Hepatitis B virus X protein promotes podocyte pyroptosis in hepatitis B virus-associated glomerulonephritis by down-regulating microRNA -223 targeting NLRP3 inflammasome / 中华肝脏病杂志

Objective: To investigate the potential function and related mechanism of microRNA-223 (miRNA-223) in the podocyte pyroptosis of hepatitis B virus (HBV)-associated glomerulonephritis induced by HBV X protein (HBx). Methods: HBx-overexpressing lentivirus was transfected into human renal podocytes to mimic the pathogenesis of HBV-GN. Real-time fluorescence quantitative PCR and Western blotting experiments were used to detect the mRNA and protein expression of pyroptosis-related proteins [nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD (ASC) and caspase-1], and inflammatory factors (interleukin-1β and interleukin-18), respectively.TUNEL staining and flow cytometry were used to detect the number of pyroptosis cells. Immunofluorescence staining was used to detect the expression of podocytes biomarkers desmin and nephrin; Hoechst 33342 staining was used to observe the morphological and quantitative changes of podocyte nuclei. Enzyme-linked immunosorbent assay was used to measure caspase-1 activity. The dual luciferase reporter gene assay was used to verify the downstream target of miRNA-223. Podocytes were divided into the following nine groups: control group (no special treatment), empty plasmid group (transfected with empty plasmid), HBx overexpression group (transfected with HBx overexpression lentivirus), HBx overexpression+miRNA-223 mimic group (transfected with HBx overexpression lentivirus and miRNA-223 mimic), HBx overexpression+miRNA-223 inhibitor group (transfected with HBx overexpression lentivirus and miRNA-223 inhibitor), HBx overexpression+miRNA-223 mimic+NLRP3 group (transfected with HBx overexpression lentivirus, miRNA-223 mimic and NLRP3 overexpression plasmid), HBx overexpression+miRNA-223 mimic+ NLRP3 siRNA group (transfected with HBx overexpression lentivirus, miRNA-223 mimic and NLRP3 siRNA), HBx overexpression+miRNA-223 inhibitor+NLRP3 group (transfected with HBx overexpression lentivirus, miRNA-223 inhibitor and NLRP3 overexpression plasmid), HBx overexpression+miRNA-223 inhibitor+NLRP3 siRNA group (transfected with HBx overexpression lentivirus, miRNA-223 inhibitor and NLRP3 siRNA). Results: miRNA-223 was down-regulated in HBx overexpression group compared with the control group (P < 0.05). TUNEL and immunofluorescence staining showed that NLRP3 knockdown attenuated podocyte injury and pyroptosis induced by HBx overexpression (P < 0.05). Dual luciferase reporter gene assay demonstrated that NLRP3 was one of the downstream targets of miRNA-223. Rescue experiments revealed that NLRP3 overexpression weakened the protective effect of miRNA-223 in podocyte injury (P < 0.05). The addition of miRNA-223 mimic and NLRP3 siRNA decreased the expression of NLRP3 inflammasome and cytokines, and reduced the number of pyroptosis cells induced by HBx overexpression (all P < 0.05); The addition of miRNA-223 inhibitor and NLRP3 overexpression plasmid significantly increased the expression of NLRP3 inflammasome and cytokines, caspase-1 activity, and the number of pyroptosis cells (all P < 0.05). Conclusion: HBx may promote podocyte pyroptosis of HBV-GN via downregulating miRNA-223 targeting NLRP3 inflammasome, suggesting that miRNA-223 is expected to be a potential target for the treatment of HBV-GN.

Effective substances and mechanism of Yishen Guluo Mixture in treatment of chronic glomerulonephritis based on metabolomics and serum pharmacochemistry / 中国中药杂志

