A novel SPE-LC-MRM strategy for serum demethylzeylasteral quantitation developed with an 18O-labeled internal standard.

Natural bioactive compounds (NBCs) are widely used in clinical treatment. For example, Tripterygium wilfordii Hook f. is commonly known in China as Lei-Gong-Teng which means thunder god vine. This herb is widely distributed in Eastern and Southern China, Korea, and Japan. The natural bioactive compounds of this herb can be extracted and made into tripterygium glycoside tablets. It is one of the most commonly used and effective traditional Chinese herbal medicines against rheumatoid arthritis (RA), nephrotic syndrome (NS), autoimmune hepatis (AIH), and so on. However, many NBCs are difficult to reliably quantify in the serum due to the effects of matrix and RSD. In addition, the targeted compound's internal standard (IS) is rarely sold due to the complex isotope internal standard synthesis pathway. In this study, a new quantitation method for 18O labeling combined with off-line SPE was formulated. We contrasted the recoveries and matrix effects of various separation methods in order to choose the best method. Furthermore, we optimized the conditions for SPE loading and washing. An isotopic internal standard was prepared by the 16O/18O exchanging reaction in order to eliminate the matrix effects. The method's accuracy and precision met the requirements for method validation. The recovery of this method was close to 60%. The relative standard deviation (RSD) of the high-concentration sample was 2%, and the limit of detection (LOD) was 1 ng/mL. This method could be used to analyze the clinical serum concentration of demethylzeylasteral. Sixty samples were collected from 10 patients with diabetes nephropathy. The quantitation results of demethylzeylasteral in patients' serum obtained using this method exhibited a correlation between therapeutic drug monitoring (TDM) and decreased urinary protein. This work may have broad implications for the study of drug metabolism in vivo and the clinical application of low-abundance and difficult-to-quantify NBCs.

Wnt1 inhibits vascular smooth muscle cell calcification by promoting ANKH expression.

AIMS: Wnt signaling plays a critical role in vascular calcification (VC). Wnt factors induce different physiological and pathological effects on cardiovascular functions. Wnt1, a ligand of Wnt/ß-catenin signaling, promotes pro-angiogenesis and reduces myocardial infarction. The role of Wnt1 on VC in chronic kidney disease (CKD) is not fully understood. METHODS AND RESULTS: We used human vascular smooth muscle cells (VSMCs) and a rat model of chronic renal failure (CRF), and observed a native protective mechanism by which VC is reduced via the activation of Wnt1 and its transcriptional target ANKH inorganic pyrophosphate transport regulator (ANKH) gene. ANKH is an essential calcification inhibitor that effluxes inorganic pyrophosphate (PPi) from VSMCs to play an inhibitory role in VC. Vascular ANKH and plasma PPi were significantly downregulated in the rat model of CRF. The knockdown or inhibition of ANKH reversed the effect of Wnt1 on VC in VSMCs. Clinical analysis revealed low plasma levels of Wnt1 and PPi were associated with CKD in patients. Applying a Wnt/ß-catenin signaling agonist can alleviate the progression of VC. CONCLUSION: This work reveals the ANKH regulation of Wnt1 in VSMCs is essential for blocking VC. Our findings may contribute to the development of medications that target Wnt signaling and/or ANKH to inhibit VC.

Associations of sclerostin with carotid artery atherosclerosis and all-cause mortality in Chinese patients undergoing maintenance hemodialysis.

BACKGROUND: Previous clinical studies found inconsistent relationship between circulating sclerostin levels and treatment outcome in patients undergoing maintenance hemodialysis (MHD). Therefore, this study aimed to assess the associations of sclerostin with carotid artery atherosclerosis and all-cause mortality in Chinese patients undergoing MHD. METHODS: This retrospective study assessed 84 patients undergoing MHD at the Nephrology Department of Beijing Hospital from January to April 2012, with a median follow-up of 61.2 months (range: 11.5 to 63 months). Carotid artery intima-media thicknesses (CIMTs) and atherosclerotic plaques were measured by B-mode Doppler ultrasound at baseline. Blood samples were collected for measuring serum sclerostin and soluble klotho (s-klotho) levels. The associations of sclerostin levels with carotid artery atherosclerosis was evaluated by correlation methods. Predictive factors of mortality were assessed by multivariate COX regression. RESULTS: Baseline serum sclerostin averaged 162.01 pmol/L, with an interquartile range of 121.69 to 225.22 pmol/L, while CIMT values were 1.35 ± 0.39 mm. Carotid artery atherosclerotic plaques were detected in 68 subjects (81%). Subjects with sclerostin levels above the median value had higher CIMT (p = 0.038) and higher prevalence of atherosclerotic plaque (p = 0.025). During follow-up, 27 patients died; Kaplan-Meier curves indicated that subjects with high sclerostin levels (above the median value at baseline) had shorter survival (log rank p = 0.011). In multivariate COX regression analysis, serum sclerostin (HR, 1.095; 95% confidence interval [CI] 1.022-1.174, p = 0.010) and albumin (HR, 0.742; 95%CI 0.612-0.900, p = 0.002) levels were independent predictors of all-cause mortality. CONCLUSIONS: Sclerostin is positively associated with CIMT. In addition, patients with low baseline serum sclerostin undergoing MHD show better survival.

