RESUMO
Hyperglycemia and increased activity of the renal angiotensin II (ANG II) system are two primary pathogenic stimuli for the onset and progression of podocyte injury in diabetic nephropathy. However, the underlying mechanisms are not fully understood. Store-operated Ca2+ entry (SOCE) is an important mechanism that helps maintain cell Ca2+ homeostasis in both excitable and nonexcitable cells. Our previous study demonstrated that high glucose (HG) enhanced podocyte SOCE (1). It is also known that ANG II activates SOCE by releasing endoplasmic reticulum Ca2+. However, the role of SOCE in stress-induced podocyte apoptosis and mitochondrial dysfunction remains unclear. The present study was aimed to determine whether enhanced SOCE mediated HG- and ANG II-induced podocyte apoptosis and mitochondrial damage. In kidneys of mice with diabetic nephropathy, the number of podocytes was significantly reduced. In cultured human podocytes, both HG and ANG II treatment induced podocyte apoptosis, which was significantly blunted by an SOCE inhibitor, BTP2. Seahorse analysis showed that podocyte oxidative phosphorylation in response to HG and ANG II was impaired. This impairment was significantly alleviated by BTP2. The SOCE inhibitor, but not a transient receptor potential cation channel subfamily C member 6 inhibitor, significantly blunted the damage of podocyte mitochondrial respiration induced by ANG II treatment. Furthermore, BTP2 reversed impaired mitochondrial membrane potential and ATP production and enhanced mitochondrial superoxide generation induced by HG treatment. Finally, BTP2 prevented the overwhelming Ca2+ uptake in HG-treated podocytes. Taken together, our results suggest that enhanced SOCE mediated HG- and ANG II-induced podocyte apoptosis and mitochondrial injury.NEW & NOTEWORTHY This study tested the hypothesis that overwhelming store-operated Ca2+ entry is a novel mechanism contributing to high glucose- and angiotensin II-induced podocyte apoptosis and mitochondrial injury.
Assuntos
Nefropatias Diabéticas , Podócitos , Camundongos , Humanos , Animais , Nefropatias Diabéticas/metabolismo , Angiotensina II/toxicidade , Angiotensina II/metabolismo , Podócitos/metabolismo , Apoptose , Glucose/toxicidade , Glucose/metabolismoRESUMO
Neogenin, a transmembrane receptor, was recently found in kidney cells and immune cells. However, the function of neogenin signaling in kidney is not clear. Mesangial cells (MCs) are a major source of extracellular matrix (ECM) proteins in glomerulus. In many kidney diseases, MCs are impaired and manifest myofibroblast phenotype. Overproduction of ECM by the injured MCs promotes renal injury and accelerates the progression of kidney diseases. The present study aimed to determine if neogenin receptor was expressed in MCs and if the receptor signaling regulated ECM protein production by MCs. We showed that neogenin was expressed in the glomerular MCs. Deletion of neogenin using CRISPR/Cas9 lentivirus system significantly reduced the abundance of fibronectin, an ECM protein. Netrin-1, a ligand for neogenin, also significantly decreased fibronectin production by MCs and decreased neogenin protein expression in MCs. Furthermore, treatment of human MCs with high glucose (HG, 25 mM) significantly increased the protein abundance of neogenin as early as 8 h. Consistently, neogenin expression in glomerulus significantly increased in the eNOS-/-db/db diabetic mice starting as early as the age of 8 wk and this increase sustained at least to the age of 24 wk. We further found that the HG-induced increase in neogenin abundance was blunted by antioxidant PEG-catalase and N-acetyl cysteine. Taken together, our results suggest a new mechanism of regulation of fibronectin production by MCs. This previously unrecognized neogenin-fibronectin pathway may contribute to glomerular injury responses during the course of diabetic nephropathy.
