Aristolochic acid I promoted clonal expansion but did not induce hepatocellular carcinoma in adult rats.

Aristolochic acid I (AAI) is a well-known nephrotoxic carcinogen, which is currently reported to be also associated with hepatocellular carcinoma (HCC). Whether AAI is a direct hepatocarcinogen remains controversial. In this study we investigated the association between AAI exposure and HCC in adult rats using a sensitive rat liver bioassay with several cofactors. Formation of glutathione S-transferase placental form-positive (GST-P+) foci was used as the marker for preneoplastic lesions/clonal expansion. We first conducted a medium-term (8 weeks) study to investigate whether AAI had any tumor-initiating or -promoting activity. Then a long-term (52 weeks) study was conducted to determine whether AAI can directly induce HCC. We showed that oral administration of single dose of AAI (20, 50, or 100 mg/kg) in combination with partial hepatectomy (PH) to stimulate liver proliferation did not induce typical GST-P+ foci in liver. In the 8-week study, only high dose of AAI (10 mg · kg-1 · d-1, 5 days a week for 6 weeks) in combination with PH significantly increased the number and area of GST-P+ foci initiated by diethylnitrosamine (DEN) in liver. Similarly, only high dose of AAI (10 mg· kg-1· d-1, 5 days a week for 52 weeks) in combination with PH significantly increased the number and area of hepatic GST-P+ foci in the 52-week study. No any nodules or HCC were observed in liver of any AAI-treated groups. In contrast, long-term administration of AAI (0.1, 1, 10 mg· kg-1· d-1) time- and dose-dependently caused death due to the occurrence of cancers in the forestomach, intestine, and/or kidney. Besides, AAI-DNA adducts accumulated in the forestomach, kidney, and liver in a time- and dose-dependent manner. Taken together, AAI promotes clonal expansion only in the high-dose group but did not induce any nodules or HCC in liver of adult rats till their deaths caused by cancers developed in the forestomach, intestine, and/or kidney. Findings from our animal studies will pave the way for further large-scale epidemiological investigation of the associations between AA and HCC.

Integrin αvß1 Modulation Affects Subtype B Avian Metapneumovirus Fusion Protein-mediated Cell-Cell Fusion and Virus Infection.

Avian metapneumovirus (aMPV) fusion (F) protein mediates virus-cell membrane fusion to initiate viral infection, which requires F protein binding to its receptor(s) on the host cell surface. However, the receptor(s) for aMPV F protein is still not identified. All known subtype B aMPV (aMPV/B) F proteins contain a conserved Arg-Asp-Asp (RDD) motif, suggesting that the aMPV/B F protein may mediate membrane fusion via the binding of RDD to integrin. When blocked with integrin-specific peptides, aMPV/B F protein fusogenicity and viral replication were significantly reduced. Specifically we identified integrin αv and/or ß1-mediated F protein fusogenicity and viral replication using antibody blocking, small interfering RNAs (siRNAs) knockdown, and overexpression. Additionally, overexpression of integrin αv and ß1 in aMPV/B non-permissive cells conferred aMPV/B F protein binding and aMPV/B infection. When RDD was altered to RAE (Arg-Ala-Glu), aMPV/B F protein binding and fusogenic activity were profoundly impaired. These results suggest that integrin αvß1 is a functional receptor for aMPV/B F protein-mediated membrane fusion and virus infection, which will provide new insights on the fusogenic mechanism and pathogenesis of aMPV.

Preventive effect of phosphodiesterase 5 inhibitor tadalafil on experimental post-pyelonephritic renal injury in rats.

BACKGROUND: To evaluate the effects of Tadalafil, a phosphodiesterase 5 enzyme inhibitor, on Escherichia coli-induced renal damage in an acute pyelonephritis (PN) rat model. METHODS: Experimental PN was induced in 32 Wistar rats, and four groups were formed: group 1 (no treatment), group 2 (antibiotic), group 3 (Tadalafil), and group 4 (antibiotic + Tadalafil). Antibiotic was given on days 3 to 8, and Tadalafil was administered between days 0 and 28 of bacterial inoculation. Half of the rats were killed on the ninth day (early period) and histopathological parameters, immunohistochemical renal fibrosis markers, and oxidant/antioxidant system activities were evaluated. The rest of the rats were killed at the sixth week of the study and evaluated for histopathological parameters and renal fibrosis markers. RESULTS: Inflammatory activity was significantly milder in rats treated with antibiotic + Tadalafil versus no treatment group both in the early and late periods. In the late period, interstitial fibrosis or tubular atrophy was lower in the antibiotic + Tadalafil group versus the no treatment and antibiotic groups, and in Tadalafil versus antibiotic group. Tadalafil administration significantly reduced renal malondialdehyde and nitric oxide levels and enhanced superoxide dismutase and catalase activities. In addition, circulating tumor necrosis factor α, interleukin 1ß was greatly reduced in Tadalafil group versus the no treatment group. CONCLUSIONS: We have provided the first evidence that phosphodiesterase 5 enzyme inhibitor Tadalafil ameliorates circulating inflammatory cytokines, reverses oxidant/antioxidant dysfunction and eventually possesses an overall protective effect on renal tissue from Escherichia coli-induced PN-related kidney injury. Phophodieterase 5 inhibitor might be a novel therapeutic target for PN.

