Ticagrelor Protects against Sepsis-Induced Acute Kidney Injury through an Adenosine Receptor-Dependent Pathway.

OBJECTIVE: Ticagrelor is a widely used anti-platelet drug. However, the mechanisms by which ticagrelor protects against sepsis-induced acute kidney injury (AKI) have not been clearly demonstrated. We designed this study to explore the protective effect of ticagrelor on sepsis-induced AKI and to explore the underlying mechanisms. METHODS: C57BL6J mice received oral ticagrelor (20 mg/kg and 50 mg/kg) for 7 days, and then caecal ligation and puncture (CLP) were performed. An adenosine receptor antagonist, CGS15943, was administered (10 mg/kg, intraperitoneal injection) to block the adenosine pathway 2 h before CLP. After 24 h, serum creatinine levels were measured. Periodic acid-Schiff (PAS) staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining were employed to analyze pathological changes and cell apoptosis. Serum concentrations of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and mRNA expression of tissue TNF-α and IL-1ß were detected. Western blotting analysis was used to determine AKT and mammalian target of rapamycin (mTOR) protein expression in the kidney. RESULTS: PAS staining showed less swelling of renal tubules, and TUNEL staining revealed less cell apoptosis in the ticagrelor group than in the CLP group. Serum creatinine levels were significantly lower in the ticagrelor group than in the CLP group. Moreover, significantly lower serum and kidney levels of TNF-α and IL-1ß were observed in the ticagrelor group. CGS15943 blocked the effects of ticagrelor. Western blotting analysis showed increased phosphorylation of AKT and mTOR in the kidneys of the 50 mg/kg ticagrelor group. The adenosine receptor antagonist inhibited the activation of AKT and mTOR. CONCLUSION: This study demonstrates that the protective effect of ticagrelor on sepsis-induced AKI depends on adenosine receptor activation and the subsequent increase of AKT and mTOR phosphorylation.

Analysis of malnutrition factors for inpatients with chronic kidney disease.

Objective: Malnutrition is a common complication of Chronic Kidney Disease (CKD), and it is the risk factor of CKD prognosis. This study aim to evaluate the nutritional status of inpatients with CKD by using the Subjective Global Assessment (SGA), and to analyze the related factors of malnutrition; and to provide effective reference for early detection of malnutrition status in patients with CKD and timely nutrition intervention. Methods: A total of 426 patients (238 male patients, 188 female patients) aged 62.62 ± 14.61 and 61.14 ± 14.82, respectively admitted to the Nephrology Department of Wannan Medical College from February 2020 to December 2020 were selected and included in to this study by convenience sampling. 426 patients with CKD were evaluated by SGA. Human body weight, hemoglobin (Hb), total protein (TP), albumin (ALB), pre-albumin (PA), qualitative analysis of urinary protein and other laboratory indexes were collected and measured. The correlation between malnutrition and age, education, gender, diet, CKD stage and other factors was analyzed by spearman correlation analysis. Results: The incidence of malnutrition was 85.7% among 426 patients with CKD. Gender, age, education level, CKD stage, diabetes mellitus, weight loss and reduced food intake were related to SGA nutritional assessment (P < 0.05). The expression levels of ALB, PA and Hb in the malnutrition group were significantly lower than those in the normal group (P < 0.05). The degree of malnutrition in CKD patients was significant negatively correlated with the expression levels of ALB (r = -0.188), PA (r = -0.262) and Hb (r = -0.176) (P < 0.05). The multivariate Logistic regression analysis model showed that female (OR = 2.155), ≥60 years old (OR = 7.671), weight loss (OR = 10.691), reduced food intake (OR = 28.953), moderate and severe serum ALB expression (OR = 3.391 and 8.326) were risk factors for malnutrition in patients with CKD (P < 0.05). Malnutrition was correlated with the results of qualitative examination of urinary protein (r = 0.268, P < 0.05). Conclusion: Gender, age, weight loss, reduced food intake, serum ALB expression were independently associated with malnutrition in patients with chronic kidney disease, Hence, the medical staff should take timely and effective nutrition intervention for the patients with malnutrition, delay the renal function damage of patients with CKD and improve the quality of life of patients. Inpatients with CKD, especially women, should increase their dietary intake, maintain normal weight and improve their nutritional status.