This study aimed to investigate the effective substances and mechanism of Yishen Guluo Mixture in the treatment of chronic glomerulonephritis(CGN) based on metabolomics and serum pharmacochemistry. The rat model of CGN was induced by cationic bovine serum albumin(C-BSA). After intragastric administration of Yishen Guluo Mixture, the biochemical indexes related to renal function(24-hour urinary protein, serum urea nitrogen, and creatinine) were determined, and the efficacy evaluations such as histopathological observation were carried out. The serum biomarkers of Yishen Guluo Mixture in the treatment of CGN were screened out by ultra-performance liquid chromatography-quadrupole time-of-flight/mass spectrometry(UPLC-Q-TOF-MS) combined with multivariate statistical analysis, and the metabolic pathways were analyzed. According to the mass spectrum ion fragment information and metabolic pathway, the components absorbed into the blood(prototypes and metabolites) from Yishen Guluo Mixture were identified and analyzed by using PeakView 1.2 and MetabolitePilot 2.0.4. By integrating metabolomics and serum pharmacochemistry data, a mathematical model of correlation analysis between serum biomarkers and components absorbed into blood was constructed to screen out the potential effective substances of Yishen Guluo Mixture in the treatment of CGN. Yishen Guluo mixture significantly decreased the levels of 24-hour urinary protein, serum urea nitrogen, and creatinine in rats with CGN, and improved the pathological damage of the kidney tissue. Twenty serum biomarkers of Yishen Guluo Mixture in the treatment of CGN, such as arachidonic acid and lysophosphatidylcholine, were screened out, involving arachidonic acid metabolism, glycerol phosphatide metabolism, and other pathways. Based on the serum pharmacochemistry, 8 prototype components and 20 metabolites in the serum-containing Yishen Guluo Mixture were identified. According to the metabolomics and correlation analysis of serum pharmacochemistry, 12 compounds such as genistein absorbed into the blood from Yishen Guluo Mixture were selected as the potential effective substances for the treatment of CGN. Based on metabolomics and serum pharmacochemistry, the effective substances and mechanism of Yishen Guluo Mixture in the treatment of CGN are analyzed and explained in this study, which provides a new idea for the development of innovative traditional Chinese medicine for the treatment of CGN.

Avances en el tratamiento de cáncer de próstata resistente a la castración: énfasis en nuevas terapias hormonales / Advances in the treatment of castration-resistant prostate cancer: emphasis in new hormonal therapies

Prostate cancer represents the second cancer-related cause of death in North American and Chilean men. The main treatment for incurable stages of disease is surgical or pharmacological castration. However, with time and despite the addition of anti-androgens, the disease progresses to a clinical state that has been commonly referred to as “hormone refractory”. In recent years, the concept of hormone refractoriness has been challenged and replaced by “castration resistance”, acknowledging that further and optimal hormonal manipulation can be attained, beyond achieving testosterone levels at castration range. The purpose of this review is to summarize the recent therapeutic breakthroughs in the management of metastatic castrate resistant prostate cancer (mCRPC), with greater emphasis in the newer hormonal therapy agents such as Abiraterone and Enzalutamide. Future combination and sequential treatment strategies are contextualized in the current era of personalized cancer medicine and genomic characterization of prostate cancer.

Reflexiones en torno a la ley N° 20.584 y sus implicancias para la investigación biomédica en Chile / Considerations on the new Chilean law about rights and duties of patients

After years of discussion by the Chilean legislature, the Law Nº 20.584, which regulates health care related rights and duties of people, entered into force in Chile in October 2012. This bill represents an important step in the recognition and protection of health care related rights, welfare, dignity and duties of persons. It also intends to protect potential participants in clinical research. However such protective measures include explicit prohibitions such as the review of clinical records or the inclusion of people with mental or psychological handicaps as research participants. We herein discuss the implications of this law in medical research.

Assessment of nitric oxide and prostaglandins levels in different pathological types of glomerular disease

The objective of this study was to evaluate plasma nitrite and nitrate as well as urinary nitrite levels in forty cases with different glomerular disorders. In addition, serum thromboxane A2 [TXA2] and prostacyclin 12 [PGI2] levels were evaluated in these cases in comparison to 20 healthy control subjects. The effect of steroid therapy on the above mentioned parameters was also evaluated. The results revealed that urinary nitrite level was significantly elevated among patients compared to control. The elevation was observed in diabetic nephropathy as well as proliferative forms of glomerular disease such as lupus nephritis and mesangioproliferative GN. However, plasma nitrite, nitrate and serum cGMP levels were similar in patients compared to the control levels. TXA2 level exhibited significant elevation in patients, whereas serum PGI2 showed significant decrease in its level among cases as compared to control subjects. Steroid therapy was demonstrated to have no effect on all of the studied parameters. The study concluded that the data indicated that NO might play a role in the pathogenesis of proliferative glomerular disease and diabetic nephropathy. The demonstration of normal level of urinary nitrite in non proliferative GN suggests that enhanced NO production is not the sole cause of proteinuria in these cases. Further studies are, therefore, needed to examine the exact role of NO in pathogenesis of glomerular injury. The significant elevation of TXA2 level together with the significant decrease in serum PGI2 should be thoroughly investigated regarding the renal hemodynamics, the extent of proteinuria and the thromboembolic tendency reported in these cases

