Background To establish a model of chronic renal fibrosis following acute kidney injury (AKI) in BALB/c mice and to observe the effect of AKI on podocyte injury and chronic fibrosis of the kidney. Additional aims included using the model to explore the role of podocyte injury in AKI and post-injury fibrosis. Methods Fifty BALB/C mice were randomly divided into control group (Ctr), sham group (sham), AKI 20 group (renal ischemia, 20 min reperfusion), AKI 30 group (renal ischemia, 30 min reperfusion) and AKI 40 group (renal ischemia, 40 min reperfusion). Mice serum and 24-h urine were collected on the 8th, 9th, 10th, 14th, and 28th days for urinary protein, serum creatinine (Scr) and blood urea nitrogen (BUN) analysis. HE staining, transmission electron microscopy (TEM), Masson staining, Q-PCR, Western Blot and immunohistochemistry were applied. Results Serum Scr and BUN levels across all AKI groups at the 9th day were significantly higher ( P < 0.05) than controls, with higher reperfusion groups maintaining that increase up to 28 days ( P < 0.05). Compared with Ctr group, the urinary protein of the AKI 40 group significantly rose on the 9th day ( P < 0.05), normalizing immediately on the 10th day ( P < 0.05). In contrast, the AKI 30 group rose significantly on the 14th day ( P < 0.05) maintaining elevated levels for two weeks ( P < 0.05). HE staining demonstrated ischemia-dependent renal tissue damage was aggravated in the mild to aggravated AKI groups. Mesangial proliferation, glomerulosclerosis, and tubulointerstitial pathology were also significantly increased in these groups ( P < 0.05). Masson staining further showed that glomerular, renal tubular, and interstitial collagen were increased by ischemia in a time-dependent manner. Transmission EM additionally that podocytes of the mild to severe AKI groups displayed extensive fusion, exfoliation and GBM exposure. Synaptopodin, Nephrin, and CD2AP mRNA and protein expression demonstrated ischemic time-dependent decreases, while the TRPC6 was increased. There was a significant difference in the levels of Synaptopodin, Nephrin, CD2AP, and TRPC6 between the mild and severe AKI groups ( P < 0.05). Conclusions 1) During the AKI process mice podocyte injury, proteinuria and the subsequent progression into chronic renal fibrosis is observed.2) Podocyte injury may be one of the causes of ischemia-reperfusion acute kidney injury and post-injury fibrosis. Electronic supplementary material The online version of this article (10.1186/s12882-019-1298-x) contains supplementary material, which is available to authorized users.

The impact of the renal microenvironment on macrophage phenotype determination can contribute to the progression or resolution of renal fibrosis. Although the complement proteins affect macrophage polarization, whether complement component 3 (C3) can induce macrophage polarization and regulate renal interstitial fibrosis remains undetermined. In the present study, we investigated the contribution of C3 on macrophage polarization and renal fibrosis in C3-deficient mice with unilateral ureteral obstruction and bone marrow-derived macrophages. C3-deficient mice exhibited attenuated renal fibrosis and ameliorated peritubular capillary rarefaction. Lack of C3 contributed to M2 macrophage polarization, increased IL-10 and VEGF164, and decreased TNF-α and soluble VEGF receptor 1 expression in the obstructed kidneys at the early stages of unilateral ureteral obstruction. C3a facilitated LPS-induced M1 polarization and inflammatory factor production in bone marrow-derived macrophages in vitro, accompanied by increased ERK, NF-κB, and STAT1 phosphorylation. The ERK-specific inhibitor PD98059 inhibited the phosphorylation of ERK, NF-κB, and STAT1 and attenuated M1 polarization-related inflammatory factor production. Furthermore, the culture supernatant from M1 macrophages and C3a-treated M2 macrophages were more detrimental to angiogenesis compared with M2 macrophage supernatants. Thus, complement C3 exacerbates renal interstitial fibrosis by facilitating macrophage M1 polarization, promoting proinflammatory cytokine expression, and deteriorating peritubular capillary rarefaction in the kidney.

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.

Complement C3 plays a prominent role in inflammatory processes, and its increase exacerbates ischemia reperfusion injury (IRI)-induced acute kidney injury (AKI). Infiltrated neutrophils can be stimulated to form neutrophil extracellular traps (NETs), leading to renal injury. However, the relationship between the increase of C3 and the release of NETs in AKI was not clear. Here we found that IRI in the mouse kidney leads to increased neutrophils infiltration and NET formation. Furthermore, neutrophils depletion by anti-Ly6G IgG (1A8) did not reduce C3 activation but reduced kidney injury and inflammation, indicating a link between neutrophils infiltration and renal tissue damage. Pretreatment with 1A8 suppressed ischemia-induced NET formation, proving that extracellular traps (ETs) in renal tissue were mainly derived from neutrophils. Renal ischemia injury also leads to increased expression of C3. Moreover, C3 KO mice (C3 KO) with IRI exhibited attenuated kidney damage and decreased neutrophils and NETs. In vitro, C3a primed neutrophils to form NETs, reflected by amorphous extracellular DNA structures that colocalized with CitH3 and MPO. These data reveal that C3 deficiency can ameliorate AKI by reducing the infiltration of neutrophils and the formation of NETs. Targeting C3 activation may be a new therapeutic strategy for alleviating the necroinflammation of NETs in AKI.

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.