Role of TFEB in autophagic modulation of ischemia reperfusion injury in mice kidney and protection by urolithin A.

Kidney ischemia reperfusion injury (IRI) is an acute kidney injury associated with high number of mortality. We have examined the molecular mechanism and found that oxidative stress and hypoxia leads to induction of autophagy. In IRI induced autophagy, TFEB translocated to nucleus in response to IRI and induced a number of target genes of Coordinated Lysosomal Expression and Regulation (CLEAR) network. Real-time PCR analyses result showed IRI dependent increase in mRNA level to lysosomal hydrolases (Ctsa, Psap), lysosomal membranes (Lamp1), lysosomal acidification (Atp6ap1) non-lysosomal proteins involved in lysosomal biogenesis (M6pr, Nagpa) and autophagy (Becn1, VPS11). Overall, both lysosomal biogenesis and autophagy pathways were induced. Two key players of TFEB dependent proteins in autophagy, LAMP1 and BECN1 were verified by protein analyses. Pretreatment with urolithin A promoted autophagy and attenuated renal injury in kidney IRI and thus inverse relationship existed between TFEB-CLEAR pathway and kidney injury. Urolithin A also attenuated IRI induced pro-inflammatory cytokines TNFα, IL1ß, MIP1α and MIP2 mRNA and associated kidney injury. Overall, our results explored the understanding of autophagy and CLEAR network to kidney IRI and those insights may help to develop new therapeutic strategies to protect against IRI.

Protective effect of urolithin a on cisplatin-induced nephrotoxicity in mice via modulation of inflammation and oxidative stress.

Limitation of widely used anti-cancer agent cisplatin for a patient is nephrotoxicity. Nephrotoxicity is presentable in mice by injecting cisplatin at 25 mg/kg with 3 days endpoint. We used the same model to understand the protective role of urolithin A. Cisplatin-induced renal damages measured by histological damage in proximal tubular cells and by the increase in serum neutrophil gelatinase-associated lipocalin (NGAL), blood urea nitrogen (BUN), creatinine and urinary Kidney Injury Molecule-1 (KIM-1). Urolithin A pretreatment reduced all the above renal damage parameters in a significant way. Urolithin A attenuated cisplatin-induced pro-inflammatory cytokine/chemokine tumor necrosis factor α (TNFα), interleukin 23 (IL-23), interleukin 18 (IL-18) and macrophage inflammatory protein 2 (MIP2). Cisplatin-induced CD11b positive macrophages in kidneys reduced by urolithin A. Urolithin A also attenuated cisplatin-induced renal oxidative/nitrative stress, which was measured by lipid peroxidation(4-hydroxy-2-nonenal or 4-HNE protein adducts) and protein nitration. Urolithin A cisplatin-induced kidney injury in mice through the down regulation of inflammatory cytokines/chemokine, immune cells, and oxidative/nitrative stress thus improving cisplatin-induced proximal tubular cell death.

Baicalin Ameliorates Experimental Liver Cholestasis in Mice by Modulation of Oxidative Stress, Inflammation, and NRF2 Transcription Factor.

Experimental cholestatic liver fibrosis was performed by bile duct ligation (BDL) in mice, and significant liver injury was observed in 15 days. Administration of baicalin in mice significantly ameliorates liver fibrosis. Experimental cholestatic liver fibrosis was associated with induced gene expression of fibrotic markers such as collagen I, fibronectin, alpha smooth muscle actin (SMA), and connective tissue growth factor (CTGF); increased inflammatory cytokines (TNFα, MIP1α, IL1ß, and MIP2); increased oxidative stress and reactive oxygen species- (ROS-) inducing enzymes (NOX2 and iNOS); dysfunctional mitochondrial electron chain complexes; and apoptotic/necrotic cell death markers (DNA fragmentation, caspase 3 activity, and PARP activity). Baicalin administration on alternate day reduced fibrosis along with profibrotic gene expression, proinflammatory cytokines, oxidative stress, and cell death whereas improving the function of mitochondrial electron transport chain. We observed baicalin enhanced NRF2 activation by nuclear translocation and induced its target genes HO-1 and GCLM, thus enhancing antioxidant defense. Interplay of oxidative stress/inflammation and NRF2 were key players for baicalin-mediated protection. Stellate cell activation is crucial for initiation of fibrosis. Baicalin alleviated stellate cell activation and modulated TIMP1, SMA, collagen 1, and fibronectin in vitro. This study indicates that baicalin might be beneficial for reducing inflammation and fibrosis in liver injury models.