Value of contrast-enhanced ultrasound for diagnosis and follow-up of renal artery stenosis in patients with chronic kidney disease.

OBJECTIVE: To evaluate whether contrast-enhanced ultrasound (CEUS) is an accurate, non-nephrotoxic diagnostic method and follow-up tool for use in patients with chronic kidney disease (CKD) and renal artery stenosis (RAS). METHODS: In this prospective and monocentric study, we compared the sensitivity and specificity of CEUS for the diagnosis of RAS in CKD patients, using digital subtraction angiography (DSA) or computed tomographic angiography (CTA) as the gold standard methods. Further, the value of CEUS for distinguishing restenosis from other diseases was assessed. The ultrasound physicians conducted the examinations and served as the CEUS report readers who were blinded to the DSA or CTA results. RESULTS: Patients with RAS (n = 60) were enrolled. Average patient age was 64.4 ± 18.0 years and median estimated glomerular filtration rate was 66.1 mL/min/1.73 m2. CEUS was used to image 94 stenotic renal arteries and DSA- or CTA-verified stenosis was present in 96 renal arteries. The kappa value for CEUS was 0.776 (P < 0.001), with an accuracy of 92.5%, a sensitivity of 94.7%, and a specificity of 84.0%. The accuracy of CEUS was the same for the diagnosis of the CKD3b-5 group as for the CKD1-3a group (100% vs. 87.5%, P = 0.148). There was no difference in CEUS accuracy for the diagnosis of Takayasu RAS compared with atherosclerotic RAS (95.8% vs. 91.7%, P = 0.795). Twenty-nine CEUS examinations were performed to follow in-stent restenosis or progression of RAS, with a median follow-up time of 5.0 months (range 1.0-20.0). Two cases of in-stent restenosis in patients suffering from deteriorating kidney function and recurrent hypertension were examined by CEUS. CONCLUSION: CEUS examination is a credible alternative for diagnosing moderate and severe RAS in patients with CKD, and is a reliable tool for follow-up surveillance after renal artery revascularization treatment. It shouldn't be thought as a color-coded duplex ultrasonography rescue in these patients.

Demethylzeylasteral reduces the level of proteinuria in diabetic nephropathy: Screening of network pharmacology and verification by animal experiment.

This study aimed to use network pharmacology to detail the natural components isolated from Triptergium wilfordii Hook F (TwHF) and examine the effect of the main component (demethylzeylasteral, DEM) on rat models of diabetic nephropathy (DN). In this study, we used network pharmacology to detail the natural components isolated from TwHF, referenced a gene library when screening for components effective in the management of DN, and DEM was confirmed in DN rats. All data were analyzed using the Discovery Studio 4.5 System and the systems Dock online docking method platform. All 24 rats were divided into 4 groups: control, DN, TwHF, and DEM. Blood and urine samples were tested at 0, 8, and 12 weeks. Renal histopathological changes were scored. Network pharmacology indicated that 370 compounds and 46 small molecules (including DEM) were biologically active constituents of TwHF, mainly affecting the inflammatory response through PI3K-Akt and Jak-STAT pathways. Proteinuria in the TwHF and DEM groups was significantly lower than in the DN group (p ≤ .001), and the decrease in proteinuria in the DEM group was more obvious than in the TwHF group (p = .004). The tubular interstitial scores were better in the DEM group than in the TwHF and DN groups. These results indicate that DEM effectively reduced proteinuria and alleviated the tubular interstitial changes in rat models of DN, which may be provide a scientific foundation for the development of novel drugs for treatment of DN.

Using Two Detection Methods to Observe the Changes and Significance of Free Light Chain in Serum and Urine in Patients with Renal Insufficiency.

