<i>In</i><i>situ</i> analysis for LAMA4⁺, TCF21⁺ and KCNJ1⁺ using confocal laser scanning microscopy.

<p>RCC tumor tissues (T) were characterized by a significant increase of LAMA4 expression (<b>B</b>) and decrease of TCF21 (<b>E</b>) and KCNJ1 (<b>H</b>) expression as compared to non-tumoral (NT) kidney portion (<b>A</b>, <b>D</b>, <b>G</b>). Nuclei are highlighted with TO-PRO in blue, Quantification of specific protein expression was obtained as described in the Methods section, (<b>C</b>, <b>F</b>, <b>I</b>). Results are expressed as mean ± S.D. For each group, all images (magnification 63X) are from a single patient and are representative of the whole group of patients.</p

Gene Expression analysis of KCNJ1, SFRP1 and TCF21 genes.

<p>Expression levels of KCNJ1 (<b>A</b>), SFRP1 (<b>B</b>) and TCF21 (<b>C</b>) genes were calculated relative to the mean expression levels of ACTB and RPL13 genes. From left to right: box plot of log₂ gene expression intensities resulting from microarray analysis (each dot stands for a sample); histogram of the expression levels (mean ± SE) of target genes in NT and ccRCC samples; histogram of the expression levels (mean ± SE) of target genes in NT and G1, G2 and G3 ccRCC samples. * = p-value < 0.05; # = p-value < 0.01; ** = p-value < 0.001. </p

qRT-PCR analysis of differentially alternative splicing exons identified in DAB2 gene.

<p>Left panel (<b>A</b>) shows the percentage of inclusion of the skipped exon in tumoral (T) and non-tumoral (NT) tissues, calculated as the average of the expression ratio of skipped exon relative to both upstream exon and the downstream one. Right panel (<b>B</b>) shows the relative expression ratio (mean ± SE) of each exon of the triplet involved in the splicing event both in NT and T samples. Expression levels were calculated relative to the mean expression levels of ACTB and RPL13 genes. * = p-value < 0.001.</p

Gene Expression analysis of PTP4A3, CAV2 and LAMA4 genes.

<p>Expression levels of PTP4A3 (<b>A</b>), CAV2 (<b>B</b>) and LAMA4 (<b>C</b>) genes were calculated relative to the mean expression levels of ACTB and RPL13 genes. From left to right: box plot of log₂ gene expression intensities resulting from microarray analysis (each dot stands for a sample); histogram of the expression levels (mean ± SE) of target genes in NT and ccRCC samples; histogram of the expression levels (mean ± SE) of target genes in NT and G1, G2 and G3 ccRCC samples. * = p-value < 0.05; # = p-value < 0.01; ** = p-value < 0.001.</p

image_4_Complement Activation During Ischemia/Reperfusion Injury Induces Pericyte-to-Myofibroblast Transdifferentiation Regulating Peritubular Capillary Lumen Reduction Through pERK Signaling.tif

<p>Pericytes are one of the principal sources of scar-forming myofibroblasts in chronic kidneys disease. However, the modulation of pericyte-to-myofibroblast transdifferentiation (PMT) in the early phases of acute kidney injury is poorly understood. Here, we investigated the role of complement in inducing PMT after transplantation. Using a swine model of renal ischemia/reperfusion (I/R) injury, we found the occurrence of PMT after 24 h of I/R injury as demonstrated by reduction of PDGFRβ⁺/NG2⁺ cells with increase in myofibroblasts marker αSMA. In addition, PMT was associated with significant reduction in peritubular capillary luminal diameter. Treatment by C1-inhibitor (C1-INH) significantly preserved the phenotype of pericytes maintaining microvascular density and capillary lumen area at tubulointerstitial level. In vitro, C5a transdifferentiated human pericytes in myofibroblasts, with increased αSMA expression in stress fibers, collagen I production, and decreased antifibrotic protein Id2. The C5a-induced PMT was driven by extracellular signal-regulated kinases phosphorylation leading to increase in collagen I release that required both non-canonical and canonical TGFβ pathways. These results showed that pericytes are a pivotal target of complement activation leading to a profibrotic maladaptive cellular response. Our studies suggest that C1-INH may be a potential therapeutic strategy to counteract the development of PMT and capillary lumen reduction in I/R injury.</p

image_1_Complement Activation During Ischemia/Reperfusion Injury Induces Pericyte-to-Myofibroblast Transdifferentiation Regulating Peritubular Capillary Lumen Reduction Through pERK Signaling.tif

