Ming-Kuen Lai

Bcl-xL augmentation potentially reduces ischemia/reperfusion induced proximal and distal tubular apoptosis and autophagy.

Background. Apoptosis and autophagy may contribute to cell homeostasis in the kidney subjected to ischemia/reperfusion injury via mitochondrial injury. Ischemia/reperfusion induces differential sensitivity between proximal and distal tubules via a dissociated Bcl-xL expression. We hypothesized Bcl-xL augmentation in the proximal and distal tubules may potentially reduce ischemia/reperfusion induced renal dysfunction. Methods. We augmented Bcl-xL protein expression in the kidney with intrarenal adenoviral bcl-xL gene transfer and evaluated the potential effect of Bcl-xL augmentation on ischemia/reperfusion induced renal oxidative stress, apoptosis, and autophagy in the rat. Results. Intrarenal arterial Adv-bcl-xL administration augmented maximal Bcl-xL protein expression of rat kidney after 7 days of transfection. The primary location of Bcl-xL augmentation was found in proximal and distal tubules, but not in glomeruli. Ischemia/reperfusion increased mitochondrial cytochrome C release, renal O2−. level and renal 3-nitrosine and 4-hydroxyneonal accumulation, potentiated tubular apoptosis and autophagy, including increase in microtubule-associated protein 1 light chain 3 (LC-3) and Beclin-1 expression, Bax/Bcl-xL ratio, caspase 3 expression and poly-(ADP-ribose)-polymerase fragments, and subsequent proximal and distal tubular apoptosis/autophagy. However, Adv-bcl-xL administration significantly reduced ischemia/reperfusion enhanced mitochondrial cytochrome C release, O2−. production, 3-nitrotyrosine and 4-hydroxynonenal accumulation, Beclin-1 expression, Bax/Bcl-xL ratio, and proximal and distal tubular apoptosis/autophagy, consequently improving renal dysfunction. Further study showed that Bcl-xL augmentation was more efficiently than Bcl-2 augmentation in amelioration of ischemia/reperfusion induced proximal and distal tubular apoptosis and renal dysfunction. Conclusions. Our results suggest that Adv-bcl-xL gene transfer significantly improves ischemia/reperfusion-induced renal dysfunction via the downregulation of renal tubular apoptosis and autophagy.

DUAL EFFECTS OF OUABAIN ON THE REGULATION OF PROLIFERATION AND APOPTOSIS IN HUMAN PROSTATIC SMOOTH MUSCLE CELLS

PURPOSE To elucidate the role of ouabain in the pathophysiology of benign prostatic hyperplasia we examined the effects of ouabain on the proliferation and apoptosis of human prostatic smooth muscle cells. MATERIALS AND METHODS Primary cultures of human prostatic smooth muscle cells were obtained from 7 patients with bladder outlet obstruction caused by benign prostatic enlargement. A cell proliferation study was performed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay method to examine the effects of different concentrations of ouabain and various inhibitors. Western blot analysis was done to determine mitogen activated protein kinase (MAPK) activation. The terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling reaction method and caspase-3 activity assay were also performed to examine the apoptotic mechanism. RESULTS Ouabain exhibited a modest but significant proliferative effect in nanomolar concentrations; whereas it induced cell apoptosis at higher concentrations. Ouabain caused rapid activation of p42/44 MAPKs. The proliferative effect of ouabain was completely flattened by W-7 and MAPK kinase (MEK) inhibitor, suggesting the requirement of Ca(2+) mobilization and the involvement of the MEK-p42/44 MAPK cascade. The cytotoxic effect by ouabain was defined as apoptosis and necrosis using the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling reaction technique and lactate dehydrogenase release assay, respectively. In addition, ouabain induced profound caspase-3 activity in the cytotoxic concentrations and DEVD-CHO reversed the cytotoxic action to ouabain, demonstrating the involvement of caspase-3 activation in the cytotoxic action. CONCLUSIONS Ouabain at different concentrations caused dual effects on proliferation and apoptosis in human prostatic smooth muscle cells. At low concentrations ouabain promoted cell proliferation via a Ca(2+) dependent mechanism and activation of the MEK-p42/44 MAPK pathway; whereas it induced cell apoptosis via the activation of caspase-3 activity at higher concentrations.