Background: Free light chains κ and λ (FLC κ, FLC λ) are of great significance in diagnostic and monitoring monoclonal gammopathy. Freelite and N-Latex methods are two common monitoring methods at present. But the two meanings are not completely equivalent, especially for patients with renal insufficiency. We analyzed the changes of serum and urine FLC in renal insufficiency patients without monoclonal gammopathy and the clinical significance of these changes. Methods: This study is an observational study. Patients ≥ 18 years old, who met the diagnostic criteria of chronic kidney disease (CKD), excluding monoclonal gammopathy, were selected. Fasting serum and 24-hour urine were taken to detect serum FLC κ, serum FLC λ, SCr, serum ß 2-microglobulin, urinary FLC κ, urinary FLC λ, urinary α 1-microglobulin, and urinary ß 2-microglobulin. Results: There was a good correlation between the two methods for determining serum/urinary FLC. No matter serum or urine, FLC showed a good correlation with renal function by the N-Latex method, but not by the Freelite method. Under the N-Latex method, FLC κ/λ remained stable, which was basically within the reference range of healthy people and was not affected by renal function. There was a good correlation between FLC detected by N-Latex and microglobulin in serum and urine. Conclusion: When the concentration of FLC is low, the N-Latex method is more recommended to monitor FLC. The FLC measured by the N-Latex method is more closely related to renal function. The ratio of FLC κ/λ determined by the N-Latex method remained stable within the recommended range.

Nomogram to predict the progression of patients with primary membranous nephropathy and nephrotic syndrome.

BACKGROUND: The outcome of patients with primary membranous nephropathy (pMN) who present with nephrotic syndrome (NS) is variable and difficult to predict. The goal of this study was to develop a nomogram to predict the risk of progression for specific individuals. METHODS: This retrospective study involved biopsy-proven patients with pMN and NS treated between January 2012 and June 2018. The primary outcome of our investigation was progression, defined as a reduction of estimated glomerular filtration rate (eGFR) that was equal to or over 20% compared with baseline at the end of follow-up or the onset of end-stage renal disease (ESRD). We used backwards stepwise logistic regression analysis to create a nomogram to predict prognosis. The model was validated internally using bootstrap resampling. RESULTS: A total of 111 patients were enrolled. After a median follow-up of 40.0 months (range 12-92 months), 18.9% (21/111) patients showed progression. Backwards stepwise selection using the Akaike information criterion (AIC) identified the following four variables as independent risk factors for progression, which were all used in the nomogram: age ≥ 65 years [odds ratio (OR) 7.004; 95% confidence interval (CI) 1.783-27.505; p = 0.005], Ln (sPLA2R-Ab) (OR 2.150; 95% CI 1.293-3.577; p = 0.003), Ln (proteinuria) (OR 5.939; 95% CI 1.055-33.436; p = 0.043) and Ln (Uα1m/Cr) (OR 2.808; 95% CI 1.035-7.619; p = 0.043). The discriminative ability and calibration of the nomogram revealed good predictive ability, as indicated by a C-index of 0.888 (95% CI 0.814-0.940) and a bootstrap-corrected C-index of 0.869; calibration curves were also well fitted. A receiver operating characteristic (ROC) curve for the nomogram score revealed significantly better discrimination than each of the three risk factors alone, including Ln (sPLA2R-Ab) [area under the curve (AUC) 0.769], Ln (proteinuria) (AUC 0.653) and Ln (Uα1m) (AUC 0.781) in the prediction of progression (p < 0.05). The optimal cutoff value of the nomogram score was 117.8 with a positive predictive value of 44.4% and a negative predictive value of 98.5%. CONCLUSION: The nomogram successfully achieved good predictive ability of progression for patients with pMN who present with NS. It can therefore help clinicians to individualize treatment plans and improve the outcome of pMN.

The relationship between sclerostin and carotid artery atherosclerosis in patients with stage 3-5 chronic kidney disease.

PURPOSE: Sclerostin is an antagonist of the Wnt/ß-catenin pathway. We previously reported that sclerostin is closely related to carotid artery atherosclerosis and long-term outcome in hemodialysis patients. The present study investigated the association between sclerostin, renal function, and carotid artery atherosclerosis in non-dialysis patients with stage 3-5 chronic kidney disease (CKD 3-5ND). METHODS: A total of 140 patients with CKD 3-5ND were enrolled in this cross-sectional study. The Chronic Kidney Disease Epidemiology Collaboration equation was used to calculate estimated glomerular filtration rate (eGFR). Atherosclerotic plaques in the carotid artery were detected by B-mode Doppler ultrasound. Blood samples were collected to assess serum sclerostin levels. Unconditional logistic regression analysis was used to identify risk factors for carotid atherosclerotic plaques. RESULTS: The median eGFR was 24.9 ml/min/1.73 m2 (interquartile range [IQR] 10.0-40.3 ml/min/1.73 m2) and median serum sclerostin level was 46.76 pmol/l (IQR 30.18-67.56 pmol/l). Carotid atherosclerotic plaques were detected in 104 subjects (74.3%). There was a negative association between sclerostin level and eGFR (r = - 0.214, p = 0.011). Unconditional logistic regression analysis revealed that sclerostin level was an independent risk factor for the occurrence of carotid plaques, with an odds ratio (95% confidence interval) of 1.026 (1.003, 1.051). CONCLUSION: Serum sclerostin increases with declining renal function in patients with CKD 3-5ND. Sclerostin is an independent risk factor for carotid atherosclerosis.

