The Role of the ADAMTS18 Gene-Induced Immune Microenvironment in Mouse Kidney Development.

The aim of this study is to investigate the role of the ADAMTS18 gene in regulating the renal development of mice. PAS staining was used to observe the kidney development of E12.5-E17.5 mice, while immunofluorescence staining and RT-PCR were used to observe the expression of ADAMTS18. Ureteric bud (UB) branches were observed using immunofluorescence staining using the UB marker E-cadherin, and the apoptosis and proliferation of posterior renal mesenchymal cells were analyzed using TUNEL and PH3 fluorescence staining. Flow cytometry was used to analyze the immune cell infiltration, and western blotting (WB) was used to analyze the expression of PD-1/PD-L1 and CTLA-4. As a result, the ADAMTS18 gene expression gradually increased as the kidney continued to mature during embryonic development. Compared with that in the control and vector groups, UB branching was significantly reduced in the ADAMTS18 deletion group (p < 0.05), but that deletion of ADAMTS18 did not affect posterior renal mesenchymal cell proliferation or apoptosis (p > 0.05). Compared with those in the control and vector groups, the proportion of embryonic kidney B cells and the proportion of CD8+ cells were significantly greater after ADAMTS18 was knocked down (p < 0.05), but the difference in neutrophil counts was not significant (p > 0.05). The WB analysis revealed that the PD-1/PD-L1 and CTLA-4 expression was significantly increased after ADAMTS18 was knocked down (p < 0.05). In conclusion, the ADAMTS18 gene may be involved in mice kidney development by regulating the immune microenvironment and activating immune checkpoints. Deletion of the ADAMTS18 gene may be unfavorable for kidney development.

The progression of obstructive renal fibrosis in rats is regulated by ADAMTS18 gene methylation in the embryonic kidney through the AKT/Notch pathway.

This study aimed to explore the mechanism by which postembryonic renal ADAMTS18 methylation influences obstructive renal fibrosis in rats. After exposure to transforming growth factor (TGF)-ß1 during the embryonic period, analysis of postembryonic renal ADAMTS18 methylation and expression levels was conducted. Histological analysis was performed to assess embryonic kidney lesions and damage. Western blot analysis was used to determine the expression of renal fibrosis markers. Rats with ureteral obstruction and a healthy control group were selected. The methylation levels of ADAMTS18 in the different groups were analyzed. Western blot analysis and immunohistochemistry were performed to analyze the expression of renal fibrosis markers, and kidney-related indicators were measured. Treatment with TGF-ß1 resulted in abnormal development of the postembryonic kidney, which was characterized by rough kidney surfaces with mild depressions and irregularities on the outer surface. TGF-ß1 treatment significantly promoted ADAMTS18 methylation and activated the protein kinase B (AKT)/Notch pathway. Ureteral obstruction was induced to establish a renal hydronephrosis model, which led to renal fibrotic injury in newborn rats. Overexpression of the ADAMTS18 gene alleviated renal fibrosis. The western blot results showed that compared to that in the control group, the expression of renal fibrosis markers was significantly decreased after ADAMTS18 overexpression, and there was a thicker renal parenchymal tissue layer and significantly reduced p-AKT/AKT and Notch1 levels. TGF-ß1 can induce ADAMTS18 gene methylation in the postembryonic kidney, and the resulting downregulation of ADAMTS18 expression has long-term effects on kidney development, potentially leading to increased susceptibility to obstructive renal fibrosis. This mechanism may involve activation of the AKT/Notch pathway. Reversing ADAMTS18 gene methylation may reverse this process.

Curcumin inhibits lipopolysaccharide-induced proximal renal tubular epithelial cell death by regulating ADAMTS18 methylation and AKT/Notch pathways.