Assuntos
Diabetes Mellitus Experimental , Nefropatias Diabéticas , Proteínas de Membrana , Animais , Células Cultivadas , Diabetes Mellitus Experimental/metabolismo , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/metabolismo , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Glucose/metabolismo , Proteínas de Membrana/genética , Células Mesangiais/metabolismo , Camundongos , Fatores de Transcrição/metabolismoRESUMO
Activation of immunological pathways and disturbances of extracellular matrix (ECM) dynamics are important contributors to the pathogenesis of chronic kidney diseases. Glomerular mesangial cells (MCs) are critical for homeostasis of glomerular ECM dynamics. Interleukin-6 (IL-6) can act as a pro/anti-inflammatory agent relative to cell types and conditions. This study investigated whether IL-6 influences ECM protein production by MCs and the regulatory pathways involved. Experiments were carried out in cultured human MCs (HMCs) and in mice. We found that overexpression of IL-6 and its receptor decreased the abundance of fibronectin and collagen type IV in MCs. ELISA and immunoblot analysis demonstrated that thapsigargin [an activator of store-operated Ca2+ entry (SOCE)], but not the endoplasmic reticulum stress inducer tunicamycin, significantly increased IL-6 content. This thapsigargin effect was abolished by GSK-7975A, a selective inhibitor of SOCE, and by silencing Orai1 (the channel protein mediating SOCE). Furthermore, inhibition of NF-κB pharmacologically and genetically significantly reduced SOCE-induced IL-6 production. Thapsigargin also stimulated nuclear translocation of the p65 subunit of NF-κB. Moreover, MCs overexpressing IL-6 and its receptor in HMCs increased the content of the glucagon-like peptide-1 receptor (GLP-1R), and IL-6 inhibition of fibronectin was attenuated by the GLP-1R antagonist exendin 9-39. In agreement with the HMC data, specific knockdown of Orai1 in MCs using the targeted nanoparticle delivery system in mice significantly reduced glomerular GLP-1R levels. Taken together, our results suggest a novel SOCE/NF-κB/IL-6/GLP-1R signaling pathway that inhibits ECM protein production by MCs.
Assuntos
Proteínas da Matriz Extracelular/metabolismo , Interleucina-6/metabolismo , Células Mesangiais/metabolismo , Receptores de Interleucina-6/metabolismo , Animais , Células Cultivadas , Regulação para Baixo , Proteínas da Matriz Extracelular/genética , Receptor do Peptídeo Semelhante ao Glucagon 1/genética , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Humanos , Interleucina-6/genética , Masculino , Camundongos Endogâmicos C57BL , NF-kappa B/genética , NF-kappa B/metabolismo , Proteína ORAI1/genética , Proteína ORAI1/metabolismo , Receptores de Interleucina-6/genética , Transdução de SinaisRESUMO
Sex is an important biological variable that impacts diverse physiological and pathological processes, including the progression of diabetic nephropathy. Diabetic nephropathy is one of the most common complications of diabetes mellitus and is the leading cause of end-stage renal disease. The endothelial nitric oxide synthase-deficient (eNOS-/-) db/db mouse is an appropriate and valuable model to study mechanisms in the development of diabetic nephropathy because of the similarities of the features of diabetic kidney disease in this model to those in humans. The aim of the present study was to determine whether there was a sex difference in renal injury in eNOS-/-db/db mice. Both male and female eNOS-/-db/db mice showed hyperglycemia, obesity, and renal hypertrophy. However, there was no significant difference in those variables between male and female mice. Furthermore, both male and female diabetic mice showed progressive albuminuria and significantly greater levels of serum creatinine and blood urea nitrogen compared with the same sex of wild-type mice (nondiabetic controls). Although all three variables in female eNOS-/-db/db mice had a tendency to be greater than those in male eNOS-/-db/db mice, those sex differences were not statistically significant. Moreover, both male and female eNOS-/-db/db mice showed significant mesangial expansion, higher glomerular injury scores, profound renal fibrosis, and substantial accumulation of fibronectin and collagen type IV proteins. However, sex differences in those structural changes were not observed. Similarly, survival rates of male and female eNOS-/-db/db mice were comparable. Taken together, the results from the present study suggest no sex difference in renal structural and functional damage in eNOS-/-db/db mice.
