas , Proteínas Motores Moleculares , Transporte Biológico , Dineínas/metabolismo , Cinesinas , Modelos Biológicos , Proteínas Motores Moleculares/metabolismoRESUMO
Extracellular adenosine triphosphate (ATP) and its receptor, P2X7 receptor (P2X7R), are playing an important role in the pathological process of renal ischemia-reperfusion injury, but their underlying mechanism remains unclear. Also, the effects of tubular epithelium-expressed P2X7 receptor on ischemia acute kidney injury is still unknown. The aim of this study is to clarify if this mechanism involves the activation of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome in the renal tubular epithelial cells. In our research, we used male C57BL/6 wild type and P2X7R (-/-) mice, cultured human proximal tubular epithelial cells, and kidneys from acute kidney injury patients. Mice underwent for unilateral nephrectomy combined with the lateral renal pedicle clamping. Cultured cells were subjected to hypoxia/reoxygenation or ATP. Apyrase and A438079 were used to block the extracellular ATP/P2X7 receptor pathway. We also constructed radiation-induced bone marrow (BM) chimeras by using P2X7R (-/-) mice and P2X7R (+/+) wild-type mice. P2X7 receptor deficiency protected from renal ischemia-reperfusion injury and attenuated the formation of NLRP3 inflammasome. By using BM chimeras, we found a partial reduction of serum creatinine and less histological impairment in group wild-type BM to P2X7R (-/-) recipient, compared with group wild-type BM to wild-type recipient. In renal tubular epithelial cells, hypoxia/reoxygenation induced ATP release and extracellular ATP depletion reduced the expression of active IL-1ß. ATP activated the NLRP3 inflammasome in renal tubular epithelial cells, which were blunted by transient silence of P2X7 receptor, as well as by P2X7 receptor blocking with A438079. In human samples, we found that patients with Stage 3 AKI had higher levels of P2X7 receptor expression than patients with Stage 1 or Stage 2. Extracellular ATP/P2X7 receptor axis blocking may protect renal tubular epithelial cells from ischemia-reperfusion injury through the regulation of NLRP3 inflammasome.
Assuntos
Injúria Renal Aguda/metabolismo , Inflamação/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Traumatismo por Reperfusão/fisiopatologia , Injúria Renal Aguda/patologia , Animais , Feminino , Humanos , Inflamação/patologia , Masculino , Camundongos , Transdução de Sinais , Análise de Sobrevida , TransfecçãoRESUMO
Podocytes are terminally differentiated cells with little proliferative capacity. The high expression levels of cell cycle inhibitory proteins, including p21, p27, and p57, play an important role in maintaining the low level of proliferation of mature podocytes. In the present study, we aimed to explore the role of yes-associated protein (YAP) signalling in adriamycin-induced podocyte re-entry into the cell cycle and dedifferentiation. Proliferating cell nuclear antigen (PCNA)-, cyclin-dependent kinase 4 (CDK4)-, and Cyclin D1-positive podocytes were found in mice with adriamycin-induced nephropathy. In vitro, adriamycin administration increased the percentage of cells in S phase and the upregulation of mesenchymal-related marker proteins. CDK4 and cyclin D1 were significantly up-regulated after incubation with adriamycin. Overexpression of YAP in podocytes promoted their entry into the cell cycle; up-regulated cyclin D1, desmin, and snail2 expression and down-regulated Wilms' tumour 1 (WT1) and nephrin production. Recombinant murine FGF-basic induced podocytes to re-enter the cell cycle, inhibited WT1 and nephrin, and increased desmin and snail2 expression. Pretreating podocytes with verteporfin, an inhibitor of YAP/ TEA domain transcription factor (TEAD), decreased the adriamycin-induced overexpression of cyclin D1 and reduced the ratio of S-phase podocytes. This result was further verified by knocking down YAP expression using RNA interference. In conclusion, adriamycin induced podocytes to re-enter the cell cycle via upregulation of CDK4 and cyclin D1 expression, which was at least partly mediated by YAP signalling. Re-entry into the cell cycle induced the over-expression of mesenchymal markers in podocytes.