MYC pathway is activated in clear cell renal cell carcinoma and essential for proliferation of clear cell renal cell carcinoma cells.

Clear cell renal cell carcinoma (ccRCC) is the major and aggressive subtype of RCC. Previously, we identified 383 differentially expressed genes by analyzing full-length cDNA libraries of ccRCC and normal kidney tissues. In this study, we applied functional network analysis to the differentially expressed genes for identifying deregulated molecular pathways in ccRCC, and the results indicated that MYC showed a prominent role in the highest scoring network. The upregulation of MYC expression was validated in ccRCC tissues and cell lines. Furthermore, Knockdown of MYC expression by MYC-specific siRNA significantly inhibited the abilities of uncontrolled proliferation, anchorage-independent growth and arrested cell cycle in the G0/G1 phase in ccRCC cells. Moreover, we found that 37 differentially expressed genes were shown to be MYC-target genes, and the upregulation of the MYC-target genes BCL2, CCND1, PCNA, PGK1, and VEGFA were demonstrated. The expression of these MYC-target genes was significantly correlated with the expression of MYC in ccRCC tissues, and knockdown of MYC also suppressed the expression of these MYC-target genes in ccRCC cells. The recruitment of MYC to the promoter regions of BCL2, CCND1, PCNA, PGK1, and VEGFA was shown by Chromatin immunoprecipitation assay. These results suggest that MYC pathway is activated and plays an essential role in the proliferation of ccRCC cells.

Risk factors for prostate carcinoma in Taiwan

Although prostate carcinoma remains a rare disease among Chinese men, its incidence is on the rise. The authors conducted a hospital‐based case–control study to identify risk factors for prostate carcinoma in northern Taiwan.

Nicotinamide N-methyltransferase induces cellular invasion through activating matrix metalloproteinase-2 expression in clear cell renal cell carcinoma cells

Nicotinamide N-methyltransferase (NNMT) was recently identified as one clear cell renal cell carcinoma (ccRCC)-associated gene by analyzing full-length complementary DNA-enriched libraries of ccRCC tissues. The aim of this study is to investigate the potential role of NNMT in cellular invasion. A strong NNMT expression is accompanied with a high invasive activity in ccRCC cell lines, and small interfering RNA-mediated NNMT knockdown effectively suppressed the invasive capacity of ccRCC cells, whereas NNMT overexpression markedly enhanced that of human embryonic kidney 293 (HEK293) cells. A positive correlation between the expression of NNMT and matrix metallopeptidase (MMP)-2 was found in ccRCC cell lines and clinical tissues. The treatment of blocking antibody or inhibitor specific to MMP-2 significantly suppressed NNMT-dependent cellular invasion in HEK293 cells. Furthermore, SP-1-binding region of MMP-2 promoter was found to be essential in NNMT-induced MMP-2 expression. The specific inhibitors of PI3K/Akt signaling markedly decreased the binding of SP1 to MMP-2 promoter as shown by chromatin immunoprecipitation assay. We also demonstrated that PI3K/Akt pathway plays a role in NNMT-dependent cellular invasion and MMP-2 activation. Moreover, short hairpin RNA-mediated knockdown of NNMT expression efficiently inhibited the growth and metastasis of ccRCC cells in non-obese diabetic severe combined immunodeficiency mice. Taken together, the present study suggests that NNMT has a crucial role in cellular invasion via activating PI3K/Akt/SP1/MMP-2 pathway in ccRCC.