Baseline Serum Cystatin C Is a Potential Predictor for Acute Kidney Injury in Patients with Acute Pancreatitis.

AIMS: The study is aimed at studying the incidence of acute kidney injury (AKI) and exploring the potential predictor for AKI in patients with acute pancreatitis. METHODS: A retrospective study adopting a stratified cohort sampling design was performed in a cohort of patients (n = 237) diagnosed with acute pancreatitis without any renal injury. The following information including age, gender, serum creatinine, serum urea nitrogen, serum uric acid, serum cystatin C, fasting serum glucose, serum amylase, serum lipase, serum choline esterase, total protein, albumin, globulin, total bilirubin, direct bilirubin, total bile acids, glutamic-pyruvic transaminase, glutamic-oxaloacetic transaminase, gamma glutamyl transpeptidase, and alkaline phosphatase were collected from each patient when they were diagnosed with acute pancreatitis. Student t-test was conducted to figure out the difference between patients with and without AKI. Univariate and multivariate logistic regression analyses were used for investigating the predictors for AKI in patients with acute pancreatitis. RESULTS: 18 (7.6%) patients in total had developed AKI among the study group. Compared with patients without AKI (1.01 ± 0.26 mg/L), the level of baseline serum cystatin C (CYS-C) was significantly higher in patients with AKI (3.64 ± 2.17 mg/L, P < 0.001). Baseline serum CYS-C (OR = 203.594, P < 0.001) was the independent and significant predictor for AKI in patients with acute pancreatitis. AKI in patients with acute pancreatitis could be identified with a sensitivity of 88.9% at specificity of 100% (AUC = 0.948, 95% CI 0.879-1.000) by baseline serum CYS-C (cut-off value = 1.865 mg/L). CONCLUSIONS: Baseline serum CYS-C shall be adopted to predict the potential risk of AKI in patients with acute pancreatitis.

Feature selection and classification of urinary mRNA microarray data by iterative random forest to diagnose renal fibrosis: a two-stage study.

Renal fibrosis is a common pathological pathway of progressive chronic kidney disease (CKD). However, kidney function parameters are suboptimal for detecting early fibrosis, and therefore, novel biomarkers are urgently needed. We designed a 2-stage study and constructed a targeted microarray to detect urinary mRNAs of CKD patients with renal biopsy and healthy participants. We analysed the microarray data by an iterative random forest method to select candidate biomarkers and produce a more accurate classifier of renal fibrosis. Seventy-six and 49 participants were enrolled into stage I and stage II studies, respectively. By the iterative random forest method, we identified a four-mRNA signature in urinary sediment, including TGFß1, MMP9, TIMP2, and vimentin, as important features of tubulointerstitial fibrosis (TIF). All four mRNAs significantly correlated with TIF scores and discriminated TIF with high sensitivity, which was further validated in the stage-II study. The combined classifiers showed excellent sensitivity and outperformed serum creatinine and estimated glomerular filtration rate measurements in diagnosing TIF. Another four mRNAs significantly correlated with glomerulosclerosis. These findings showed that urinary mRNAs can serve as sensitive biomarkers of renal fibrosis, and the random forest classifier containing urinary mRNAs showed favourable performance in diagnosing early renal fibrosis.

Are Urinary Tubular Injury Markers Useful in Chronic Kidney Disease? A Systematic Review and Meta Analysis.

