The Association Between Oxidative Stress Alleviation via Sulforaphane-Induced Nrf2-HO-1/NQO-1 Signaling Pathway Activation and Chronic Renal Allograft Dysfunction Improvement.

BACKGROUND/AIMS: Chronic renal allograft dysfunction (CRAD) is a leading cause of long-term renal allograft loss. Oxidative stress may account for the nonspecific interstitial fibrosis and tubular atrophy that occur in CRAD. An antioxidant intervention via Nrf2 signaling pathway activation might be a promising therapy for some kidney diseases. The present paper investigates whether there is an association between oxidative stress alleviation via sulforaphane-induced Nrf2-HO-1/NQO-1 signaling pathway activation and CRAD improvement. METHODS: F344 rat kidneys were orthotopically transplanted into Lewis rat recipients to establish CRAD models. Sulforaphane was administered at 1.5 mg/kg intraperitoneally once daily. Renal function and 24-hour urinary protein were monitored for variations for 24 weeks after transplantation. After 24 weeks, renal histopathology was evaluated according to the Banff criteria after hematoxylin and eosin, Masson's trichrome and periodic acid-Schiff stainings. Additionally, intrarenal oxidative stress was assessed by the indicators malondialdehyde, 8-isoprostane, oxidized-low density lipoprotein and 8-hydroxy-2'-deoxyguanosine, as well as the activity levels of the antioxidant enzymes total superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and γ-glutamylcysteine synthetase. Nrf2, HO-1 and NQO-1 expression levels were determined via immunohistochemical and Western blot analyses. RESULTS: The sulforaphane-induced Nrf2-HO-1/NQO-1 signaling pathway activation, as demonstrated by immunohistochemical and Western blot analyses, delayed the progression of serum creatinine and blood urea nitrogen, particularly lowering the 24-hour urinary protein levels of CRAD. The semi-quantified histopathological changes were also alleviated. Evidence of oxidative stress alleviation, as indicated by a concurrent decrease in the indicators and sustained levels of antioxidant enzymes activity, was found in the renal allografts after sulforaphane intervention. CONCLUSION: Oxidative stress alleviation caused by continuous sulforaphane-induced Nrf2-HO-1/NQO-1 signaling pathway activation is associated with functional and morphological improvements of CRAD.

Complement induces podocyte pyroptosis in membranous nephropathy by mediating mitochondrial dysfunction.

Podocyte damage mediated by in situ complement activation in the glomeruli is a key factor in the pathogenesis of membranous nephropathy (MN), but the molecular mechanism has not been fully elucidated. Pyroptosis is a special type of programmed cell death, mediate inflammatory response and induce tissue injury. However, it is not clear whether pyroptosis is involved in the development and progression of MN. Here, we report that pyroptosis plays an important role in promoting podocyte injury in MN. We first observed the occurrence of pyroptosis in the kidneys of MN patients and validated that complement stimulation triggered pyroptosis in podocytes and that inhibiting pyroptosis reversed complement-induced podocyte damage in vitro. In addition, stimulation of complement caused mitochondrial depolarization and reactive oxygen species (ROS) production in podocytes, and inhibition of ROS reversed complement-induced pyroptosis in podocytes. Interestingly, inhibition of pyroptosis in turn partially alleviated these effects. Furthermore, we also found the involvement of pyroptosis in the kidneys of passive Heymann nephritis (PHN) rats, and inhibitors of pyroptosis-related molecules relieved PHN-induced kidney damage in vivo. Our findings demonstrate that pyroptosis plays a critical role in complement-induced podocyte damage in MN and mitochondrial dysfunction is an important mechanism underlying this process. It provides new insight that pyroptosis may serve as a novel therapeutic target for MN treatment in future studies.

Treatment of Membranous Nephropathy by Disulfiram through Inhibition of Podocyte Pyroptosis.

Introduction: Membranous nephropathy (MN) is a common chronic kidney disease in adults and a major challenge of clinical practice for its treatment. Despite major advances, since the discovery of the phospholipase A2 receptor as the major autoantigen of podocytes in MN, the mechanisms leading to glomerular damage remain elusive. Pyroptosis, a newly discovered type of programed necrotic cell death mainly mediated by gasdermin, was found to be responsible for podocyte injury in MN in our recent work. Objectives: The aim of this study was to explore the therapeutic effect of an FDA-approved drug, disulfiram (DSF), in the treatment of MN by inhibiting pyroptosis. Methods and Results: DSF significantly alleviated C3a/C5a-induced podocyte injury in vitro and renal lesions in passive Heymann nephritis (PHN) rats, as reflected by the decreased percentage of propidium iodide staining podocytes, decreased lactate dehydrogenase release from cultured podocytes and improvement in 24-h urine protein, serum albumin, serum creatinine, abnormal alterations of podocyte injury markers Desmin and WT-1 and podocyte foot process fusion in PHN rats. The protective effect of DSF on podocyte injury in vitro and in vivo can be ascribed to its inhibition of the activation and membrane translocation of the pyroptosis executor gasdermin D (GSDMD) in podocytes. DSF also inhibited the increase and activation of the pyroptosis signaling pathway NLRP3-ASC-Caspase-1/IL-18/GSDMD in C3a/C5a-treated podocytes and renal tissue of PHN rats. Conclusion: DSF is a potential drug for MN treatment, and its clinical application needs to be further investigated.