Novel evidence demonstrates that epithelial-mesenchymal transition contributes to nephrolithiasis-induced renal fibrosis.

PURPOSE: To investigate fibrotic lesions in renal tissues obtained from patients with large calculi, and to selectively evaluate the expression and clinical significance of Twist and E-cadherin in nephrolithiasis patients. METHODS: We recruited 50 patients with kidney stone and 32 matched healthy controls. We determined plasma creatinine (Cr) and corrected Cr clearance (CCr). For the 50 patients, we detected daily urine protein excretion. At the end of percutaneous nephroscopic lithotomy, we performed puncture biopsy to acquire kidney tissue. We obtained normal control kidney tissues from non-nephrolithiasis patients who received a surgical biopsy during open surgery. We determined the expression of Twist and E-cadherin by immunohistochemical staining and scored it with clinical parameters. In addition, we analyzed the degree of expression of Twist and its correlation with long-term renal survival. RESULTS: Overall, the renal function of patients significantly decreased, as indicated by Cr and reduced CCr compared with healthy controls. Activated Twist was strongly expressed in tubular epithelial cells from kidneys of nephrolithiasis patients, whereas we found little positive staining of Twist in normal kidneys. Meanwhile, the expression of E-cadherin was significantly suppressed in kidneys of nephrolithiasis patients. Twist expression was inversely correlated with E-cadherin expression; using multivariate analysis, data showed that the factors influencing renal survival in patients were CCr (relative ratio, 4.39; 95% confidence interval, 1.34-14.38; P = 0.013) and the extent of Twist expression (relative ratio, 3.45; 95% confidence interval, 1.10-10.68; P = 0.033). CONCLUSIONS: Our data suggest that the possible novel EMT marker molecule Twist and Twist staining might be a valuable index predicting renal fibrosis progression in human nephrolithiasis.

RNA interference of pax2 inhibits growth of transplanted human endometrial cancer cells in nude mice.

The development of human endometrial carcinoma (HEC) is a complex pathologic process involves several oncogenes and tumor suppressor genes. The full-length paired-box gene 2 (pax2), a recently discovered oncogene, promotes cell proliferation and growth and inhibits apoptosis of HEC cells. Here, we examined the effect of pax2 small interfering RNA (siRNA) on the growth of transplanted HEC cells in nude mice. The expression of Pax2 in 21 cases of normal endometrium and 38 cases of HEC was examined by immohistochemistry (IHC). HEC models were developed by subcutaneously transferring HEC cells into nude mice, followed by treatment with empty lentivirus vector, lentivirus vector-based pax2 siRNA, and phosphate buffered saline, respectively. Four weeks later, tumor size was measured, tumor inhibition rate was calculated, and histological analyses were conducted after staining with hematoxylin and eosin. The expression of Pax2 and Bcl-2 was detected by Western blot; proliferating cell nuclear antigen (PCNA) was detected by IHC. Significant differences were observed in the positive rate of Pax2 between normal endometrium and HEC (14.2% vs. 60.5%, P < 0.01). The expression index of Pax2 in well differentiated tumors was 1.88 ± 1.68, much lower than that in tumors of moderate (3.07 ± 1.96, P < 0.05) or poor differentiation (5.45 ± 2.76, P <0.01). Tumor necrosis increased, nuclear basophilia stain decreased, tumor growth was inhibited, and PCNA, Pax2, and Bcl-2 expression was reduced in HEC models treated with pax2 siRNA. These results indicate that Pax2 expression is related to HEC tumor biology with the increased expression of Pax2 correlated to malignancy. pax2 siRNA down-regulates Pax2 expression and inhibits tumorigenesis of HEC in nude mice, possibly due to cell apoptosis and the inhibition of tumor proliferation induced by down-regulation of Bcl-2.