Ischemic conditioning by short periods of reperfusion attenuates renal ischemia/reperfusion induced apoptosis and autophagy in the rat

Prolonged ischemia amplified iscehemia/reperfusion (IR) induced renal apoptosis and autophagy. We hypothesize that ischemic conditioning (IC) by a briefly intermittent reperfusion during a prolonged ischemic phase may ameliorate IR induced renal dysfunction. We evaluated the antioxidant/oxidant mechanism, autophagy and apoptosis in the uninephrectomized Wistar rats subjected to sham control, 4 stages of 15-min IC (I15 × 4), 2 stages of 30-min IC (I30 × 2), and total 60-min ischema (I60) in the kidney followed by 4 or 24 hours of reperfusion. By use of ATP assay, monitoring O2-. amounts, autophagy and apoptosis analysis of rat kidneys, I60 followed by 4 hours of reperfusion decreased renal ATP and enhanced reactive oxygen species (ROS) level and proapoptotic and autophagic mechanisms, including enhanced Bax/Bcl-2 ratio, cytochrome C release, active caspase 3, poly-(ADP-ribose)-polymerase (PARP) degradation fragments, microtubule-associated protein light chain 3 (LC3) and Beclin-1 expression and subsequently tubular apoptosis and autophagy associated with elevated blood urea nitrogen and creatinine level. I30 × 2, not I15 × 4 decreased ROS production and cytochrome C release, increased Manganese superoxide dismutase (MnSOD), Copper-Zn superoxide dismutase (CuZnSOD) and catalase expression and provided a more efficient protection than I60 against IR induced tubular apoptosis and autophagy and blood urea nitrogen and creatinine level. We conclude that 60-min renal ischemia enhanced renal tubular oxidative stress, proapoptosis and autophagy in the rat kidneys. Two stages of 30-min ischemia with 3-min reperfusion significantly preserved renal ATP content, increased antioxidant defense mechanisms and decreased ischemia/reperfusion enhanced renal tubular oxidative stress, cytosolic cytochrome C release, proapoptosis and autophagy in rat kidneys.

Hypoxia preconditioning attenuates bladder overdistension‐induced oxidative injury by up‐regulation of Bcl‐2 in the rat

We explored whether hypoxic preconditioning minimizes oxidative injury induced by overdistension/emptying in the rat bladder. For hypoxic preconditioning, female Wistar rats were placed in a hypobaric chamber (380 Torr) 15 h day−1 for 28 days. Overdistension was induced by infusion of two times the threshold volume of saline into the bladder and was maintained for 1 or 2 h, followed by drainage/emptying. During overdistension (ischaemia) and emptying (reperfusion) periods, a bursting increase of reactive oxygen species (ROS) from the bladder was originated from the large numbers of infiltrating leucocytes and scattered resident cells, including urothelial, submucosal, and smooth muscle cells. ROS impaired the voiding function by a reduction of bladder afferent and efferent nerve activity and bethanecol‐ or ATP‐induced detrusor contraction. ROS enhanced pro‐apoptotic mechanisms, including increases in the Bax/Bcl‐2 ratio, CPP32 expression, and poly(ADP‐ribose) polymerase (PARP) fragments with subsequent apoptotic cell formation in the insulted bladders. Hypoxia preconditioning up‐regulated Bcl‐2 expression in the bladder and significantly reduced the levels of ROS and apoptosis detected in the overdistension/emptying bladders and preserved partial voiding function. Bcl‐2 up‐regulation by hypoxia preconditioning contributes protection against overdistension/emptying‐induced oxidative stress and injury in the bladder.

Expanding the donor pool: use of renal transplants from non-heart-beating donors supported with extracorporeal membrane oxygenation