Ameliorative Effect of Daidzein on Cisplatin-Induced Nephrotoxicity in Mice via Modulation of Inflammation, Oxidative Stress, and Cell Death.

Oxidative stress and inflammation are part and parcel of cisplatin-induced nephrotoxicity. The purpose of this work is to study the role of soy isoflavone constituent, daidzein, in cisplatin-induced renal damage. Cisplatin-induced nephrotoxicity was evident by the histological damage in proximal tubular cells and by the increase in serum neutrophil gelatinase-associated lipocalin (NGAL), blood urea nitrogen (BUN), creatinine, and urinary kidney injury molecule-1 (KIM-1). Cisplatin-induced cell death was shown by TUNEL staining and caspase-3/7 activity. Daidzin treatment reduced all kidney injury markers (NGAL, BUN, creatinine, and KIM-1) and attenuated cell death (apoptotic markers). In cisplatin-induced kidney injury, renal oxidative/nitrative stress was manifested by the increase in lipid peroxidation and protein nitration. Cisplatin induced the reactive oxygen species-generating enzyme NOX-2 and impaired antioxidant defense enzyme activities such as glutathione peroxidase (GPX) and superoxide dismutase (SOD) activities. Cisplatin-induced oxidative/nitrative stress was attenuated by daidzein treatment. Cisplatin induced CD11b-positive macrophages in kidneys and daidzein attenuated CD11b-positive cells. Daidzein attenuated cisplatin-induced inflammatory cytokines tumor necrosis factor α (TNFα), interleukin 10 (IL-10), interleukin 18 (IL-18), and monocyte chemoattractant protein-1 (MCP-1). Daidzein attenuated cell death in vitro. Our data suggested that daidzein attenuated cisplatin-induced kidney injury through the downregulation of oxidative/nitrative stress, immune cells, inflammatory cytokines, and apoptotic cell death, thus improving kidney regeneration.

Hsp27 Acts as a Master Molecular Chaperone and Plays an Essential Role in Hepatocellular Carcinoma Progression.

AIMS: Hsp27, a master molecular chaperone, plays an important role in cancer. However, the specific co-chaperones that partner with Hsp27 and the role of Hsp27 in hepatocellular carcinoma (HCC) are not fully enumerated. The present study focuses on the role of Hsp27 in HCC and explores its potential co-chaperones in HCC development. METHODS: Gene overexpression or knockdown was used to observe the role of Hsp27 in HCC. Co-immunoprecipitation and mass spectrometry were used to explore apoptosis resistance by regulating multiple co-chaperones of Hsp27. Hsp27 protein-protein interaction (PPI) networks were constructed by the MetaCore software. RESULTS: Hsp27 was upregulated in HCC tissues, and Hsp27 overexpression significantly facilitated formation of HCC cell colony and invasion in normoxia and tolerance in hypoxia by interacting with HIF-1α. Next, the analysis of microarrays revealed that Hsp27 regulated several cellular signaling pathways, including Wnt, ErbB and TGF-ß signaling. Moreover, we characterized the Hsp27 PPI map, which indicated that Hsp27 along with its co-chaperones formed different complexes and exerts transcription regulation activity by activating sp1, c-Myc, p53 and ESR1. CONCLUSIONS: Hsp27 along with its co-chaperones was related to the development of HCC by regulating multiple signaling pathways, and drugs that target Hsp27 along with its co-chaperones may be a potential therapy for HCC.

Pharmacological inhibition of NADPH oxidase protects against cisplatin induced nephrotoxicity in mice by two step mechanism.