Introduction: This study aimed to explore the effect of curcumin on the activity of lipopolysaccharide (LPS)-induced proximal renal tubular epithelial cells (PRTECs) and to analyze the molecular mechanism by which curcumin regulates their occurrence. Material and methods: LPS-induced PRTECs were used to construct an inflammatory cell model. RT-qPCR and western blot (WB) were used to detect ADAMTS18 expression. Methylation-specific PCR was used to detect ADAMTS18 methylation levels. After curcumin treatment, MTT assay was used to analyze cell viability, flow cytometry was used to analyze apoptosis, and ADAMTS18 expression and methylation levels were detected again. After transfection with siADAMTS18, cell viability and apoptosis were analyzed again. The levels of inflammatory factors were measured by enzyme-linked immunosorbent assay, and the expression levels of AKT, Notch-1 and Notch-2 were analyzed by WB. Results: Curcumin strongly inhibited LPS-induced PRTEC inflammatory lesions, restored normal cell proliferation, and reduced the apoptosis rate by downregulating ADAMTS18 methylation and restoring ADAMTS18 expression. After siADAMTS18, the ability of curcumin to improve cell viability was reduced, and the ability of curcumin to downregulate inflammatory factors was significantly reduced. Curcumin could also inhibit the expression of AKT, Notch-1 and Notch-2 simultaneously. siADAMTS18 attenuated the abovementioned effects of curcumin. Conclusions: Curcumin inhibits LPS-induced PRTEC death by regulating ADAMTS18 methylation and AKT/Notch pathways.

[Diagnostic efficacy of prostate cancer using targeted biopsy with 6-core systematic biopsy for patients with PI-RADS 5].

OBJECTIVE: To investigate the diagnostic efficacy of targeted biopsy (TBx), systematic biopsy (SBx), TBx+6-core SBx in prostate cancer (PCa) / clinically significant prostate cancer (cs-PCa) for patients with prostate imaging reporting and data system (PI-RADS) score of 5, and thereby to explore an optimal sampling scheme. METHODS: The data of 585 patients who underwent multiparametric magnetic resonance imaging (mpMRI) with at least one lesion of PI-RADS score 5 at Peking University First Hospital from January 2019 to June 2022 were retrospectively analyzed. All patients underwent mpMRI / transrectal ultrasound (TRUS) cognitive guided biopsy (TBx+SBx). With the pathological results of combined biopsy as the gold standard, we compared the diagnostic efficacy of TBx only, SBx only, and TBx+6-core SBx for PCa/csPCa. The patients were grouped according to mpMRI T-stage (cT2, cT3, cT4) and the detection rates of different biopsy schemes for PCa/csPCa were compared using Cochran's Q and McNemar tests. RESULTS: Among 585 patients with a PI-RADS score of 5, 560 (95.7%) were positive and 25(4.3%) were negative via TBx+SBx. After stratified according to mpMRI T-stage, 233 patients (39.8%) were found in cT2 stage, 214 patients (36.6%) in cT3 stage, and 138 patients (23.6%) in cT4 stage. There was no statistically significant difference in the detection rate of PCa/csPCa between TBx+6-core SBx and TBx+SBx (all P>0.999). Also, there was no statistically significant difference in the detection rate of PCa/csPCa between TBx and TBx+SBx in the cT2, cT3, and cT4 subgroups (PCa: P=0.203, P=0.250, P>0.999; csPCa: P=0.700, P=0.250, P>0.999). The missed diagnosis rate of SBx for PCa and csPCa was 2.1% (12/560) and 1.8% (10/549), and that of TBx for PCa and csPCa was 1.8% (10/560) and 1.4% (8/549), respectively. However, the detection rate of TBx+6-core SBx for PCa and csPCa was 100%. Compared with TBx+SBx, TBx and TBx+6-core SBx had a fewer number of cores and a higher detection rate per core (P < 0.001). CONCLUSION: For patients with a PI-RADS score of 5, TBx and TBx+6-core SBx showed the same PCa/csPCa detection rates and a high detection rates per core as that of TBx+SBx, which can be considered as an optimal scheme for prostate biopsy.