Assuntos
Nefropatias Diabéticas/enzimologia , Rim/enzimologia , Óxido Nítrico Sintase Tipo III/deficiência , Animais , Glicemia/metabolismo , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/patologia , Nefropatias Diabéticas/fisiopatologia , Modelos Animais de Doenças , Progressão da Doença , Proteínas da Matriz Extracelular/metabolismo , Feminino , Fibrose , Predisposição Genética para Doença , Hiperglicemia/sangue , Hiperglicemia/enzimologia , Hiperglicemia/genética , Rim/patologia , Rim/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico Sintase Tipo III/genética , Obesidade/enzimologia , Obesidade/genética , Obesidade/fisiopatologia , Receptores para Leptina/deficiência , Receptores para Leptina/genética , Fatores Sexuais , Micção , Aumento de PesoRESUMO
The present study was to examine sex and strain differences in glomerular filtration rate (GFR) and renal blood flow (RBF) in C57BL6, 129/Sv, and C57BLKS/J mice, three commonly used mouse strains in renal research. GFR was measured by transdermal measurement of FITC-sinitrin clearance in conscious mice. RBF was measured by a flow probe placed in the renal artery under an anesthetic state. In C57BL6 mice, there were no sex differences in both GFR and RBF. In 129/Sv mice, females had significantly greater GFR than males at age of 24 weeks, but not at 8 weeks. However, males had higher RBF and lower renal vascular resistance (RVR). Similar to 129/Sv, female C57BLKS/J had significantly greater GFR at both 8 and 24 weeks, lower RBF, and higher RVR than males. Across strains, male 129/Sv had lower GFR and higher RBF than male C57BL6, but no significant difference in GFR and greater RBF than male C57BLKS/J. No significant difference in GFR or RBF was observed between C57BL6 and C57BLKS/J mice. Deletion of eNOS in C57BLKS/J mice reduced GFR in both sexes, but decreased RBF in males. Furthermore, there were no sex differences in the severity of renal injury in eNOS-/- dbdb mice. Taken together, our study suggests that sex differences in renal hemodynamics in mice are strain and age dependent. eNOS was not involved in the sex differences in GFR, but in RBF. Furthermore, the sexual dimorphism did not impact the severity of renal injury in diabetic nephropathy.
Assuntos
Hemodinâmica , Rim , Camundongos , Masculino , Animais , Feminino , Camundongos Endogâmicos C57BL , Rim/irrigação sanguínea , Hemodinâmica/fisiologia , Circulação Renal/fisiologia , Resistência Vascular , Taxa de Filtração Glomerular/fisiologiaRESUMO
Mutations in canonical transient receptor potential-6 (TRPC6) channels give rise to rare familial forms of focal and segmental glomerulosclerosis (FSGS). Here we examined a possible role for TRPC6 in the progression of chronic puromycin aminonucleoside (PAN) nephrosis in Sprague-Dawley rats, a classic model of acquired nephrotic syndromes. We used CRISPR/Cas9 technology to delete a 239-bp region within exon 2 of the Trpc6 gene (Trpc6del allele). Trpc6del/del rats expressed detectable Trpc6 transcripts missing exon 2, and TRPC6 proteins could be detected by immunoblot of renal cortex. However, the abundance of Trpc6 transcripts and TRPC6 protein in renal cortex was much lower than in Trpc6wt/wt littermates, and functional TRPC6 channels could not be detected in whole-cell recordings from glomerular cells cultured from Trpc6del/del animals, possibly because of disruption of ankyrin repeats 1 and 2. During the chronic phase of PAN nephrosis, Trpc6del/del rats had reduced urine albumin excretion, reduced serum cholesterol and triglycerides, and improved azotemia compared to wild-type Trpc6wt/wt littermates. Glomerulosclerosis was severe during chronic PAN nephrosis in Trpc6wt/wt rats but was markedly reduced in Trpc6del/del littermates. Trpc6del/del animals also had less severe tubulointerstitial fibrosis as assessed by several biochemical and histological analyses, as well as reduced foot process effacement and glomerular basement thickening compared to Trpc6wtt/wt controls. None of the manipulations in this study affected the abundance of TRPC5 channels in renal cortex. TRPC3 was increased in PAN nephrosis and in Trpc6del/del rats. These data support a role for TRPC6 channels in driving an acquired form of secondary FSGS. KEY MESSAGES: We examined aminonucleoside nephrosis in rats with wild type and inactivated TRPC6. TRPC6 channels were inactivated by CRISPR/Cas9 editing of the Trpc6 gene. TRPC6 inactivation reduced albuminuria in the chronic but not the acute phase. TRPC6 inactivation reduced glomerulosclerosis and ultrastructural changes. TRPC6 inactivation also reduced interstitial changes and renal fibrosis.