BACKGROUND: Cisplatin induced nephrotoxicity is primarily caused by ROS (Reactive Oxygen Species) induced proximal tubular cell death. NADPH oxidase is major source of ROS production by cisplatin. Here, we reported that pharmacological inhibition of NADPH oxidase by acetovanillone (obtained from medicinal herb Picrorhiza kurroa) led to reduced cisplatin nephrotoxicity in mice. METHODS: In this study we used various molecular biology and biochemistry methods a clinically relevant model of nephropathy, induced by an important chemotherapeutic drug cisplatin. RESULTS: Cisplatin-induced nephrotoxicity was evident by histological damage from loss of the tubular structure. The damage was also marked by the increase in blood urea nitrogen, creatinine, protein nitration as well as cell death markers such as caspase 3/7 activity and DNA fragmentation. Tubular cell death by cisplatin led to pro-inflammatory response by production of TNFα and IL1ß followed by leukocyte/neutrophil infiltration which resulted in new wave of ROS involving more NADPH oxidases. Cisplatin-induced markers of kidney damage such as oxidative stress, cell death, inflammatory cytokine production and nephrotoxicity were attenuated by acetovanillone. In addition to that, acetovanillone enhanced cancer cell killing efficacy of cisplatin. CONCLUSION: Thus, pharmacological inhibition of NADPH oxidase can be protective for cisplatin-induced nephrotoxicity in mice.

Epigallocatechin 3-gallate ameliorates bile duct ligation induced liver injury in mice by modulation of mitochondrial oxidative stress and inflammation.

Cholestatic liver fibrosis was achieved by bile duct ligation (BDL) in mice. Liver injury associated with BDL for 15 days included significant reactive oxygen/nitrogen species generation, liver inflammation, cell death and fibrosis. Administration of Epigallocatechin 3-Gallate (EGCG) in animals reduced liver fibrosis involving parenchymal cells in BDL model. EGCG attenuated BDL-induced gene expression of pro-fibrotic markers (Collagen, Fibronectin, alpha 2 smooth muscle actin or SMA and connective tissue growth factor or CTGF), mitochondrial oxidative stress, cell death marker (DNA fragmentation and PARP activity), NFκB activity and pro-inflammatory cytokines (TNFα, MIP1α, IL1ß, and MIP2). EGCG also improved BDL induced damages of mitochondrial electron transport chain complexes and antioxidant defense enzymes such as glutathione peroxidase and manganese superoxide dismutase. EGCG also attenuated hydrogen peroxide induced cell death in hepatocytes in vitro and alleviate stellate cells mediated fibrosis through TIMP1, SMA, Collagen 1 and Fibronectin in vitro. In conclusion, the reactive oxygen/nitrogen species generated from mitochondria plays critical pathogenetic role in the progression of liver inflammation and fibrosis and this study indicate that EGCG might be beneficial for reducing liver inflammation and fibrosis.

Mitochondrial modulation by Epigallocatechin 3-Gallate ameliorates cisplatin induced renal injury through decreasing oxidative/nitrative stress, inflammation and NF-kB in mice.

Cancer chemotherapy drug cisplatin is known for its nephrotoxicity. The aim of this study is to investigate whether Epigallocatechin 3-Gallate (EGCG) can reduce cisplatin mediated side effect in kidney and to understand its mechanism of protection against tissue injury. We used a well-established 3-day cisplatin induced nephrotoxicity mice model where EGCG were administered. EGCG is a major active compound in Green Tea and have strong anti-oxidant and anti-inflammatory properties. EGCG protected against cisplatin induced renal dysfunction as measured by serum creatinine and blood urea nitrogen (BUN). EGCG improved cisplatin induced kidney structural damages such as tubular dilatation, cast formation, granulovaculoar degeneration and tubular cell necrosis as evident by PAS staining. Cisplatin induced kidney specific mitochondrial oxidative stress, impaired activities of mitochondrial electron transport chain enzyme complexes, impaired anti-oxidant defense enzyme activities such as glutathione peroxidase (GPX) and manganese superoxide dismutase (MnSOD) in mitochondria, inflammation (tumor necrosis factor α and interleukin 1ß), increased accumulation of NF-κB in nuclear fraction, p53 induction, and apoptotic cell death (caspase 3 activity and DNA fragmentation). Treatment of mice with EGCG markedly attenuated cisplatin induced mitochondrial oxidative/nitrative stress, mitochondrial damages to electron transport chain activities and antioxidant defense enzyme activities in mitochondria. These mitochondrial modulations by EGCG led to protection mechanism against cisplatin induced inflammation and apoptotic cell death in mice kidney. As a result, EGCG improved renal function in cisplatin mediated kidney damage. In addition to that, EGCG attenuated cisplatin induced apoptotic cell death and mitochondrial reactive oxygen species (ROS) generation in human kidney tubular cell line HK-2. Thus, our data suggest that EGCG may represent new promising adjunct candidate for cisplatin.

