C3a/C3aR synergies with TGF-ß to promote epithelial-mesenchymal transition of renal tubular epithelial cells via the activation of the NLRP3 inflammasome.

BACKGROUND: Complement component 3a and its receptor (C3a/C3aR) and the nucleotide-binding oligomerization domain-like receptor protein-3 (NLRP3) inflammasome contribute to epithelial-mesenchymal transition (EMT). However, the relationship between C3a/C3aR and the NLRP3 inflammasome in EMT remains unclear. This study aimed to elucidate the roles of C3a/C3aR and the NLRP3 inflammasome involved in TGF-ß-induced EMT. METHOD: Mouse renal tubular epithelial cells (TCMK-1) were exposed to C3a and TGF-ß for 48 h. C3aR antagonist, MCC950, an inhibitor of the NLRP3 inflammasome and PD98059, an inhibitor of ERK signaling, were respectively applied to pretreat the cells at 30 min before C3a and TGF-ß administration.The cells were collected for western blot, immunofluorescence staining and ELISA. Unilateral ureteral obstruction (UUO) models were established using male C57BL/6 wild-type (WT) mice and age-matched C3aR-deficient mice. MCC950 was intraperitoneally injected in UUO mice. Kidney samples were collected for immunohistochemistry staining. RESULTS: In vitro, C3a synergized with TGF-ß to promote EMT and the activation of the NLRP3 inflammasome. Inhibition of C3aR attenuated EMT and the activation of the NLRP3 inflammasome. Inhibition of the NLRP3 inflammasome alleviated EMT but didn't affect the expression of C3aR. Inhibition of ERK signaling inhibited the activation of the NLRP3 inflammasome. In vivo, the expression of IL-1ß was significantly higher in UUO mice compared to the sham-operated mice. C3aR deficiency and inhibition of the NLRP3 Inflammasome contributed to decreased IL-1ß in UUO mice. CONCLUSION: Our data revealed that C3a/C3aR synergies with TGF-ß to activate the NLRP3 inflammasome to promote epithelial-mesenchymal transition of renal tubular epithelial cells through ERK signaling, and the way in which C3aR activates the inflammasome is to promote the assembly of the NLRP3 inflammasome.

Interstitial complement C3 activation and macrophage infiltration in patients with hypertensive nephropathy
.

BACKGROUND: The interplay between interstitial complement C3 activation and macrophage infiltration might play an important role in the pathogenesis of hypertensive nephropathy (HN), but human data are limited. We sought to investigate interstitial complement C3 expression and macrophage infiltration in HN as well as the relationships between C3 activation and macrophage infiltration, their association with clinicopathologic data, and changes in renal function. MATERIALS AND METHODS: Using immunohistochemistry, we analyzed 20 renal tissue specimens from HN patients and 40 control specimens for complement C3, angiotensin (AGT), angiotensin II, and macrophage marker CD68 levels. Serum creatinine levels, estimated glomerular filtration rate (eGFR), annual rates of change in eGFR, and the interstitial fibrosis, glomerulosclerosis, and arteriolar lesion scores were recorded. RESULTS: Patients with HN showed elevated levels of interstitial C3 expression, AGT, angiotensin II, and interstitium-infiltrating macrophages compared to controls. The enhanced interstitial expression of C3 was correlated significantly with interstitial macrophage density, serum creatinine level as well as interstitial fibrosis, glomerulosclerosis, and arteriolar lesion scores, but was inversely correlated with eGFR and annual rates of change in eGFR. CONCLUSION: In human HN, inflammation involving complement C3 activation and macrophage infiltration as well as interactions between them, may play important roles in the pathogenesis and progression of interstitial fibrosis and kidney damage.
.

Bone marrow-derived mesenchymal stem cells ameliorate nephrosis through repair of impaired podocytes.