BACKGROUND: Adverse outcome of chronic kidney disease, such as end stage renal disease, is a significant burden on personal health and healthcare costs. Urinary tubular injury markers, such as NGAL, KIM-1 and NAG, could provide useful prognostic value for the early identification of high-risk patients. However, discrepancies between recent large prospective studies have resulted in controversy regarding the potential clinical value of these markers. Therefore, we conducted the first meta-analysis to provide a more persuasive argument to this debate. METHODS: In the current meta-analysis, based on ten prospective studies involving 29366 participants, we evaluated the role of urinary tubular injury markers (NGAL, KIM-1 and NAG) in predicting clinical outcomes including CKD stage 3, end stage renal disease and mortality. The prognostic values of these biomarkers were estimated using relative risks and 95% confidence interval in adjusted models. All risk estimates were normalized to those of 1 standard deviation increase in log-scale concentrations to minimize heterogeneity. Fixed-effects models were adopted to combine risk estimates. The quality of the research and between-study heterogeneity were evaluated. The level of research evidence was identified according to the GRADE profiler. RESULTS: uNGAL was identified as an independent risk predictor of ESRD (pooled adjusted relative risk: 1.40[1.21 to 1.61], p<0.001) and of overall mortality (pooled adjusted relative risk: 1.10[1.03 to 1.18], p = 0.001) in patients with chronic kidney disease. A borderline significance of uKIM-1 in predicting CKD stage 3 independently in the community-based population was observed (pooled adjusted relative risk: 1.13[1.00 to 1.27], p = 0.057). Only the prognostic value of uNGAL for ESRD was supported by a grade B level of evidence. CONCLUSION: The concentration of uNGAL can be used in practice as an independent predictor of end stage renal disease among patients with chronic kidney disease, but it may be not useful in predicting disease progression to CKD stage 3 among community-based population.

Urinary vimentin mRNA as a potential novel biomarker of renal fibrosis.

Renal fibrosis is a histological outcome of chronic kidney disease (CKD) progression. However, the noninvasive detection of renal fibrosis remains a challenge. Here we constructed a renal fibrosis target mRNA array and used it to detect urinary mRNAs of CKD patients for investigating potential noninvasive biomarkers of renal fibrosis. We collected urine samples from 39 biopsy-proven CKD patients and 11 healthy controls in the training set. Urinary mRNA profiles of 86 genes showed a total of 21 mRNAs that were differentially expressed between CKD patients and controls (P < 0.05), and vimentin (VIM) mRNA demonstrated the highest change fold of 9.99 in CKD vs. controls with robust correlations with decline of renal function and severity of tubulointerstitial fibrosis. Additionally, VIM mRNA further differentiated patients with moderate-to-severe fibrosis from none-to-mild fibrosis group with an area of the curve of 0.796 (P = 0.008). A verification of VIM mRNA in the urine of an additional 96 patients and 20 controls showed that VIM is not only well correlated with renal function parameters but also correlated with proteinuria and renal fibrosis scores. Multiple logistic regression and receiver-operating characteristics analysis further showed that urine VIM mRNA is the best predictive parameter of renal fibrosis compared with estimated glomerular filtration rate, serum creatinine, and blood urea nitrogen. In addition, there is no improved predictive performance for the composite biomarkers to predict renal fibrosis severity compared with a single gene of VIM. Overall, urinary VIM mRNA might serve as a novel independent noninvasive biomarker to monitor the progression of kidney fibrosis.

CD2AP mRNA in urinary exosome as biomarker of kidney disease.

AIMS: Podocyte injury plays an important role in the pathogenesis of kidney disease. Urinary exosomes are microvesicles released by tubular epithelial cells and podocytes containing information of their originated cells. This study investigated for the first time whether podocyte related mRNA in urinary exosome could serve as novel biomarkers for kidney disease. METHODS: Urine samples were collected from 32 patients of kidney disease who underwent kidney biopsy and 7 controls. CD2AP, NPHS2 and synaptopodin were detected by real-time RT-PCR on RNA isolated from urinary exosome. RESULTS: The pellet microvesicles were positively stained with exosome and podocyte marker, AQP2, CD9 and nephrin. CD2AP mRNA was lower (p=0.008) in kidney disease patients compared with controls and decreased with the increasing severity of proteinuria (p=0.06). CD2AP correlated with serum creatinine (r=-0.373, p=0.035), BUN (r=-0.445, p=0.009) and eGFR (r=0.351, p=0.046). Neither NPHS2 nor synaptopodin correlated with parameters of renal function. CD2AP mRNA correlated negatively with 24 hour urine protein (r=-0.403, p=0.022), severity of tubulointerstitial fibrosis (r=-0.394, p=0.026) and glomerulosclerosis (r=-0.389, p=0.031) and could discriminate kidney disease from controls with AUC of 0.821 (p=0.008). CONCLUSIONS: Urinary exosome mRNA of CD2AP might be a non-invasive tool for detecting both renal function and fibrosis of kidney disease.