Nanosecond pulsed electric field (nsPEF) treatment for hepatocellular carcinoma: a novel locoregional ablation decreasing lung metastasis.

Hepatocellular carcinoma (HCC) is a highly aggressive malignancy. Nanosecond pulsed electric field (nsPEF) is a new technology destroying tumor cells with a non-thermal high voltage electric field using ultra-short pulses. The study's aim was to evaluate the ablation efficacy of nsPEFs with human HCC cell lines and a highly metastatic potential HCC xenograft model on BALB/c nude mice. The in vivo study showed nsPEFs induced HCC cell death in a dose dependent manner. On the high metastatic hepatocellular carcinoma cell line (HCCLM3) xenograft mice model, tumor growth was inhibited significantly in nsPEF-treated- groups (single dose and multi-fractionated dose). Besides a local effect, the nsPEF treatment reduced pulmonary metastases. The nsPEFs also enhanced HCC cell phagocytosis by human macrophage cell (THP1) in vitro. The nsPEF is efficient in controlling HCC progression and reducing its metastasis. NsPEF treatment may elicit a host immune response against tumor cells. This study suggests nsPEF therapy could be used as a potential locoregional therapy for hepatocellular carcinoma.

Protective effect of metalloporphyrins against cisplatin-induced kidney injury in mice.

Oxidative and nitrative stress is a well-known phenomenon in cisplatin-induced nephrotoxicity. The purpose of this work is to study the role of two metalloporphyrins (FeTMPyP and MnTBAP), water soluble complexes, in cisplatin-induced renal damage and their ability to scavenge peroxynitrite. In cisplatin-induced nephropathy study in mice, renal nitrative stress was evident by the increase in protein nitration. Cisplatin-induced nephrotoxicity was also evident by the histological damage from the loss of the proximal tubular brush border, blebbing of apical membranes, tubular epithelial cell detachment from the basement membrane, or intra-luminal aggregation of cells and proteins and by the increase in blood urea nitrogen and serum creatinine. Cisplatin-induced apoptosis and cell death as shown by Caspase 3 assessments, TUNEL staining and DNA fragmentation Cisplatin-induced nitrative stress, apoptosis and nephrotoxicity were attenuated by both metalloporphyrins. Heme oxygenase (HO-1) also plays a critical role in metalloporphyrin-mediated protection of cisplatin-induced nephrotoxicity. It is evident that nitrative stress plays a critical role in cisplatin-induced nephrotoxicity in mice. Our data suggest that peroxynitrite is involved, at least in part, in cisplatin-induced nephrotoxicity and protein nitration and cisplatin-induced nephrotoxicity can be prevented with the use of metalloporphyrins.

Administration of minocycline ameliorates damage in a renal ischemia/reperfusion injury model.

PURPOSE: The purpose of this study was to investigate the effects of minocycline on the renal dysfunction and injury caused by bilateral ischemia/reperfusion (I/R) of murine kidneys in vivo. METHODS: Male C57BL/6 mice were administered minocycline (45 mg/kg i.v.) or saline (0.9%, v/v, NaCl) 36 hours prior to I/R. Mice were subjected to bilateral renal ischemia (35 min) followed by reperfusion (6 hours). Serum creatinine (sCr) and blood urea nitrogen (BUN) levels were measured. Additionally, renal superoxide dismutase (SOD) levels, malondialdehyde (MDA) levels and myeloperoxidase (MPO) activity were determined. The expression of intercellular adhesion molecule-1 (ICAM-1), caspase-3, caspase-8 and caspase-9 was determined using real time RT-PCR and Western blot analysis. RESULTS: Minocycline administration significantly reduced the increases in sCr and BUN caused by I/R, indicating attenuation of renal dysfunction and injury, and reduced histological evidence of renal damage caused by I/R. Minocycline administration also markedly reduced the evidence of oxidative stress (MPO activity, SOD and MDA levels), inflammation (ICAM-1 expression and MPO activity) and apoptosis (caspase-3, caspase-8 and caspase-9 expression) in mouse kidneys subjected to I/R. CONCLUSION: These findings provide good evidence that minocycline can reduce the renal dysfunction and injury caused by I/R of the kidney. Its mechanism may involve suppression of apoptosis, inflammatory response and oxidative stress.