PURPOSE: The purpose of this study was to investigate the effects of bone marrow-derived mesenchymal stem cells (BMSC) on podocytes of puromycin amino nuclear glucoside (PAN) -induced nephrosis in mice. METHODS: Mice were randomly divided into Control, PAN and BMSC groups. Mice were injected with PAN (0.5 mg/g weight) via the tail vein. The 24-h urinary protein was obtained after modelling, and urinary protein excretion was determined. The blood and kidney specimens were isolated after the tenth day of modelling. Blood samples were collected for measuring serum creatinine (SCr) and blood urea nitrogen (BUN). A sample of kidney was taken for observing pathological changes through hematoxylin-eosin staining and electron microscopy, and the rest of the kidney was used for detecting the protein and mRNA expression of nephrin, CD2AP, synaptopodin, TRPC6 by real-time quantitative PCR, Western-blot and immunohistochemistry. RESULTS: After PAN injection, podocyte foot process fusion was detected by electron microscopy, and the 24 h urinary protein excretion increased compared with control mice on days 3, 7 and 10 post-PAN injection (P.

Role of complement 3 in TNF-α-induced mesenchymal transition of renal tubular epithelial cells in vitro.

Injured renal tubular epithelial cells (RTECs) have been recently thought to directly contribute to the accumulation of myofibroblasts in renal tubulointerstitial fibrosis through a process of epithelial to mesenchymal transition (EMT). However, the factors inducing RTECs to undergo EMT and the underlying mechanisms need to be further elucidated. This study aimed to determine the EMT-inducing activity of proinflammatory cytokine TNF-α and the role for complement 3 (C3) in this activity in an in vitro model of human RTECs (HK-2 cells). Wild type HK-2 cells were treated with TNF-α, IFN-γ or C3a; C3 siRNA- or control siRNA-carrying HK-2 cells were treated with TNF-α. Changes in the cell morphology and phenotype were assessed by microscopy, RT-PCR, western blotting, and immunostaining. TNF-α effectively induced HK-2 cells to express C3 and to transform into morphologically myofibroblast-like cells that lost E-cadherin (a classical epithelial cell marker) expression but acquired alpha-smooth muscle actin (α-SMA, a classical myofibroblast differentiation marker) expression. C3 siRNA robustly attenuated all the morphologic and phenotypic changes induced by TNF-α but the control siRNA showed no effect. Our preliminary observations suggest that TNF-α may induce EMT in RTECs through inducing C3 expression.

Bone marrow-derived mesenchymal stem cells differentiation into tubular epithelial-like cells in vitro.

The possibility of differentiating bone marrow-derived mesenchymal stem cells (BMSCs) into tubular epithelial-like cells is explored in vitro. Purified BMSCs from Sprague-Dawley rats were obtained by density gradient centrifugation. Third generation BMSCs were divided into six groups and were cultured under different conditions. The expression of alkaline phosphatase and cytokeratin (CK)-18 protein was detected through staining and immunocytochemistry, respectively, and the expression of E-cadherin proteins was recorded through immunofluorescence. Some cells in ischemia/reperfusion (I/R), all-trans retinoic acid (ATRA), epidermal growth factor (EGF) and bone morphogenetic protein-7 (BMP-7) groups turned positive, whereas the positive cells in the combined group significantly increased compared with the other groups. Compared with the control group, the positive expression rates of CK-18 in the I/R, ATRA, EGF, BMP-7 and the combined group were 11·50% ± 3·84%, 27·40% ± 2·70%, 29·60% ± 4·51%, 26·80% ± 5·00% and 44·00% ± 3·16%, respectively, and CK-18 mRNA expression in the combined group was obviously higher than that in the other groups (P < 0·01). Immunofluorescence detection showed that E-cadherin expression was not detectable in the control group, whereas the positive expression rates of E-cadherin in the I/R, ATRA, EGF, BMP-7 and the combined group were 6·75% ± 2·13%, 16·40% ± 2·69%, 18·25% ± 3·50%, 16·06% ± 2·00% and 30·26% ± 5·16%, respectively. The addition of ATRA, EGF and BMP-7 induces BMSCs differentiation into tubular epithelial-like cells in stimulated acute renal failure microenvironment in vitro.