[Application of dynamic contrast enhanced status in multiparametric magnetic resonance imaging for prostatic cancer with PI-RADS 4 lesion].

OBJECTIVE: To evaluate the diagnostic value of dynamic contrast enhanced (DCE) of multiparametric magnetic resonance imaging (mpMRI) for prostate imaging reporting and data system (PI-RADS) 4 lesion in prostate peripheral zone. METHODS: The clinical data of patients with PI-RADS 4 lesion in prostate peripheral zone who underwent prostate biopsy from January 2018 to September 2021 in Peking University First Hospital were retrospectively included. According to DCE status, the patients were divided into the conventional group (4 points for diffusion-weighted imaging) and the comprehensive group (3 points for diffusion-weighted imaging + 1 point for DCE positive). Pearson's chi-square test or Fisher's exact test for comparison was conducted between prostate cancer and non-cancer patients. Univariate and multivariate Logistic regression were performed to analyze the correlation of positive biopsy with age, total prostate specific antigen (PSA), free PSA/total PSA (f/tPSA), prostate volume (PV), PSA density (PSAD) and DCE status. RESULTS: Among the 267 prostate biopsy patients, 217 cases were diagnosed as prostatic cancer (81.27%) and 50 cases were non-cancer (18.73%). Statistical analysis between the prostatic cancer group and the non-cancer group showed that there were significant differences in age, tPSA, PV and PSAD (all P < 0.05), but no significant differences in f/tPSA between the two groups. About different PI-RADS 4 lesion groups, the conventional group and the comprehensive group showed significant difference in biopsy results (P=0.001), and the conventional group had a higher positive rate. The PV of comprehensive group was larger than that of the conventional group. Among the prostate cancer patients diagnosed by biopsy, statistical analysis between the conventional group and comprehensive group showed that there were not significant differences in International Society of Urological Pathology (ISUP) grade and distinguishing clinically significant prostate cancer (all P > 0.05). Logistic univariate analysis showed that the diagnosis of prostate cancer was related to age, tPSA, f/tPSA, PV and DCE group status (all P < 0.05). Multivariate analysis showed that age, tPSA, PV and DCE group status (all P < 0.05) were independent risk factors for the diagnosis of prostatic cancer. CONCLUSION: tPSA, f/tPSA, PV and PSAD are the indicators to improve the diagnosis of prostatic cancer with PI-RADS 4 lesion in peripheral zone lesions. DCE status is worth considering, so that we can select patients for biopsy more accurately, reduce the rate of missed diagnosis of prostate cancer as well as avoid unnecessary prostate puncture.

Curcumin Interferes with TGF- ß 1-Induced Fibrosis in NRK-49F Cells by Reversing ADAMTS18 Gene Methylation.