<p>Pericytes are one of the principal sources of scar-forming myofibroblasts in chronic kidneys disease. However, the modulation of pericyte-to-myofibroblast transdifferentiation (PMT) in the early phases of acute kidney injury is poorly understood. Here, we investigated the role of complement in inducing PMT after transplantation. Using a swine model of renal ischemia/reperfusion (I/R) injury, we found the occurrence of PMT after 24 h of I/R injury as demonstrated by reduction of PDGFRβ⁺/NG2⁺ cells with increase in myofibroblasts marker αSMA. In addition, PMT was associated with significant reduction in peritubular capillary luminal diameter. Treatment by C1-inhibitor (C1-INH) significantly preserved the phenotype of pericytes maintaining microvascular density and capillary lumen area at tubulointerstitial level. In vitro, C5a transdifferentiated human pericytes in myofibroblasts, with increased αSMA expression in stress fibers, collagen I production, and decreased antifibrotic protein Id2. The C5a-induced PMT was driven by extracellular signal-regulated kinases phosphorylation leading to increase in collagen I release that required both non-canonical and canonical TGFβ pathways. These results showed that pericytes are a pivotal target of complement activation leading to a profibrotic maladaptive cellular response. Our studies suggest that C1-INH may be a potential therapeutic strategy to counteract the development of PMT and capillary lumen reduction in I/R injury.</p

image_2_Complement Activation During Ischemia/Reperfusion Injury Induces Pericyte-to-Myofibroblast Transdifferentiation Regulating Peritubular Capillary Lumen Reduction Through pERK Signaling.tif

<p>Pericytes are one of the principal sources of scar-forming myofibroblasts in chronic kidneys disease. However, the modulation of pericyte-to-myofibroblast transdifferentiation (PMT) in the early phases of acute kidney injury is poorly understood. Here, we investigated the role of complement in inducing PMT after transplantation. Using a swine model of renal ischemia/reperfusion (I/R) injury, we found the occurrence of PMT after 24 h of I/R injury as demonstrated by reduction of PDGFRβ⁺/NG2⁺ cells with increase in myofibroblasts marker αSMA. In addition, PMT was associated with significant reduction in peritubular capillary luminal diameter. Treatment by C1-inhibitor (C1-INH) significantly preserved the phenotype of pericytes maintaining microvascular density and capillary lumen area at tubulointerstitial level. In vitro, C5a transdifferentiated human pericytes in myofibroblasts, with increased αSMA expression in stress fibers, collagen I production, and decreased antifibrotic protein Id2. The C5a-induced PMT was driven by extracellular signal-regulated kinases phosphorylation leading to increase in collagen I release that required both non-canonical and canonical TGFβ pathways. These results showed that pericytes are a pivotal target of complement activation leading to a profibrotic maladaptive cellular response. Our studies suggest that C1-INH may be a potential therapeutic strategy to counteract the development of PMT and capillary lumen reduction in I/R injury.</p

image_3_Complement Activation During Ischemia/Reperfusion Injury Induces Pericyte-to-Myofibroblast Transdifferentiation Regulating Peritubular Capillary Lumen Reduction Through pERK Signaling.tif

<p>Pericytes are one of the principal sources of scar-forming myofibroblasts in chronic kidneys disease. However, the modulation of pericyte-to-myofibroblast transdifferentiation (PMT) in the early phases of acute kidney injury is poorly understood. Here, we investigated the role of complement in inducing PMT after transplantation. Using a swine model of renal ischemia/reperfusion (I/R) injury, we found the occurrence of PMT after 24 h of I/R injury as demonstrated by reduction of PDGFRβ⁺/NG2⁺ cells with increase in myofibroblasts marker αSMA. In addition, PMT was associated with significant reduction in peritubular capillary luminal diameter. Treatment by C1-inhibitor (C1-INH) significantly preserved the phenotype of pericytes maintaining microvascular density and capillary lumen area at tubulointerstitial level. In vitro, C5a transdifferentiated human pericytes in myofibroblasts, with increased αSMA expression in stress fibers, collagen I production, and decreased antifibrotic protein Id2. The C5a-induced PMT was driven by extracellular signal-regulated kinases phosphorylation leading to increase in collagen I release that required both non-canonical and canonical TGFβ pathways. These results showed that pericytes are a pivotal target of complement activation leading to a profibrotic maladaptive cellular response. Our studies suggest that C1-INH may be a potential therapeutic strategy to counteract the development of PMT and capillary lumen reduction in I/R injury.</p