MicroRNA-29c in urinary exosome/microvesicle as a biomarker of renal fibrosis.

Micro (mi)RNAs are frequently dysregulated in the development of renal fibrosis. Exosomes are small membrane vesicles that could be isolated from urine secreted from all nephron segments. Here we sought to observe for the first time whether miRNA in urine exosome could serve as a potential biomarker of renal fibrosis. Urine samples were collected from 32 chronic kidney disease (CKD) patients who underwent kidney biopsy and 7 controls. Exosome was isolated and confirmed by immunogold staining of exosome marker. Members of miR-29, miR-200, and RNU6B as endogenous control were detected by RT quantitative PCR. Electronic microscopy verified a typical shape of exosome with average size of 65.1 nm and labeled it with anti-CD9 and anti-aquaporin 2 antibody. Members of miR-29 and miR-200 are readily measured with reduced levels compared with controls (P < 0.05) and can robustly distinguish CKD from controls [area under the curve (AUC) varied from 0.902 to 1 by receiver operating characteristics analysis]. miR-29c correlated with both estimated glomerular filtration rate (r = 0.362; P < 0.05) and degree of tubulointerstitial fibrosis (r = -0.359; P < 0.05) for CKD patients. Moreover, miRNA in exosome was decreased in mild fibrosis group compared with moderated to severe group. miR-29a and miR-29c could predict degree of tubulointerstitial fibrosis with AUC of 0.883 and 0.738 (P < 0.05). The sensitivity and specificity for distinguishing mild from moderate to severe fibrosis were 93.8 and 81.3% with the use of miR-29a and 68.8 and 81.3% for miR-29c. Overall, miR-29c in urinary exosome correlates with both renal function and degree of histological fibrosis, suggesting it as a novel, noninvasive marker for renal fibrosis.

Urinary mRNA expression of CCN2/CCN3 as a noninvasive marker for monitoring glomerular structure changes in nondiabetic chronic kidney disease.

BACKGROUND: We investigated whether urinary mRNA of connective tissue growth factor (CCN2) and nephroblastoma overexpressed gene (CCN3) can provide clinical insight into the management of patients with nondiabetic CKD. METHODS: Urinary mRNA expression of CCN2 and CCN3 were measured by Real-time PCR in 35 CKD patients and 12 controls. RESULTS: Urinary mRNA of CCN2 and CCN3 were distinctively greater in CKDs than healthy controls. Urinary CCN3/CCN2 mRNA ratio correlated to the degree of glomerular histological changes in those who received renal biopsy. CONCLUSION: Urinary CCN3/CCN2 mRNA ratio may be a useful noninvasive biomarker for evaluating patients with nondiabetic CKD prior to renal biopsy.

Application of systems biology to the study of chronic kidney disease.

Chronic kidney disease (CKD) is a major public health problem that affects about 10% of the general population. Current approaches to characterize the category and progression of CKD are normally based on renal histopathological results and clinical parameters. However, this information is not sufficient to predict CKD progression risk reliably or to guide preventive interventions. Nowadays, the appearance of systems biology has brought forward the concepts of "-omics" technologies, including genomics, transcriptomics, proteomics, and metabolomics. Systems biology, together with molecular analysis approaches such as microarray analysis, genome-wide association studies (GWAS), and serial analysis of gene expression (SAGE), has provided the framework for a comprehensive analysis of renal disease and serves as a starting point for generating novel molecular diagnostic tools for use in nephrology. In particular, analysis of urinary mRNA and protein levels is rapidly evolving as a non-invasive approach for CKD monitoring. All these systems biological molecular approaches are required for application of the concept of "personalized medicine" to progressive CKD, which will result in tailoring therapy for each patient, in contrast to the "one-size-fits-all" therapies currently in use.

