5/6th Nephrectomy in Combination with High Salt Diet and Nitric Oxide Synthase Inhibition to Induce Chronic Kidney Disease in the Lewis Rat

Chronic kidney disease (CKD) is a global problem. Slowing CKD progression is a major health priority. Since CKD is characterized by complex derangements of homeostasis, integrative animal models are necessary to study development and progression of CKD. To study development of CKD and novel therapeutic interventions in CKD, we use the 5/6th nephrectomy ablation model, a well known experimental model of progressive renal disease, resembling several aspects of human CKD. The gross reduction in renal mass causes progressive glomerular and tubulo-interstitial injury, loss of remnant nephrons and development of systemic and glomerular hypertension. It is also associated with progressive intrarenal capillary loss, inflammation and glomerulosclerosis. Risk factors for CKD invariably impact on endothelial function. To mimic this, we combine removal of 5/6th of renal mass with nitric oxide (NO) depletion and a high salt diet. After arrival and acclimatization, animals receive a NO synthase inhibitor (NG-nitro-L-Arginine) (L-NNA) supplemented to drinking water (20 mg/L) for a period of 4 weeks, followed by right sided uninephrectomy. One week later, a subtotal nephrectomy (SNX) is performed on the left side. After SNX, animals are allowed to recover for two days followed by LNNA in drinking water (20 mg/L) for a further period of 4 weeks. A high salt diet (6%), supplemented in ground chow (see time line Figure 1), is continued throughout the experiment. Progression of renal failure is followed over time by measuring plasma urea, systolic blood pressure and proteinuria. By six weeks after SNX, renal failure has developed. Renal function is measured using ''gold standard'' inulin and para-amino hippuric acid (PAH) clearance technology. This model of CKD is characterized by a reduction in glomerular filtration rate (GFR) and effective renal plasma flow (ERPF), hypertension (systolic blood pressure>150 mmHg), proteinuria (> 50 mg/24 hr) and mild uremia (>10 mM). Histological features include tubulo-interstitial damage reflected by inflammation, tubular atrophy and fibrosis and focal glomerulosclerosis leading to massive reduction of healthy glomeruli within the remnant population (<10%). Follow-up until 12 weeks after SNX shows further progression of CKD.     

无创呼吸机在慢性阻塞性肺疾病急性加重期应用的疗效评价

摘要目的：观察无创呼吸机对不伴有呼吸衰竭的慢性阻塞性肺疾病急性加重期（AECOPD）患者症状、体征、血气分析和肺功能的影响。方法：80例AECOPD患者随机分为对照组和试验组,对照组给予常规治疗,如常规抗感染、化痰、平喘等治疗。试验组在常规治疗的基础上给予无创呼吸机正压通气（NIPPV）治疗。评价两组患者治疗前和治疗一周后临床症状体征、血气分析和肺功能的变化。结果：两组患者治疗后临床症状及体征评分较治疗前均呈下降趋势（P〈0．05）,但试验组下降更为明显（P〈0．05）,并且试验组症状改善时间明显缩短（P〈0．05）;治疗后两组的pH,PO2和SaO2均较治疗前升高,PC02较治疗前下降（P〈0．05）,和对照组相比,治疗后试验组的PO：和PC02改善更明显（P〈0．05）,而pH和Sa02无显著差异（P〉0．05）;治疗后两组的FEV1,FVC,FEV1实测值／预计值和FEVI％均较治疗前升高（P〈0．05）,试验组升高更明显（P〈0．05）。结论：对不伴有呼吸衰竭的AECOPD患者早期应用NIPPV治疗能有效改善临床症状和体征,缩短症状改善时间,改善通气,缓解呼吸肌疲劳,预防呼吸衰竭的发生。     

Virulence of infecting Helicobacter pyloristrains and intensity of mononuclear cell infiltration are associated with levels of DNA hypermethylation in gastric mucosae

DNA methylation changes are known to occur in gastric cancers and in premalignant lesions of the gastric mucosae. In order to examine variables associated with methylation levels, we quantitatively evaluated DNA methylation in tumors, non-tumor gastric mucosae, and in gastric biopsies at promoters of 5 genes with methylation alterations that discriminate gastric cancers from non-tumor epithelia (EN1, PCDH10, RSPO2, ZIC1, and ZNF610). Among Colombian subjects at high and low risk for gastric cancer, biopsies from subjects from the high-risk region had significantly higher levels of methylation at these 5 genes than samples from subjects in the low risk region (p ≤ 0.003). When results were stratified by Helicobacter pylori infection status, infection with a cagA positive, vacA s1m1 strain was significantly associated with highest methylation levels, compared with other strains (p = 0.024 to 0.001). More severe gastric inflammation and more advanced precancerous lesions were also associated with higher levels of DNA methylation (p ≤ 0.001). In a multivariate model, location of residence of the subject and the presence of cagA and vacA s1m1 in the H. pylori strain were independent variables associated with higher methylation in all 5 genes. High levels of mononuclear cell infiltration were significantly related to methylation in PCDH10, RSPO2, and ZIC1 genes. These results indicate that for these genes, levels of methylation in precancerous lesions are related to H. pylori virulence, geographic region and measures of chronic inflammation. These genes seem predisposed to sustain significant quantitative changes in DNA methylation at early stages of the gastric precancerous process.     

microRNA-34b/c on chromosome 11q23 is aberrantly methylated in chronic lymphocytic leukemia