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Ciclo Celular , Desdiferenciação Celular , Nefropatias/induzido quimicamente , Nefropatias/patologia , Podócitos/metabolismo , Podócitos/patologia , Animais , Ciclo Celular/efeitos dos fármacos , Desdiferenciação Celular/efeitos dos fármacos , Ciclina D1/metabolismo , Quinase 4 Dependente de Ciclina/metabolismo , Desmina/metabolismo , Regulação para Baixo/efeitos dos fármacos , Doxorrubicina , Fator 2 de Crescimento de Fibroblastos/farmacologia , Rim/metabolismo , Rim/patologia , Nefropatias/metabolismo , Masculino , Camundongos Endogâmicos BALB C , Podócitos/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição da Família Snail/metabolismo , Regulação para Cima/efeitos dos fármacos , Proteínas de Sinalização YAPRESUMO
BACKGROUND This study was designed to observe incidence and risk factors of low oxygenation after orthotropic liver transplantation (OLT). MATERIAL AND METHODS We retrospectively evaluated all adult patients who underwent living-donor OLT between January 1, 2017 and December 31, 2017. Postoperative low oxygenation was defined as PaO2/FiO2 <300 mmHg within 24 hours after surgery. Early acute kidney injury (AKI) after OLT was also defined when AKI was happened with 24 hours after operative. RESULTS A total of 301 patients, aged 50.35±10.29 years were enrolled. Of these patients, 100 patients (33.2%) suffered postoperative low oxygenation (PaO2/FiO2=251.80±35.84). Compared with the normal oxygenation group, body mass index (BMI) (24.48±3.53 versus 23.1±3.27 kg/m², P=0.001), preoperative hemoglobin (115.79±29.27 versus 111.52±29.80 g/L, P=0.033), preoperative MELD (22.25±6.54 versus 20.24±5.74, P=0.008), and intraoperative urinary volume (1.25 [0.76, 1.89] versus 2.04 [1.49, 3.68] mL/kg/h, P=0.003) were higher in low oxygenation group. There were more cases of earlier AKIs that occurred after OLT in low oxygenation patients than that in normal group (47% versus 23.4%, P<0.001). Logistic analysis showed that the preoperative BMI (hazard ration [HR]=1.107, [1.010, 1.212], P=0.029) and early AKI after OLT (HR=2.115, [1.161, 3.855], P=0.014) were independent risk factors for postoperative low oxygenation. CONCLUSIONS The incidence of postoperative low oxygenation after liver transplantation in adults was 33.2%. BMI and early AKI after OLT were correlated with postoperative hypoxemia.
Assuntos
Doença Hepática Terminal/cirurgia , Hipóxia/epidemiologia , Hipóxia/etiologia , Transplante de Fígado/efeitos adversos , Feminino , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Complicações Pós-Operatórias/epidemiologia , Estudos Retrospectivos , Fatores de RiscoRESUMO
Previous work showed that the activation of protein kinase A (PKA) signaling promoted mitochondrial fusion and prevented podocyte apoptosis. The cAMP response element binding protein (CREB) is the main downstream transcription factor of PKA signaling. Here we show that the PKA agonist 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate-cyclic AMP (pCPT-cAMP) prevented the production of adriamycin (ADR)-induced reactive oxygen species and apoptosis in podocytes, which were inhibited by CREB RNA interference (RNAi). The activation of PKA enhanced mitochondrial function and prevented the ADR-induced decrease of mitochondrial respiratory chain complex I subunits, NADH-ubiquinone oxidoreductase complex (ND) 1/3/4 genes, and protein expression. Inhibition of CREB expression alleviated pCPT-cAMP-induced ND3, but not the recovery of ND1/4 protein, in ADR-treated podocytes. In addition, CREB RNAi blocked the pCPT-cAMP-induced increase in ATP and the expression of peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1-α). The chromatin immunoprecipitation assay showed enrichment of CREB on PGC1-α and ND3 promoters, suggesting that these promoters are CREB targets. In vivo, both an endogenous cAMP activator (isoproterenol) and pCPT-cAMP decreased the albumin/creatinine ratio in mice with ADR nephropathy, reduced glomerular oxidative stress, and retained Wilm's tumor suppressor gene 1 (WT-1)-positive cells in glomeruli. We conclude that the upregulation of mitochondrial respiratory chain proteins played a partial role in the protection of PKA/CREB signaling.