Integrin ß3 Induction Promotes Tubular Cell Senescence and Kidney Fibrosis.

Tubular cell senescence is a common biologic process and contributes to the progression of chronic kidney disease (CKD); however, the molecular mechanisms regulating tubular cell senescence are poorly understood. Here, we report that integrin ß3 (ITGB3) expression was increased in tubular cells and positively correlated with fibrosis degree in CKD patients. ITGB3 overexpression could induce p53 pathway activation and the secretion of TGF-ß, which, in turn, resulted in senescent and profibrotic phenotype change in cultured tubular cells. Moreover, according to the CMAP database, we identified isoliquiritigenin (ISL) as an agent to inhibit ITGB3. ISL treatment could suppress Itgb3 expression, attenuate cellular senescence, and prevent renal fibrosis in mice. These results reveal a crucial role for integrin signaling in cellular senescence, potentially identifying a new therapeutic direction for kidney fibrosis.

Decreased expression of sirtuin 3 protein correlates with early stage chronic renal allograft dysfunction in a rat kidney model.

Chronic renal allograft dysfunction (CRAD) is the primary factor affecting the long-term survival of patients who have undergone renal transplantation. Oxidative stress and inflammation serve an important role in the pathological damage caused by CRAD in the early post-transplantation phase. Previous studies have demonstrated that sirtuin 3 (sirt3) protects cells from oxidative stress and inflammation. A model of renal orthotopic transplantation was established in the current study and kidney samples were harvested from the rats 12 weeks following surgery. Compared with the control groups, there were significantly increased levels of serum creatinine, blood urea nitrogen and 24 h urinary protein in the allograft group (P<0.05). Pathological examinations indicated mononuclear cell infiltration and intimal proliferation in the allograft group, which had a higher Banff score compared with the control groups. There were increased levels of malondialdehyde, decreased sirt3 protein expression and decreased superoxide dismutase enzyme activity in the allograft group compared with the control groups (P<0.05). In addition, there was a negative correlation between the expression of sirt3 and 24 h urinary protein excretion, serum creatinine levels, tubulointerstitial mononuclear cell infiltration, smooth muscle cell migration in the vascular wall and Banff scores. Thus, sirt3 may serve an important protective role in the early stage of CRAD.

Zebrafish z-otu, a novel Otu and Tudor domain-containing gene, is expressed in early stages of oogenesis and embryogenesis.

Several studies have suggested that Otu domain had de-ubiquitinating activity and Tudor domain was important for the formation of germ cells. Here, we reported a novel zebrafish ovary-specific gene containing Otu and Tudor domain, z-otu, which was expressed at stages I-III oocytes and embryonic stages from zygotes to early blastula during embryonic cells maintained their totipotency. Therefore, z-otu might link the ubiquitin signaling pathway to early oogenesis and maintaining the totipotency of embryonic cell.

Sulforaphane Attenuates Contrast-Induced Nephropathy in Rats via Nrf2/HO-1 Pathway.

Background. Oxidative stress plays an important role in the pathogenesis of contrast-induced nephropathy (CIN). The aim of this study was to investigate the antioxidant effects of sulforaphane (SFN) in a rat model of CIN and a cell model of oxidative stress in HK2 cells. Methods. Rats were randomized into four groups (n = 6 per group): control group, Ioversol group (Ioversol-induced CIN), Ioversol + SFN group (CIN rats pretreated with SFN), and SFN group (rats treated with SFN). Renal function tests, malondialdehyde (MDA), and reactive oxygen species (ROS) were measured. Western blot, real-time polymerase chain reaction analysis, and immunohistochemical analysis were performed for nuclear factor erythroid-derived 2-like 2 (Nrf2) and heme oxygenase-1 (HO-1) detection. Results. Serum blood urea nitrogen (BUN), creatinine, and renal tissue MDA were increased after contrast exposure. Serum BUN, creatinine, and renal tissue MDA were decreased in the Ioversol + SFN group as compared with those in the Ioversol group. SFN increased the expression of Nrf2 and HO-1 in CIN rats and in Ioversol-induced injury HK2 cells. SFN increased cell viability and attenuated ROS level in vitro. Conclusions. SFN attenuates experimental CIN in vitro and in vivo. This effect is suggested to activate the Nrf2 antioxidant defenses pathway.

[Value of serum IgA/C3 ratio in the diagnosis of IgA nephropathy and its correlation with the clinicopathological features].