A commonly deleted region in chronic lymphocytic leukemia (CLL) is the 11q22–23 region, which encompasses the ATM gene. Evidence suggests that tumor suppressor genes other than ATM are likely to be involved in CLL with del(11q). A microRNA (miR) cluster including the miR-34b and miR-34c genes is located, among other genes, within the commonly deleted region (CDR) at 11q. Interestingly, these miRs are part of the TP53 network and have been shown to be epigenetically regulated. In this study, we investigated the expression and methylation status of these miRs in a well-characterized cohort of CLL, including cases with/without 11q-deletion. We show that the miR-34b/c promoter was aberrantly hypermethylated in a large proportion of CLL cases (48%, 25/52 cases). miR-34b/c expression correlated inversely to DNA methylation (P = 0.003), and presence of high H3K37me3 further suppressed expression regardless of methylation status. Furthermore, increased miR-34b/c methylation inversely correlated with the presence of 11q-deletion, indicating that methylation and del(11q) independently silence these miRs. Finally, 5-azacytidine and trichostatin A exposure synergistically increased the expression of miR-34b/c in CLL cells, and transfection of miR-34b or miR-34c into HG3 CLL cells significantly increased apoptosis. Altogether, our novel data suggest that miR-34b/c is a candidate tumor suppressor that is epigenetically silenced in CLL.     

KIBRA gene methylation is associated with unfavorable biological prognostic parameters in chronic lymphocytic leukemia

Ras-association domain family (RASSF) members are a family of genes containing an RA domain in either the C-terminus (RASSF1-RASSF6) or in the N-terminus (RASSF7-RASSF10). Members of this gene family are core members of the Salvador/Warts/Hippo (SWH) tumor suppressor network and have been shown to be involved in human tumorigenesis. Among the RASSF genes, RASSF1A is one of the most frequently methylated genes in a wide range of epithelial cancers, and we previously demonstrated that RASSF6 and RASSF10 genes are frequently epigenetically inactivated in acute leukemias, particularly in those of the B cell type. We here determined the methylation profiles of all members of the RASSF gene family as well as two recently identified (KIBRA, CRB3) upstream members of the SWH pathway in the leukemic B cells obtained from a well-characterized cohort of 95 patients with chronic lymphocytic leukemia (CLL). Among the RASSF genes, RASSF10 (50%) was the most frequently methylated gene, followed by RASSF6 (16%). The remaining RASSF genes were either unmethylated or showed a frequency of methylation < 10%. The upstream SWH member KIBRA was also frequently methylated in CLL (35%) in contrast to CRB3. Interestingly, the analysis of clinical-pathological parameters showed that KIBRA methylation was associated with unfavorable biological prognostic parameters, including unmutated IGHV genes (p = 0.007) and high CD38 expression (p < 0.05).     

Penalized survival models for the analysis of alternating recurrent event data

Recurrent event data are widely encountered in clinical and observational studies. Most methods for recurrent events treat the outcome as a point process and, as such, neglect any associated event duration. This generally leads to a less informative and potentially biased analysis. We propose a joint model for the recurrent event rate (of incidence) and duration. The two processes are linked through a bivariate normal frailty. For example, when the event is hospitalization, we can treat the time to admission and length-of-stay as two alternating recurrent events. In our method, the regression parameters are estimated through a penalized partial likelihood, and the variance-covariance matrix of the frailty is estimated through a recursive estimating formula. Moreover, we develop a likelihood ratio test to assess the dependence between the incidence and duration processes. Simulation results demonstrate that our method provides accurate parameter estimation, with a relatively fast computation time. We illustrate the methods through an analysis of hospitalizations among end-stage renal disease patients.     

Variable selection in joint frailty models of recurrent and terminal events

Recurrent event data are commonly encountered in biomedical studies. In many situations, they are subject to an informative terminal event, for example, death. Joint modeling of recurrent and terminal events has attracted substantial recent research interests. On the other hand, there may exist a large number of covariates in such data. How to conduct variable selection for joint frailty proportional hazards models has become a challenge in practical data analysis. We tackle this issue on the basis of the “minimum approximated information criterion” method. The proposed method can be conveniently implemented in SAS Proc NLMIXED for commonly used frailty distributions. Its finite-sample behavior is evaluated through simulation studies. We apply the proposed method to model recurrent opportunistic diseases in the presence of death in an AIDS study.     

A possible role for inducible arginase isoform (AI) in the pathogenesis of chronic venous leg ulcer

Chronic venous ulcer (CVU) is a major cause of chronic wounds of lower extremities and presents a significant financial and resource burden to health care systems worldwide. Defects in the vasculature, matrix deposition, and re-epithelialization are the main histopathological changes believed to impede healing. Supplementation of the amino acid arginine that plays a crucial role in the interactions that occur during inflammation and wound healing was proven clinically to improve acute wound healing probably through enhancing activity of inducible arginase (AI) locally in the wounds. However, the possible mechanism of arginine action and the potential beneficial effects of AI/arginine in human chronic wounds remain unclear. In the present study, using biopsies, taken under local anesthesia, from adult patients (n = 12, mean age 55 years old) with CVUs in lower extremities, we investigated the correlation between AI distribution in CVUs and the histopathological changes, mainly proliferative and vascular changes. Our results show a distinct spatial distribution of AI along the ulcer in the epidermis and in the dermis with the highest level of expression being at the ulcer edge and the least expression towards the ulcer base. The AI cellular immunoreactivity, enzymatic activity, and protein levels were significantly increased towards the ulcer edge. Interestingly, a similar pattern of expression was encountered in the proliferative and the vascular changes with strong correlations between AI and the proliferative activity and vascular changes. Furthermore, AI cellular distribution was associated with increased proliferative activity, inflammation, and vascular changes. Our findings of differential expression of AI along the CVU base, edge, and nearby surrounding skin and its associations with increased proliferative activity and vascular changes provide further support to the AI implication in CVU pathogenesis. The presence of high levels of AI in the epidermis of chronic wounds may serve as a molecular marker of impaired healing and may provide future targets for therapeutic intervention.