Interplay of hepatic and myeloid signal transducer and activator of transcription 3 in facilitating liver regeneration via tempering innate immunity.

UNLABELLED: Liver regeneration triggered by two-thirds partial hepatectomy is accompanied by elevated hepatic levels of endotoxin, which contributes to the regenerative process, but liver inflammation and apoptosis remain paradoxically limited. Here, we show that signal transducer and activator of transcription 3 (STAT3), an important anti-inflammatory signal, is activated in myeloid cells after partial hepatectomy and its conditional deletion results in an enhanced inflammatory response. Surprisingly, this is accompanied by an improved rather than impaired regenerative response with increased hepatic STAT3 activation, which may contribute to the enhanced liver regeneration. Indeed, conditional deletion of STAT3 in both hepatocytes and myeloid cells results in elevated activation of STAT1 and apoptosis of hepatocytes, and a dramatic reduction in survival after partial hepatectomy, whereas additional global deletion of STAT1 protects against these effects. CONCLUSION: An interplay of myeloid and hepatic STAT3 signaling is essential to prevent liver failure during liver regeneration through tempering a strong innate inflammatory response mediated by STAT1 signaling.

Activation of innate immunity (NK/IFN-gamma) in rat allogeneic liver transplantation: contribution to liver injury and suppression of hepatocyte proliferation.

Liver transplantation is presently the only curative treatment for patients with end-stage liver disease. However, the mechanisms underlying liver injury and hepatocyte proliferation posttransplantation remain obscure. In this investigation, liver injury and hepatocyte proliferation in syngeneic and allogeneic animal models were compared. Male Lewis and Dark Agouti (DA) rats were subjected to orthotopic liver transplantation (OLT). Rat OLT was performed in syngeneic (Lewis-Lewis) and allogeneic (Lewis-DA or DA-Lewis) animal models. Allogeneic liver grafts exhibited greater injury and cellular apoptosis than syngeneic grafts but less hepatocyte proliferation after OLT. Expression of IFN-gamma mRNA and activation of the downstream signal transducer and activator of transcription 1 (STAT1) and genes (interferon regulatory factor-1 and cyclin-dependent kinase inhibitor p21(CDKN1A)) were also greater in the allogeneic grafts compared with the syngeneic grafts. In contrast, STAT3 activation was lower in the allogeneic grafts. Furthermore, in the allogeneic grafts, depletion of natural killer (NK) cells decreased IFN-gamma/STAT1 activation but enhanced hepatocyte proliferation. These findings suggest that, compared with syngeneic transplantation, innate immunity (NK/IFN-gamma) is activated after allogeneic transplantation, which likely contributes to liver injury and inhibits hepatocyte proliferation.

Loss of signal transducer and activator of transcription 5 leads to hepatosteatosis and impaired liver regeneration.

UNLABELLED: Growth hormone controls many facets of a cell's biology through the transcription factors Stat5a and Stat5b (Stat5). However, whole body deletion of these genes from the mouse does not provide portentous information on cell-specific cytokine signaling. To explore liver-specific functions of Stat5, the entire Stat5 locus was deleted in hepatocytes using Cre-mediated recombination. Notably, Stat5-mutant mice developed fatty livers and displayed impaired proliferation of hepatocytes upon partial hepatectomy (PHx). Loss of Stat5 led to molecular consequences beyond the reduced expression of Stat5 target genes, such as those encoding suppressor of cytokine signaling 2 (SOCS2), Cish, and insulin-like growth factor 1 (IGF-1). In particular, circulating growth hormone levels were increased and correlated with insulin resistance and increased insulin levels. Aberrant growth hormone (GH)-induced activation of the transcription factors Stat1 and Stat3 was observed in mutant livers. To test whether some of the defects observed in liver-specific Stat5 deficient mice were due to aberrant Stat1 expression and activation, we generated Stat1(-/-) mice with a hepatocyte-specific deletion of Stat5. Concomitant loss of both Stat5 and Stat1 restored cell proliferation upon PHx but did not reverse fatty liver development. Thus the molecular underpinnings of some defects observed in the absence of Stat5 are the consequence of a deregulated activation of other signal transducers and activators of transcription (STAT) family members. CONCLUSION: Aberrant cytokine-Stat5 signaling in hepatocytes alters their physiology through increased activity of Stat1 and Stat3. Such cross-talk between different pathways could add to the complexity of syndromes observed in disease.