OBJECTIVE: To investigate the value of serum IgA/C3 ratio in the diagnosis of IgA nephropathy and explore its relationship with the clinicopathological features of the patients. METHODS: Sixty-six patients with IgA nephropathy, 111 with other glomerular diseases, and 40 healthy control subjects without kidney disease were tested for serum IgA and C3 levels using CRM470 adjusted standardized immune turbidimetric method, and the IgA/C3 ratio was calculated. According to Oxford and Lee's classification criteria, we analyzed the pathological grades of the renal biopsy samples from patients with IgA nephropathy. The ROC curve was used to assess the value of serum IgA and IgA/C3 ratio in predicting IgA nephropathy. RESULTS: Patients with IgA nephropathy had an elevated serum IgA/C3 ratio than those with other glomerular diseases and the control subjects, with an area under the ROC curve of 0.776. An elevated serum IgA/C3 ratio was not found to significantly correlate with the pathological grade of renal biopsy samples in patients with IgA nephropathy. CONCLUSION: In the absence of renal biopsy findings, serum IgA/C3 ratio can help in the diagnosis of IgA nephropathy.

Foxo3a inhibits cardiomyocyte hypertrophy through transactivating catalase.

The forkhead transcription factor Foxo3a is able to inhibit cardiomyocyte hypertrophy. However, its underlying molecular mechanism remains to be fully understood. Our present study demonstrates that Foxo3a can regulate cardiomyocyte hypertrophy through transactivating catalase. Insulin was able to induce cardiomyocyte hypertrophy with an elevated level of reactive oxygen species (ROS). The antioxidant agents, including catalase and N-acetyl-L-cysteine, could inhibit cardiomyocyte hypertrophy induced by insulin, suggesting that ROS is necessary for insulin to induce hypertrophy. Strikingly, we observed that the levels of catalase were decreased in response to insulin treatment. The transcriptional activity of Foxo3a depends on its phosphorylation status with the nonphosphorylated but not phosphorylated form to be functional. Insulin treatment led to an increase in the phosphorylated levels of Foxo3a. To understand the relationship between Foxo3a and catalase in the hypertrophic pathway, we characterized that catalase was a transcriptional target of Foxo3a. Foxo3a bound to the promoter region of catalase and stimulated its activity. The inhibitory effect of Foxo3a on cardiomyocyte hypertrophy depended on its transcriptional regulation of catalase. Finally, we identified that myocardin was a downstream mediator of ROS in conveying the hypertrophic signal of insulin or insulin-like growth factor-1. Foxo3a could negatively regulate myocardin expression levels through up-regulating catalase and the consequent reduction of ROS levels. Taken together, our results reveal that Foxo3a can inhibit hypertrophy by transcriptionally targeting catalase.

Cloning and characterization of a rice field eel vasa-like gene cDNA and its expression in gonads during natural sex transformation.

The vasa (vas)-related gene encodes an RNA helicase protein member of the DEAD-box family and plays key roles in germ-cell formation in higher metazoans. Using degenerate PCR and RACE, we cloned the vasa gene of the rice field eel (Monopterus albus), which is homologous to the Drosophila vasa gene. We named it ma-vas (Monopterus albus vas). Ma-vas encodes a protein of 618 amino acids, which contains all of the known characteristics of vasa homologs. RT-PCR analysis revealed that ma-vas was exclusively expressed in the gonads of the female, intersex, and male. During gonadal natural sex reversal, ma-vas is expressed in oocytes at all stages of oogenesis, in degenerating oocytes of ovotestis, and in spermatogonia and spermatocytes at early stages. The vasa positive signal was also observed in the peripheral layer of late ovary. It was not found, however, in that layer of the testis. Alkaline phosphatase (AKP) staining on the ovary and testis also indicated that some cells had differentiational potential in the peripheral layer of the ovary, suggesting that spermatogonia might arise from cells with AKP and vasa-positive staining in the peripheral layer of the female gonad.

Cloning and characterization of full length of a novel zebrafish gene Zsrg abundantly expressed in the germline stem cells.

Using the digital differential display program of the National Center for Biotechnology Information, we identified a contig of expression sequence tags (ESTs) (Accession No. BM316936), which came from zebrafish ovary and testis libraries. The full-length cDNA of this transcript was cloned and further confirmed by polymerase chain reaction and sequencing. The full-length cDNA of the novel gene is 807bp and encodes a novel protein of 187 amino acids, which shares no significant homology with any other known proteins. Characterization of genomic sequences of the gene revealed that it spans 6kb on the linkage group 3 and is composed of five exons and four introns. RT-PCR analysis showed that it was expressed in mature oocytes and one-cell stage, and persisted until 24h of development. RT-PCR also revealed that it is expressed in gonad and kidney, with the highest level of expression in the testis. The expression sites of the novel gene in adult gonad were further localized by in situ hybridization to oogonia and growing oocytes in ovary and to spermatogonia, spermatocytes but not to spermatids in testis. Based on its abundance in testis and the germline stem cell-spermatogonia and oogonia, we hypothesize that it may function as a testicular development and gametogenesis related gene that plays important roles in spermatogenesis, and named it Zsrg (zebrafish testis spermatogenesis related gene, Zsrg).