ABSTRACT
Podocyte dysfunction plays a crucial role in renal injury and is identified as a key contributor to proteinuria in Fabry disease (FD), primarily impacting glomerular filtration function (GFF). The α3ß1 integrins are important for podocyte adhesion to the glomerular basement membrane, and disturbances in these integrins can lead to podocyte injury. Therefore, this study aimed to assess the effects of chloroquine (CQ) on podocytes, as this drug can be used to obtain an in vitro condition analogous to the FD. Murine podocytes were employed in our experiments. The results revealed a dose-dependent reduction in cell viability. CQ at a sub-lethal concentration (1.0 µg/mL) induced lysosomal accumulation significantly (p < 0.0001). Morphological changes were evident through scanning electron microscopy and immunofluorescence, highlighting alterations in F-actin and nucleus morphology. No significant changes were observed in the gene expression of α3ß1 integrins via RT-qPCR. Protein expression of α3 integrin was evaluated with Western Blotting and immunofluorescence, demonstrating its lower detection in podocytes exposed to CQ. Our findings propose a novel in vitro model for exploring secondary Fabry nephropathy, indicating a modulation of α3ß1 integrin and morphological alterations in podocytes under the influence of CQ.
Subject(s)
Fabry Disease , Integrin alpha3beta1 , Kidney Diseases , Podocytes , Animals , Mice , Fabry Disease/metabolism , Integrin alpha3beta1/genetics , Integrin alpha3beta1/metabolism , Kidney Diseases/metabolism , Podocytes/metabolism , Renal InsufficiencyABSTRACT
Uremic toxins, such as p-cresyl sulfate (PCS) and indoxyl sulfate (IS), contribute to endothelial dysfunction in chronic kidney disease (CKD). This process is mediated by several cellular pathways, but it is unclear whether cAMP-responsive element-binding protein (CREB) and activating transcription factor 1 (ATF1) participate in endothelial dysfunction in uremic conditions despite playing roles in inflammatory modulation. This study aimed to evaluate the expression, activation, and transcriptional activity of CREB/ATF1 in endothelial cells exposed to PCS, IS, and uremic serum (US). In vitro, ATF1 protein levels were increased by PCS and IS, whereas CREB levels were enhanced only by IS. Activation through CREB-Ser133 and ATF1-Ser63 phosphorylation was induced by PCS, IS, and US. We evaluated the CREB/ATF1 transcriptional activity by analyzing the expression of their target genes, including ICAM1, PTGS2, NOX1, and SLC22A6, which are related to endothelial dysfunction through their roles in vascular inflammation, oxidative stress, and cellular uptake of PCS and IS. The expression of ICAM1, PTGS2 and NOX1 genes was increased by PCS, IS, and US, whereas that of SLC22A6 was induced only by IS. KG-501, a CREB inhibitor, restored the inductive effects of PCS on ICAM1, PTGS2, and NOX1 expression; IS on ICAM1, PTGS2 and SLC22A6 expression; and US on NOX1 expression. The presence of CREB and ATF1 was observed in healthy arteries and in arteries of patients with CKD, which were structurally damaged. These findings suggest that CREB/ATF1 is activated by uremic toxins and may play a relevant role in endothelial dysfunction in CKD.
Subject(s)
Renal Insufficiency, Chronic , Vascular Diseases , Cyclooxygenase 2/metabolism , Endothelial Cells/metabolism , Female , Humans , Indican/metabolism , Indican/toxicity , Male , Renal Insufficiency, Chronic/genetics , Renal Insufficiency, Chronic/metabolism , Uremic Toxins , Vascular Diseases/metabolismABSTRACT
p-Cresyl sulfate (PCS), indoxyl sulfate (IS), and inorganic phosphate (Pi) are uremic toxins found in chronic kidney disease (CKD) that are closely related to endothelial extracellular vesicles (EVs) formation. The present study aimed to understand the role of EVs and their role in cell adhesion and migration, inflammation, and oxidative stress. Human endothelial cells were treated with PCS, IS, and Pi in pre-established uremic and kinetic recommendations. EVs were characterized using scanning electron microscopy, flow cytometry, and NanoSight assays. The concentrations of EVs were established using Alamar Blue and MTT assays. Cell adhesion to extracellular matrix proteins was analyzed using an adhesion assay. Inflammation and oxidative stress were assessed by vascular cell adhesion molecule-1 expression/monocyte migration and reactive oxygen species production, respectively. The capacity of EVs to stimulate endothelial cell migration was evaluated using a wound-healing assay. Our data showed that endothelial cells stimulated with uremic toxins can induce the formation of EVs of different sizes, quantities, and concentrations, depending on the uremic toxin used. Cell adhesion was significantly (P < 0.01) stimulated in cells exposed to PCS-induced extracellular vesicles (PCSEVs) and inorganic phosphate-induced extracellular vesicles (PiEVs). Cell migration was significantly (P < 0.05) stimulated by PCSEVs. VCAM-1 expression was evident in cells treated with PCSEVs and IS-induced extracellular vesicles (ISEVs). EVs are not able to stimulate monocyte migration or oxidative stress. In conclusion, EVs may be a biomarker of endothelial injury and the inflammatory process, playing an important role in cell-to-cell communication and pathophysiological processes, although more studies are needed to better understand the mechanisms of EVs in uremia.