Abstract:  In response to organ shortage, we used the renal grafts from non‐heart‐beating donors (NHBDs). Extracorporeal membrane oxygenation (ECMO) was used to maintain NHBDs before organ procurement. We compared the results of renal transplantation from different donors, including heart‐beating donors (HBDs), living‐related donors (LDs), and NHBDs supported with ECMO. From February 1998 to June 2003, we recruited 219 patients receiving renal transplantation at National Taiwan University Hospital. Among them, 31 received kidneys from NHBDs supported with ECMO, 120 from HBDs, and 68 from LDs. Multiple organ transplant recipients were not included in this study. We compared the graft survival, serum creatinine levels, and estimated glomerular filtration rates of the three groups. The rate of delayed graft function was higher in NHBD recipients (41.9%) than in HBD recipients (27.0%) and LD recipients (10.9%) (p = 0.003). In the NHBD group, the recipients of grafts with delayed function had significantly longer ECMO runs (63.1 ± 3.0 min) than those without delayed function (53.7 ± 2.5 min) (p = 0.024). Estimated glomerular filtration rate (p = 0.472) and mean serum creatinine level (p = 0.286) were not significantly different between the three groups using a longitudinal approach. The 5‐yr graft survival rates for NHBD (88.4%, 95% CI: 0.680–0.962), HBD (83.2%, 95% CI: 0.728–0.899), and LD transplant recipients (89.3%, 95% CI: 0.619–0.974) were not significantly different (p = 0.239). The 5‐yr patient survival rates for NHBD, HBD, and LD transplant recipients were 100, 93.0 (95% CI: 0.859–0.966) and 100% respectively. The long‐term allograft survival and function of kidneys from NHBDs supported by ECMO, HBD, and LD did not differ significantly. Long ECMO running time tended to delay graft function.

Laparoscopic Adrenalectomy Using Needlescopic Instruments for Adrenal Tumors Less Than 5 cm in 112 Cases

OBJECTIVE To examine the safety and efficacy of laparoscopic adrenalectomy with needlescopic instruments for most adrenal tumors less than 5cm. METHODS Transperitoneal laparoscopic adrenalectomy with needlescopic instruments for 112 patients with presumptively benign adrenal tumors < 5cm were enrolled from July 2000 to February 2005. Operative time, blood loss, conversion and complication rates, and postoperative data were analyzed by appropriate statistical methods. RESULTS All 112 operations were completed without any mortality or reoperation. Mean operative time was 151min and mean blood loss was 30ml. Only one patient required a blood transfusion and application of a hand-assisted device. Conversion to conventional laparoscopic instruments was necessary in another five patients (4.5%). The operative time of the latter 100 cases (147+/-5.1min, mean+/-standard error of mean) was significantly shorter than that of the initial 12 cases (183+/-8.8min, p=0.001). Larger tumors, previous abdominal surgery, and pheochromocytoma group were independent risk factors of a longer operative time. Except for one leiomyosarcoma, all other tumors were benign adrenal pathologies (57 aldosterone-producing adenomas, 23 Cushings adenomas, 12 pheochromocytomas, and 20 incidentalomas). CONCLUSION The safety and effectiveness of laparoscopic adrenalectomy employing needlescopic instruments for most adrenal tumors less than 5cm was feasible with acceptable operative time. Pheochromocytomas can also be managed with a longer operative time. Patients with previous upper midline or ipsilateral upper quadrant open surgery might not be suitable candidates for such a technique.

Modified approach of hand-assisted laparoscopic nephroureterectomy for transitional cell carcinoma of the upper urinary tract

Objectives. To report a modified approach for hand-assisted laparoscopic nephroureterectomy (HALNU).Methods. Seven patients with localized transitional cell carcinoma of the upper urinary tract underwent unilateral HALNU. Patients were placed in a 60 degrees oblique position during the entire procedure. Via a 7-cm Gibson incision on the lesion side, the distal ureterectomy and bladder cuff excision were done by an open method without opening the bladder. Then, with the surgeons hand inserted into the peritoneal cavity by way of the same wound, HALNU was performed with two to three additional laparoscopic ports. The perioperative parameters were compared with those of 15 cases of conventional open nephroureterectomy.Results. Patients in the HALNU group had significantly less mean blood loss (140 versus 455 mL) and earlier resumption of oral intake (33 versus 61 hours), required fewer narcotics (38 versus 70 mg of morphine sulfate equivalent), and were discharged earlier (7.33 versus 9.1 days), with a faster convalescence to normal activity (3.7 versus 5.6 weeks; all P < 0.05). The total mean surgical time was 3.7 hours for the HALNU group.Conclusions. Our approach used the same incision to both excise the distal ureter and apply the hand-assist device. It also preserved the benefits of the minimal invasiveness of laparoscopic surgery compared with its open counterpart.