[Organogenesis of developing metanephroi allografted into nonimmunosuppressed adult rats].

OBJECTIVE: To explore novel methods of possible donor organ supply and immunologic tolerance induction of organ transplantation. METHODS: Whole metanephroi from d14-19 (E14-E19) embryos of pregnant rats were grouped and allografted into the omenta or near remnants of renal vessels of nonimmunosupressed adult rats. At the time of implantation, host rats underwent unilateral nephrectomy. Four weeks after implantation, allografted metanephroi in host rats were removed for gross, biochemical and histopathological examination. RESULT: Four weeks post-implantation, (1) E19 and E18 metanephroi had enlarged,but were replaced by connective tissues. (2) E17 and E16 metanephroi showed the signs of acute rejection such as hypercellular glomeruli and lymphocyte infiltration in peritubular spaces. E16 grafted metanephroi underwent mild acute rejection of Banff schema, while E17 had moderate or severe acute rejection. When Cyclosporine A was administrated, E17 metanephroi formed mature nephrons and collecting ducts with few lymphocyte infiltration. (3) Metanephroi from E15 and E14 embryos allografted into the omentum or near remnants of renal vessels of uninephrectomized adult rats were enlarged and vascularized, and formed mature tubules and glomeruli. (4) The concentrations of urea nitrogen and creatinine in cyst fluid of E15 and E16 metanephroi were increased 40-fold and 50-fold, which were comparable to those in bladder urine. (5) In contrast, rat metanephroi did not grow or differentiate in rats without host kidney resection. CONCLUSION: E14 and E15 metanephroi allografted into nonimmunosuppressed adult rats or E17 into cyclosporine-treated hosts undergo growth and differentiation and become vascularized. A variety of factors affect the growth and development of allografted metanephroi, while rejection is the main one.

Activation pattern of mitogen-activated protein kinases in early phase of different size liver isografts in rats.

Mitogen-activated protein kinases (MAPK) play a pivotal role in ischemia reperfusion injuries of heart and liver, but the activation pattern of MAPKs in the early phase of different size liver isografts remains unclear. The experiment is designed to investigate the activation pattern and role of MAPKs in isografts of the rat with different size liver transplantation. The animal models of different size graft liver transplantation (whole graft, 50% size, or 30% size, respectively) were established and the sham operation group served as a control. The recipients were sacrificed at 0.5-, 2-, 6-, and 24-hour time points after transplantation to harvest the graft specimens and blood samples. The serum aspartate amino transferase (AST), alanine amino transferase (ALT) and tumor necrosis factor-alpha (TNF-alpha) levels, and histological findings were evaluated. The expressions of the total and phosphorylated p46/p54 JNKs, p38 MAPK, and p42/p44 ERKs were detected by Western blot. The serum ALT and AST levels increased significantly at the 0.5-hour time point and maintained high with the peak levels at the 6-hour time point after liver transplantation. The different sizes of liver isografts did not change the expressions of total p46/p54JNKs, p38MAPK, and p42/p44 ERKs. While the expressions of phosphorylated p46/p54JNKs, p38 MAPK, and p42/p44 ERKs were either negative or mildly up-regulated in the sham operation group, they were significantly activated in the transplanted liver at the 0.5-hour time point, especially in the 30% size liver transplantation group. In conclusion, the activation of three MAPKs in liver isografts correlates with graft size and the JNK and p38 MAPK are responsible for the graft injury while the ERK signal pathway maybe participate in the regulation of cell growth and differentiation after small-for-size liver transplantation.

