Effect of Lacking ZKSCAN3 on Autophagy, Lysosomal Biogenesis and Senescence.

Transcription factors can affect autophagy activity by promoting or inhibiting the expression of autophagic and lysosomal genes. As a member of the zinc finger family DNA-binding proteins, ZKSCAN3 has been reported to function as a transcriptional repressor of autophagy, silencing of which can induce autophagy and promote lysosomal biogenesis in cancer cells. However, studies in Zkscan3 knockout mice showed that the deficiency of ZKSCAN3 did not induce autophagy or increase lysosomal biogenesis. In order to further explore the role of ZKSCAN3 in the transcriptional regulation of autophagic genes in human cancer and non-cancer cells, we generated ZKSCAN3 knockout HK-2 (non-cancer) and Hela (cancer) cells via the CRISPR/Cas9 system and analyzed the differences in gene expression between ZKSCAN3 deleted cells and non-deleted cells through fluorescence quantitative PCR, western blot and transcriptome sequencing, with special attention to the differences in expression of autophagic and lysosomal genes. We found that ZKSCAN3 may be a cancer-related gene involved in cancer progression, but not an essential transcriptional repressor of autophagic or lysosomal genes, as the lacking of ZKSCAN3 cannot significantly promote the expression of autophagic and lysosomal genes.

IgA nephropathy patients with partial crescent formation benefit from immunosuppressants: evidence from repeat renal biopsy.

BACKGROUND: Crescent formation is a serious pathological change in the IgA nephropathy (IgAN) which is believed to be primarily mediated by a mixture of parietal epithelial cells, macrophages, and myofibroblasts. It was recommended that IgAN patients with rapid renal hypofunction with a crescent body >50% should begin treatment with corticosteroids combined with cyclophosphamide. However, for patients with partial crescent formation, whether immunosuppressive therapy is necessary is a contested topic. MATERIALS AND METHODS: Data from IgAN patients with partial crescent formation who underwent repeat renal biopsy were retrospectively analyzed. RESULTS: From the first to the second renal biopsy, the mean hemoglobin level and albumin level increased significantly (P < 0.05), and uric acid and triglyceride levels decreased significantly (P < 0.05). Also, the 24-hour urinary protein excretion decreased significantly (P < 0.001), but no differences in blood pressure, creatinine level, or estimated glomerular filtration rate. For pathological indices, there were no differences in fluorescence intensity of IgA or C3 deposition (P > 0.05), but the mesangial cell proliferation decreased significantly (P < 0.05), and the proportions of global glomerulosclerosis and tubulointerstitial fibrosis increased significantly (P < 0.05, respectively). In addition, a decreased tendency in the proportion of crescent formation was observed in the second renal biopsy. CONCLUSIONS: Immunosuppressive therapy for IgAN patients with partial crescent formation can reduce proteinuria, stabilize renal function, improve anemia, and mitigate acute kidney injury in the short term.

The expression of renal Epstein-Barr virus markers in patients with lupus nephritis.

The aim of this study was to investigate the role of renal Epstein-Barr virus (EBV) infection in the pathogenesis of lupus nephritis (LN). A total of 58 renal tissue samples from patients with LN, seven normal renal tissue samples from patients with non-glomerular hematuria and 37 renal tissue samples from patients with minimal change nephropathy were collected. The expression of EBV-latent membrane protein-1 (EBV-LMP1) and EBV-encoded RNA 1 (EBER-1) in the renal tissue was examined by immunohistochemistry (IHC) and in situ hybridization (ISH), respectively. The sera levels of anti-nuclear antibody as well as antibodies to extractable nuclear antigen in patients with LN were also measured. An equivalence test showed that the results from the IHC and the ISH analyses had strong agreement. The positive rates of renal EBER-1 and EBV-LMP1 in the LN patients were significantly higher than those of the normal and minimal change nephropathy patients (P<0.001), while no significant difference was identified between those of the normal and minimal change nephropathy groups (P>0.05). The positive rates of EBV-LMP1 and EBER-1 in the renal tissues of patients with LN were not determined to be significantly different between the relapse (immunosuppressant-treated) and initial onset (non-treated) patients, between the patients with and without concurrent infection, and among the patients with different age ranges (P>0.05). The proportion of LN patients positive for anti-Sm antibody was significantly higher in the renal EBV-positive group than in the EBV-negative group (P<0.05), while the proportions of LN patients positive for the other autoantibodies that were examined were not identified to be significantly different between these two groups (P>0.05). The present study shows that renal EBV infection may contribute to the pathogenesis of LN by inducing anti-Sm antibody production.