OBJECTIVE: To explore the molecular mechanism by which curcumin affects renal interstitial fibrosis (RIF) progression by regulating ADAM metallopeptidase with thrombospondin type 1 motif 18 (ADAMTS18) methylation. METHODS: NRK-49F cells RIF model were induced with transforming growth factor ß 1 (TGF- ß 1). Effects of different concentrations of curcumin (0, 10, 20, and 30 µmol/L) on cell proliferation, cell cycle, cell apoptosis as well as cyclin D1 expression were analyzed by cell counting kit-8, flow cytometry and Western blot, respectively. ADAMTS18 methylation levels were determined by methylation-specific polymerase chain reaction. ADAMTS18, fibronectin (FN), type I collagen (Col- I) and alpha-smooth muscle actin (α -SMA) mRNA and protein expressions were analyzed by real-time PCR (RT-PCR) and Western blot, respectively. Meanwhile, cells were treated with 50 mmol/L 5-aza-2'-deoxycytidine (5-aza-dC, demethylation agent) for 72 h. Effect of curcumin on extracellular matrix (ECM) deposition was evaluated by immunochemical staining and Western blot. NRK-49F cells were transfected with ADAMTS18 small interfering RNA and grouped into a normal control, ADAMTS18-knock-out (KO), and ADAMTS18-KO+ 30 µmol/L curcumin groups, and whether curcumin can reverse the effect of ADAMTS18 knockdown on RIF was evaluated. RESULTS: Compared with the control group, TGF-ß 1 significantly inhibited the proliferation of NRK-49F cells, blocked the G1/G0 phase, promoted cell apoptosis and inhibited cyclin D1 expression (P<0.01). Among the different concentrations of curcumin, 30 µmol/L curcumin significantly reversed these processes (P<0.01). Immunochemical staining and Western blot results showed that curcumin significantly inhibited the deposition of FN, Col- I and α-SMA (P<0.01). Curcumin and 5-zaz-dC had synergistic effects, promoting ADAMTS18 expression, removing ADAMTS18 methylation, and reducing ECM deposition. ADAMTS18 knockdown promoted ECM accumulation, and curcumin reversed this process (P<0.01). CONCLUSION: TGF-ß 1-induced fibrosis in NRK-49F cells. Curcumin promoted ADAMTS18 expression, reduced ECM accumulation, and alleviated RIF progression by inhibiting ADAMTS18 methylation.

Evaluation of the diagnostic efficiency of voided urine fluorescence in situ hybridization for predicting the pathology of preoperative "low-risk" upper tract urothelial carcinoma.

Objectives: To evaluate the clinical utility of voided urine fluorescence in situ hybridization (FISH) for predicting the pathology of preoperative "low-risk" upper tract urothelial carcinoma (UTUC). Methods: Information of patients preoperatively diagnosed with "low-risk" UTUC receiving radical nephroureterectomy (RNU) between May 2014 and October 2019 were retrospectively collected. All of the patients accepted the FISH test and then were divided into two groups according to the results of FISH. The diagnostic value of FISH was assessed through the receiver operating characteristics (ROC) curve and area under the curve. Logistic regression analysis was applied to examine FISH as a predictive factor of tumor final stage and grade of preoperative "low-risk" UTUC. Results: In total, 129 patients were included. Of them, 70 (54.2%) were marked with positive FISH result. The difference at final pathology in tumor stage and tumor grade between these two groups of FISH (-) and FISH (+) had significantly statistical significance (p<0.001). Regarding to the tumor stage at final pathology, the sensitivity, specificity, positive predictive value and negative predictive value of FISH were 70.7 (58.9-80.3)68.5 (54.3-80.1)75.7 (63.7-84.8) and 62.7 (49.1-74.7), respectively. Regarding to the tumor grade at final pathology, the sensitivity, specificity, positive predictive value and negative predictive value of FISH were 64.7 (53.5-74.6), 65.9 (50.0-79.1), 78.6 (66.8-87.1) and 49.1 (36.5-62.3), respectively. The results of logistic regression analysis indicated that FISH could predict the pathologic characteristics of preoperative "low-risk" UTUC independently. Conclusions: FISH was qualified with relatively high diagnostic estimates for predicting tumor stage and grade of preoperative "low-risk" UTUC, and could be an independent predictive factor in clinical practice. For preoperative "low-risk" UTUC patients but with positive FISH result, choosing nephron-sparing surgery may require special caution.

Curcumin Inhibits Proliferation of Renal Cell Carcinoma in vitro and in vivo by Regulating miR-148/ADAMTS18 through Suppressing Autophagy.