A pilot trial assessing urinary gene expression profiling with an mRNA array for diabetic nephropathy.

BACKGROUND: The initiation and progression of diabetic nephropathy (DN) is complex. Quantification of mRNA expression in urinary sediment has emerged as a novel strategy for studying renal diseases. Considering the numerous molecules involved in DN development, a high-throughput platform with parallel detection of multiple mRNAs is needed. In this study, we constructed a self-assembling mRNA array to analyze urinary mRNAs in DN patients with aims to reveal its potential in searching novel biomarkers. METHODS: mRNA array containing 88 genes were fabricated and its performance was evaluated. A pilot study with 9 subjects including 6 DN patients and 3 normal controls were studied with the array. DN patients were assigned into two groups according to their estimate glomerular rate (eGFR): DNI group (eGFR>60 ml/min/1.73 m(2), n = 3) and DNII group (eGFR<60 ml/min/1.73 m(2), n = 3). Urinary cell pellet was collected from each study participant. Relative abundance of these target mRNAs from urinary pellet was quantified with the array. RESULTS: The array we fabricated displayed high sensitivity and specificity. Moreover, the Cts of Positive PCR Controls in our experiments were 24±0.5 which indicated high repeatability of the array. A total of 29 mRNAs were significantly increased in DN patients compared with controls (p<0.05). Among these genes, α-actinin4, CDH2, ACE, FAT1, synaptopodin, COL4α, twist, NOTCH3 mRNA expression were 15-fold higher than those in normal controls. In contrast, urinary TIMP-1 mRNA was significantly decreased in DN patients (p<0.05). It was shown that CTGF, MCP-1, PAI-1, ACE, CDH1, CDH2 mRNA varied significantly among the 3 study groups, and their mRNA levels increased with DN progression (p<0.05). CONCLUSION: Our pilot study demonstrated that mRNA array might serve as a high-throughput and sensitive tool for detecting mRNA expression in urinary sediment. Thus, this primary study indicated that mRNA array probably could be a useful tool for searching new biomarkers for DN.

Urinary mRNA markers of epithelial-mesenchymal transition correlate with progression of diabetic nephropathy.

OBJECTIVE: Epithelial-mesenchymal transition (EMT) plays an important role in the pathogenesis and progression of diabetic nephropathy (DN). Quantification of messenger RNA (mRNA) expression in urinary sediment is emerging as a noninvasive method of screening DN-associated biomarkers. The aim of our study was to examine whether urinary mRNA profile of EMT-associated genes may provide valuable clinical insight into the different stages of DN. DESIGN AND METHODS: Diabetic nephropathy patients (n = 44) and healthy controls (n = 12) were enrolled in this study. DN patients were divided into three groups according to the levels of estimated glomerular filtration rate (eGFR): Group A (eGFR > 60 ml/min per 1·73 m(2), n = 27), Group B (45 < eGFR < 60 ml/min per 1·73 m(2), n = 9), and Group C (eGFR < 45 ml/min per 1·73 m(2), n = 8). Relative mRNA abundance of α-smooth muscle actin (α-SMA), fibronectin, FSP1 and matrix metalloproteinase-9 (MMP-9) were quantified, and correlations between target mRNAs and clinical parameters were examined. RESULTS: The urinary mRNA levels of α-SMA, fibronectin and MMP-9 were significantly higher in the DN group compared with controls (P < 0·05), and mRNA levels increased with DN progression. Urinary mRNA levels of all target genes positively correlated with both urinary albumin excretion (UAE) and blood urea nitrogen (BUN). Moreover, the expression of α-SMA, fibronectin and MMP-9 mRNA correlated with serum creatinine levels (r = 0·514, r = 0·53 and r = 0·469, all P < 0·001) and GFR levels (r = -0·374, r = -0·392 and r = -0·487, all P < 0·01). CONCLUSIONS: Quantification of EMT-associated genes in urinary sediment may be a novel approach for searching new biomarkers of DN.