Epithelial-mesenchymal transdifferentiation of renal tubular epithelial cells induced by urinary proteins requires the activation of PKC-α and ßI isozymes.

Proteinuria is a common feature for almost all glomerular diseases and reflects the severity of the glomerular lesion. The presence of a large amount of proteins in tubular fluid, however, may also contribute to the development of RIF (renal interstitial fibrosis). Endocytosis of albumin in proximal tubular cells triggers PKC (protein kinase C)-dependent generation of reactive oxygen species and secretion of chemokines. As a family including 12 isozymes, which PKC isozymes participate in RIF is still unclear. EMT (epithelial-mesenchymal transdifferentiation) of RTECs (renal tubular epithelial cells) plays a crucial role in the progress of RIF induced by proteinuria. In the present study, we investigated the role of classical PKC isozymes in the proteinuria-induced EMT of RTECs. Employing immunochemical staining, we found that PKC-α, -ßI and -ßII were expressed in glomerulus and in RTECs in both normal and diseased renal tissues, while PKC-γ was only expressed in podocytes in the glomerulus. Treatment of HK-2 cells with extracted urinary proteins resulted in EMT, as evidenced by morphological changes, decreased E-cadherin expression, increased α-SMA (α-smooth muscle actin) expression, as well as production of type I collagen and fibronectin. Western blot analysis of PKC isozymes in the cytosolic compared with membrane fraction revealed translocation of PKC-α and -ßI, but not PKC-ßII, in HK-2 cells undergoing EMT. Pretreatment with selective PKC-α inhibitor G-6976 or PKC-ß inhibitor significantly attenuated EMT induced by urinary proteins. In summary, the present study suggested that PKC-α and -ßI play critical roles in the EMT of RTECs in response to urinary proteins.

Thiazolidinedione attenuate proteinuria and glomerulosclerosis in Adriamycin-induced nephropathy rats via slit diaphragm protection.

AIM: The slit diaphragm (SD) of podocyte impairment contributes to massive proteinuria and progressive glomerulosclerosis in many human glomerular diseases. The aim of the study was to determine if thiazolidinedione (TZD) reduce proteinuria and glomerulosclerosis in focal segmental glomerulosclerosis (FSGS) by preserving the structure and function of SD. METHODS: Adriamycin-induced FSGS rat models were employed. Urinary protein content was measured dynamically during the experiment. Additional biochemical parameters in serum samples were measured after the animals were killed. Glomerular sclerosis index (SI) and podocyte foot processes fusion rate (PFR) were evaluated. The protein and mRNA expressing levels of nephrin, podocin and CD2-associated protein (CD2AP) in glomeruli were assessed by immunohistochemistry and real-time quantitative polymerase chain reaction, respectively. The density of podocytes was also evaluated after anti-Wilms' tumour-1 immunohistochemical staining. RESULTS: Rosiglitazone treatment partially reduced proteinuria, but did not significantly affect the serum levels of triglyceride, cholesterol, albumin, glucose, urea nitrogen and creatinine in Adriamycin-induced FSGS rats. Glomerular SI and podocyte foot PFR were significantly attenuated by rosiglitazone treatment. Rosiglitazone prevented the reduction of nephrin, podocin and CD2AP protein expression induced by Adriamycin, however, the mRNA expression levels of these SD-related markers did not change significantly. Rosiglitazone therapy did not reverse Adriamycin-mediated reduction of the density of podocytes. CONCLUSIONS: The study data suggest that TZD are promising therapeutic agents on FSGS, and the mechanism may be mediated in part by directly protecting the structure and function of SD.