[Effect of ciclosporin A on the organogenesis and function of embryonic metanephroi allografted into adult rats].

OBJECTIVE: To evaluate the effect of ciclosporin A (CsA) on the organogenesis and function of embryonic metanephroi allografted into adult rats. METHODS: The whole metanephroi from the 15, 16 and 17 embryonic day-old (E15, E16, E17) embryos of Sprague-Dawley (SD) rat were allografted into the omenta of SD adult rats with their left kidneys resected, which were divided into 2 categories: 3 CsA-treated groups of 10 rats (E15CsASD, E16CsASD, and E17CsASD) and 3 non-CsA-treated groups of 10 rats (E15SD, E16SD, and E17SD). Thirty SD rats without kidney resection were divided into 6 equal groups and underwent allografting embryonic metanephroi in the same manner as mentioned above. The E15 metanephroi of Lewis rats were allografted into the omenta of Thirty adult Brown Norway (BN) rats with one kidney resected. Were divided into 6 equal groups, received allografting of embryonic metanephroi, and injected with CsA of normal saline in the manner as mentioned above (E15CsABN and E15BN). Two to 4 weeks after implantation, the metanephroi allografted in host rats were removed for histopathological examination or anastomosed for renal function measurement 4 weeks later. RESULTS: (1) Four weeks after implantation, the E17SD and E16SD metanephroi showed signs of acute rejection as hypercellular glomeruli and mononuclear cell infiltration in the interstitium. The E16CsASD and E17CsASD metanephroi formed mature nephrons and collecting ducts with few lymphocytic infiltrates. After CsA was discontinued, the E16CsASD and E17CsASD metanephroi were rejected fully within 21 days. (2) 4 weeks after implantation, the E15SD metanephroi were enlarged, became vascularized, and developed mature tubules and glomeruli; however, they were rejected by 100 days after implantation. The E15 Lewis metanephroi were fully rejected within two weeks in the BN adult rats. With CsA administrated, the E15 Lewis metanephroi developed normal mature nephrons and collecting ducts within the adult BN rats. If CsA was discontinued, the E15CsABN metanephroi were rejected. (3) The E15CsASD metanephroi had significantly lower values of wet weight (P = 0.006) and higher values of creatinine clearances (P = 0.007) than the E15SD metanephros transplants, but were identical to those of the E16CsASD metanephroi (P = 0.948, P = 0.840). (4) The metanephroi did not grow or differentiate in the rats without host kidney resection. CONCLUSIONS: (1) Cyclosporin A may suppress graft rejection, thus normalizing the growth and function of fetal metanephroi in the omenta of host rats. (2) A variety of factors affect the growth and development of allografted metanephroi, whereas rejection remains the major one.

Specific COX-2 inhibitor NS398 induces apoptosis in human liver cancer cell line HepG2 through BCL-2.

AIM: To evaluate the effects of NS-398, a cyclooxygenase-2 (COX-2) inhibitor, on the proliferation and apoptosis of HepG2 cells. METHODS: The effects of NS-398 on the proliferation of HepG2 cells were evaluated by MTT. DNA fragmentation gel analysis was used to analyze the apoptotic cells. DNA ploidy and apoptotic cell percentage were calculated by flow cytometry. The expression of COX-2 and Bcl-2 mRNA was identified by competitive RT-PCR. Furthermore, expression level of Bcl-2 was detected using Western blot in HepG2 after treated with NS-398. RESULTS: NS-398 inhibited cell proliferation and induced apoptosis of HepG2 cells in a concentration-dependent manner. DNA ploidy analysis showed that S phase cells were significantly decreased with increase of NS-398 concentration. The quiescent G0/G1 phase was accumulated with decrease of Bcl-2 mRNA. Whereas NS-398 had no effect on the expression of COX-2 mRNA, and no correlations were found between COX-2 mRNA and HepG2 cell proliferation and apoptosis induced by NS-398 (r = 0.056 and r = 0.119, respectively). Bcl-2 protein level was inhibited after treated with NS-398. CONCLUSION: NS-398 significantly inhibits the proliferation and induces apoptosis of HepG2 cells. Mechanisms involved may be accumulation of quiescent G0/G1 phase and decrease of Bcl-2 expression.