Assuntos
Apoptose/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/genética , Complexo I de Transporte de Elétrons/genética , Podócitos/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Apoptose/efeitos dos fármacos , Células Cultivadas , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Doxorrubicina/farmacologia , Complexo I de Transporte de Elétrons/metabolismo , Masculino , Camundongos Endogâmicos BALB C , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Podócitos/efeitos dos fármacos , Interferência de RNA , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/genética , Regulação para Cima/efeitos dos fármacosRESUMO
BACKGROUND: Our previous in vitro studies suggested that cyclin AMP (cAMP) signaling protects against podocyte injury. However, the molecular mechanisms remain unknown. The aim of the present study was to explore the role of forskolin, an agonist for adenylate cyclase, on ezrin/radixin/moesin (ERM) phosphorylation and chloride intracellular channel 5 (CLIC5) expressions in injured podocytes. METHODS: ADR nephrosis model were induced by adriamycin (ADR) injection in BalB/C mice. Parts of ADR nephrosis mice were pretreated with forskolin. Albuminuria was estimated by urine Coomassie blue stain. Nephrin, synaptopodin, CLIC5, phosphorylated ERM and podocalyxin were measured by confocal microscopy. CLIC5 and phosphorylated ERM also were studied using western blotting. RhoA and Rac1 were estimated by G-Lisa kit. RESULTS: We found that forskolin partially alleviated albuminuria and width of foot processes. Nephrin, synaptopodin, phosphorylated-ERM (p-ERM) and CLIC5 expression were decreased in ADR mice, which were improved by forskolin pretreatment. In vitro studies, pretreatment of podocytes with pCPT-cAMP(PKA-selective cAMP analogue)prevented puromycin aminonucleoside (PAN)-induced CLIC5 downregulation. 8-pCPT-2'-O-Me-cAMP (2Me-cAMP, an Epac-selective cAMP analogue) blocked PAN-induced p-ERM downregulation. PAN inhibited RhoA activation in podocytes, which could be prevented by pCPT-cAMP pretreatment. Y-27632, a Rho inhibitor, decreased CLIC5 expression in podocytes. CONCLUSION: Activation cAMP signaling might attenuate albuminuria in ADR-induced nephrosis mice. Different downstream signaling pathway might mediate cAMP protection on CLIC5 and p-ERM expression, respectively.
Assuntos
Canais de Cloreto/metabolismo , AMP Cíclico/farmacologia , Proteínas do Citoesqueleto/metabolismo , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Fosforilação/efeitos dos fármacos , Podócitos/metabolismo , Animais , Antibacterianos , Canais de Cloreto/efeitos dos fármacos , Colforsina/farmacologia , Proteínas do Citoesqueleto/efeitos dos fármacos , Doxorrubicina , Masculino , Proteínas de Membrana/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Proteínas dos Microfilamentos/efeitos dos fármacos , Nefrose/induzido quimicamente , Nefrose/metabolismo , Nefrose/patologia , Podócitos/efeitos dos fármacos , Vasodilatadores/farmacologia , Proteínas rho de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTPRESUMO
Our previous in vitro studies suggested that cyclic AMP (cAMP) signaling prevents adriamycin (ADR) and puromycin aminonucleoside (PAN)-induced apoptosis in podocytes. As cAMP is an important second messenger and plays a key role in cell proliferation, differentiation and cytoskeleton formation via protein kinase A (PKA) or exchange protein directly activated by cAMP (Epac) pathways, we sought to determine the role of PKA or Epac signaling in cAMP-mediated protection of podocytes. In the ADR nephrosis model, we found that forskolin, a selective activator of adenylate cyclase, attenuated albuminuria and improved the expression of podocyte marker WT-1. When podocytes were treated with pCPT-cAMP (a selective cAMP/PKA activator), PKA activation was increased in a time-dependent manner and prevented PAN-induced podocyte loss and caspase 3 activation, as well as a reduction in mitochondrial membrane potential. We found that PAN and ADR resulted in a decrease in Mfn1 expression and mitochondrial fission in podocytes. pCPT-cAMP restored Mfn1 expression in puromycin or ADR-treated podocytes and induced Drp1 phosphorylation, as well as mitochondrial fusion. Treating podocytes with arachidonic acid resulted in mitochondrial fission, podocyte loss and cleaved caspase 3 production. Arachidonic acid abolished the protective effects of pCPT-cAMP on PAN-treated podocytes. Mdivi, a mitochondrial division inhibitor, prevented PAN-induced cleaved caspase 3 production in podocytes. We conclude that activation of cAMP alleviated murine podocyte caused by ADR. PKA signaling resulted in mitochondrial fusion in podocytes, which at least partially mediated the effects of cAMP.