Possible new role for angiotensin-converting enzyme inhibitors in treating glomerulonephritis

Serum transforming growth factor-beta [TGF-beta1] production was estimated for 10 patients with essential hypertension, 12 patients with glomerulonephritis [5 hypertensive and 7 normotensive] and 10 healthy controls. The glomerulonephritis group received angiotensin-converting enzyme inhibitor captopril 25-75 mg/day for 4 weeks. Blood urea, serum creatinine, 24-hour urinary protein and serum TGF-beta1 were then re-estimated. Urea and creatinine were significantly higher in the hypertension and glomerulonephritis groups than in the controls and also higher in the glomerulonephritis group than the hypertension group. TGF-beta1 was significantly higher in the glomerulonephritis groups than in the control and hypertension groups. TGF-beta1 and 24-hour urinary protein were significantly reduced in the glomerulonephritis group

Immunohistochemical study of the expression of alpha-smooth muscle actin and the proliferation marker Ki-67 of glomerulonephritis

The aim of this study was to determine the relationship of alpha-smooth muscle actin (ASMA) and the proliferation marker Ki-67 of glomerulonephritis (GN). Immunohistochemical stainings with the usual streptavidin-biotin peroxidase method were performed on 86 renal biopsies using monoclonal 1A4 and Ki-67. The results of the quantitative evaluation of ASMA and Ki-67 were analyzed for the correlation between positive value of ASMA and Ki-67 in different GN. ASMA expressions of glomeruli were highest in acute post-infectious GN [APGN; 16.9 Fraction Volume (FV)%], followed by unclassified proliferative GN (UnGN; 12.5 FV%), membranoproliferative GN (MPGN; 8.5 FV%), lupus nephritis (LupusN; 6.3 FV%), IgA nephritis (IgAN; 5.6 FV%), and normal control (0.1 FV%). The Ki-67 staining was considerably elevated in lupusN (4.3 Ki-67 positives/glomerulus), APGN (2.7), MPGN (2.5), UnGN (1.66), IgAN (0.5), compared with that in normal control group (0.1 Ki-67 positives/glomerulus). Ki-67 value in each category of glomerular diseases was significantly different from that in the control biopsies (p<0.004). The relationship between morphometric results of ASMA and Ki-67 was statistically significant regardless of the diagnosis. (rs=0.425, p=0.000, ASMA= 0.1113+0.1665 Ki-67). In conclusion, the immunohistochemical assessment of ASMA and Ki-67 expression in GN might be a reliable indicator for the progression of GN. This study indicates that active cellular proliferation is associated with increased actin deposition in glomeruli.

Inherited structural and quantitative polymorphisms of C3b receptor (CR1) in normals and patients with glomerular diseases.

The erythrocyte C3b receptor (CR1) has been studied for its structural and quantitative polymorphisms in normal Indian individuals and in patients with glomerular diseases. In the normal Indian population, purification of CR1 by immunoprecipitation or C3b-Sepharose affinity column and subjecting it to electrophoresis showed the existence of two types of structural polymorphic patterns with M(r) of 190 kDa and 220 kDa, and with gene frequencies of 0.975 and 0.025, respectively. The gene frequencies of these alleles remain unaltered in the patient population. Evaluation of CR1 levels in the normal Indian population revealed a trimodal distribution of CR1 number suggesting a co-dominant allelic pattern (L and H alleles) for the quantitative expression of CR1 with gene frequencies of 0.523 and 0.477, respectively. In our earlier study we have shown that there is a decreased expression of CR1 on the erythrocytes of patients with acute glomerulonephritis. Since this decrease in the CR1 level in patients is an acquired characteristic, it may not be the level controlled by the LL homozygous alleles. The discrepancy in the gene frequencies of the structural and quantitative polymorphic alleles in normal individuals show that they are not linked to each other. In our earlier study, we showed that the affinity constant of C3b-CR1 binding in different individuals remains the same irrespective of the number of CR1 on the erythrocyte surface. Comparison of this result with the present investigation shows that there is no functional difference among various structural polymorphic forms of CR1 and the susceptibility to glomerular diseases is not associated with any of the CR1 polymorphic patterns.