Apoptosis of glomerular mesangial cells induced by sublytic C5b‐9 complexes in rats with Thy‐1 nephritis is dependent on Gadd45γ upregulation

Abstract

The complement C5b-9 complexes can result in cell apoptosis, but the mechanism of sublytic C5b-9-mediated glomerular mesangial cell (GMC) apoptosis in Thy-1 nephritis (Thy-1N) remains largely unclear. The Gadd45 gene is involved in the cellular response to DNA damage and can promote cell apoptosis. In this study, both Gadd45c expression patterns and pathologic changes of renal tissue were examined in rat Thy-1N. Both Gadd45c expression and GMC apoptosis were significantly decreased in Thy-1N rats upon the depletion of complement with cobra venom factor. Our in vitro studies showed that Gadd45c over-expression increased sublytic C5b-9-induced GMC apoptosis, while Gadd45c gene knockdown by siRNA greatly reduced GMC apoptosis. Moreover, Gadd45c gene silencing in vivo markedly inhibited the pathologic changes in the renal tissue of Thy-1N rats. These data suggest that Gadd45c gene expression is involved in regulating GMC apoptosis mediated by sublytic C5b-9 in Thy-1N.Key words: Apoptosis . Gadd45c . Glomerular mesangial cells . Sublytic C5b-9 .Thy-1 nephritis Supporting Information available online IntroductionMesangioproliferative glomerulonephritis (MsPGN) is a disease with high incidence in humans. Proliferation of glomerular mesangial cells (GMC) in MsPGN appears to be crucial for a subsequent increase in mesangial matrix and development of glomerulosclerosis [1,2]. Rat Thy-1 nephritis (Thy-1N), namely anti-thymocyte serum (ATS) induced nephritis, is a widely used animal model for studying human MsPGN [3,4]. ATS administered in rats binds to Thy-1 antigen on the membrane of GMC and then activates the complement system resulting in immunopathologic injury [5][6][7][8]. Complement activation results in generation of chemotactic factors, such as C5a, and formation of the C5b-9 complex [6,9]. Because GMC damage in Thy-1N is complement-dependent and neutrophil-independent, complement is generally regarded as the principal mediator of GMC lesions in the rats with Thy-1N. Assembly of C5b-9 can result in formation of transmembrane channels or rearrangement of membrane lipid with loss of membrane integrity. However, injury to nucleated cells by C5b-9 is almost non-lytic (sublytic) because the cell surface has many homologous restriction factors such as CD59 and MCP. Sublytic C5b-9 complexes can trigger [4,6,9,10]. Complement deposits are observed in the glomeruli of patients with MsPGN [1,2]. Our previous studies have also revealed the deposition of sublytic C5b-9 complexes on GMC surfaces in Thy-1N rats [9], but the role of sublytic C5b-9 complexes in Thy-1N has not been fully elucidated. During the progression of Thy-1N, GMC undergo two phases of change: early apoptosis/necrosis and secondary proliferation [4,9,11]. The mechanisms governing GMC apoptosis during the early stage of Thy-1N, including the genes involved in GMC apoptosis, remain unclear [12,13]. The growtharrest-and DNA-damage-inducible protein 45 (Gadd45) gene family, which encodes proteins that play important roles in controlling cell growth, has been im...

Role of activating transcription factor 3 (ATF3) in sublytic C5b‐9‐induced glomerular mesangial cell apoptosis

“…The formation of complement sublytic C5b-9 complexes on the GMC membrane was identified as previously described. 10,13,20 Briefly, the selection of the anti-Thy-1 antibody (Ab) and complement concentration used in the study was based on a checkerboard titration study to define the maximal amounts of anti-Thy-1 Ab and complement that could be used without producing rat GMC lysis. 10,20 To ensure that C5b-9 attack was insufficient to induce marked cell lysis, lactate dehydrogenase (LDH) was measured in the supernatants of cultured GMCs using the LDH assay kit.…”

“…The selection of anti-Thy-1 Ab and complement concentration used in our experiment was 5% anti-Thy-1 Ab and 4% normal human serum, which resulted in less than 5% LDH release. 13 In addition, to demonstrate that the effects on GMCs following various treatments were in fact due to sublytic C5b-9 stimulation, GMCs were divided into five groups (five treatments), namely MEM, Thy-1 Ab, Thy-1 Ab1HIS, Thy-1 Ab1C6DS and sublytic C5b-9 groups. 10 After timecourse analysis of ATF3 expression upon attack of sublytic C5b-9, another human serum incubation time of 3 h was decided.…”

“…The constructed plasmid was sequenced across both junctions to confirm the nucleotide sequence and the predicted orientation. 13,21 ATF3 shRNA vector generation The nucleotide sequences of shRNAs targeting the rat ATF3 gene were designed and chemically synthesized by Genechem Co. Ltd (Shanghai, China). These segments of DNA were searched in the NCBI sequence bank using the BLAST program, which confirmed no match to any genes other than the target gene, verifying the specificity of the target region by the shRNAs.…”

“…11,12 As for GMC apoptosis, there are many problems that remain unclear. 13 Thus, the molecular mechanism of GMC apoptosis mediated by sublytic C5b-9 complexes is worth exploring.…”

“…13,14 Overwhelming evidence confirms that the ATF3 gene is activated in many tissues by a variety of stress signals, including proinflammatory cytokines, ischemia and hypoxia. 15,16 Several studies have revealed that ATF3 can induce apoptosis in ovarian cancer cells 17,18 and enhance etoposide-or camptothecin-induced apoptosis in HeLa cells.…”