OBJECTIVE: To explore the effect of curcumin on the proliferation of renal cell carcinoma and analyze its regulation mechanism. METHODS: In RCC cell lines of A498 and 786-O, the effects of curcumin (2.5, 5, 10 µ mo/L) on the proliferation were analyzed by Annexin V+PI staining. Besides, A498 was inoculated into nude mice to establish tumorigenic models, and the model mice were treated with different concentrations of curcumin (100, 200, and 400 mg/kg), once daily for 30 days. Then the tumor diameter was measured, the tumor cells were observed by hematoxylin-eosin staining, and the protein expressions of miR-148 and ADAMTS18 were detected by immunohistochemistry. In vitro, after transfection of miR-148 mimics, miR-148 inhibitor or si-ADAMTS18 in cell lines, the expression of ADAMTS18 was examined by Western blotting and the cell survival rate was analyzed using MTT. Subsequently, Western blot analysis was again used to examine the autophagy phenomenon by measuring the relative expression level of LC3-II/LC3-I; autophagy-associated genes, including those of Beclin-1 and ATG5, were also examined when miR-148 was silenced in both cell lines with curcumin treatment. RESULTS: Curcumin could inhibit the proliferation of RCC in cell lines and nude mice. The expression of miR-148 and ADAMTS18 was upregulated after curcumin treatment both in vitro and in vivo (P<0.05). The cell survival rate was dramatically declined upon miR-148 or ADAMTS18 upregulated. However, si-ADAMTS18 treatment or miR-148 inhibitor reversed these results, that is, both of them promoted the cell survival rate. CONCLUSION: Curcumin can inhibit the proliferation of renal cell carcinoma by regulating the miR-148/ ADAMTS18 axis through the suppression of autophagy in vitro and in vivo. There may exist a positive feedback loop between miR-148 and ADAMTS18 gene in RCC.

Effectiveness and Safety of Anlotinib with or without PD-1 Blockades in the Treatment of Patients with Advanced Primary Hepatocellular Carcinoma: A Retrospective, Real-World Study in China.

Purpose: Anlotinib, a novel multi-target tyrosine kinase inhibitor, has shown encouraging antitumor effects in advanced hepatocellular carcinoma (HCC). This study evaluated the effectiveness and safety of anlotinib with or without programmed death-1 (PD-1) blockades for patients with advanced primary HCC in a real-world setting in China. Patients and Methods: Between July 2019 and May 2021, 27 patients with advanced primary HCC who received at least 2 cycles of anlotinib were included in this retrospective study. Primary endpoint was objective response rate (ORR). Secondary endpoints were disease control rate (DCR), progression-free survival (PFS), overall survival (OS), and safety. Results: Of the 27 patients, ORR and DCR were 25.93% and 74.07%, respectively. The median follow-up time was 6.27 months (range: 1.30-17.40) with a median PFS and OS of 3.29 months (95% CI: 1.31-15.47) and 6.21 months (95% CI: 2.23-15.87), respectively. A total of 14 patients received anlotinib and PD-1 blockade combination therapy, and 13 received anlotinib monotherapy. No significant differences were observed in ORR (28.57 vs 23.08%), DCR (71.43 vs 76.92%), PFS (3.38 [95% CI: 2.66-13.14] vs 11.86 months [95% CI: 4.27-15.93]) and OS (4.90 [95% CI: 2.56-13.60] vs 11.04 months [95% CI: 1.31-17.18]) between the two groups (all p>0.05). Treatment-related AEs were reported in 88.89% of patients. Grade 3 AE was bleeding, which occurred in 3 patients (11.11%). Conclusion: Anlotinib yielded a promising efficacy and manageable safety in patients with advanced primary HCC irrespective of whether patients received PD-1 blockades, indicating that anlotinib might be a promising treatment option for this patient population.

[A review of WHO Laboratory Manual for the Examination and Processing of Human Semen (5th edition)].

This paper presents a review of the contribution, revision and defects of the 5th edition of WHO Laboratory Manual for the Examination and Processing of Human Semen published in 2010. The pity is that the book lacks the data from China. The normal spermatozoon morphology as 4% seems incredible and hard to be applied in practice. The reference values provided by WHO are important records of past findings, but do not hold good for all time. The Manual cannot be looked upon as a "Bible" but a new starting point. It is an inescapable responsibility of Chinese andrologists to establish a set of criteria for human semen analysis specifically applicable to Chinese males.