[Effects of induction of tubular epithelial-myofibroblast transition by monocyte chemoattractant protein-1 and mechanism thereof: an in vitro experiment].

OBJECTIVE: To examine the effects of monocyte chemoattractant protein-1 (MCP-1) on epithelial-myofibroblast transition (EMT) of renal proximal tubular epithelial cells and the possible mechanism involved. METHODS: Human renal proximal tubular epithelial cells of the line HK-2 were cultured and divided into three groups: negative control group; positive control group, treated with transforming growth factor (TGF)-beta1 5 microg/L, and MPC-1 group, treated with MCP-1 0.1, 1, 10, and 100 microg/L for 24 h, 36 h, 48 h, and 72 h respectively. The expressions of alpha-smooth muscle actin (alpha-SMA) mRNA and protein of cells were detected by s. Western blotting and RT-PCR were used to detect the expression of P-Erk1/2, Erk1/2, P-P38MAPK, P38MAPK, and RhoA protein levels of the HK-2 cells, and RT-PCR was used to detect the expression of RhoC and Snail mRNA. Specific inhibitors of the Erk, P38MAPK, and Rho signal transduction pathways PD98059, SB203580, and Y27632 were added into the culture fluid of HK-2 cells respectively, 1 h later MCP-1 microg/L was added for 48 h, and Western blotting was used to detect the protein expression of alpha-small muscle actin (SMA). RESULTS: The expression levels of alpha-SMA protein and mRNA significantly increased in the MCP-1 treated HK-2 cells,and these expression levels were the highest in the HK-2 cells treated with MCP-1 1 microg/L for 48 h. The ratios of (P-Erk1/2)/ (Erk1/2) and P-P38MAPK/P38MAPK were significantly increased (all P < 0.05) in the MCP-1 treated HK-2 cells with the highest ratios seen in the HK-2 cells treated by 100 microg/L of MCP-1. The expression levels of RhoA protein and RhoC mRNA of the HK-2 cells stimulated with MCP-1 of different concentrations were not significantly different (all P > 0.05). MCP-1 induced expression of a-SMA was only partly inhibited by PD98059 but not by SB203587 or Y27632. MCP-1 dose-dependently increased the expression of Snail mRNA of the HK-2 cells compared with those of the negative control cells. The level of Snail mRNA was the highest in the HK-2 cells treated with 100 microg/L MCP-1. CONCLUSION: MCP-1 may induce the EMT of renal proximal tubular epithelial cells in vitro, and this effect may involve upregulation of Erk1/2 Map kinase signal pathways and Snail mRNA expression. P38MAPK or Rho kinase signal pathways can not be proven to be involved in MCP-1 induced EMT.

Path Analysis on Medical Expenditures of 855 Patients with Chronic Kidney Disease in a Hospital in Beijing.

BACKGROUND: Investigate into the medical expenditures of chronic kidney disease (CKD) patients through path analysis method of three consecutive years within a Grade-A tertiary hospital in Beijing to conduct the main influencing factors in diagnosis-related groups (DRGs) grouping of the diagnosis, and reassess the present grouping process to provide information and reference on cost control for hospitals and medical management departments. METHODS: Eight hundred and fifty-five inpatient cases whose first diagnosis were defined as CKD in the year 2014-2016 within the hospital were selected as the sample of the study, multiple linear regression and path analysis method were adopted in DRGs grouping process to investigate the main influencing factors of total medical expenditures and DRGs grouping process. RESULTS: The maximum proportion of the medical costs within CKD patients was the costs on treatment, with the highest of 35.3% on the year 2014, the second was the costs on drug, which accounted for <30% during consecutive years, and the third was the costs on examination, which accounted for about 20% on average. The main influencing factors of medical expenditures included the type of dialysis, length of hospitalization, the admission of Intensive Care Unit (ICU), and so on. The coefficients toward the effect for total costs were 0.416, 0.376, and 0.094, respectively. CONCLUSIONS: It is suggested that the type of dialysis and the admission of ICU were the major influencing factors of inpatient medical expenditures on CKD patients, and should be taken into consideration into the reassessment of DRGs grouping process to realize the localization and generalization of prospective payment system based on DRGs within the regional area and promote the implementation of medical cost control measures to reduce the economic burdens among patients and the society.

The Ratio of Plasma and Urinary 8-oxo-Gsn Could Be a Novel Evaluation Index for Patients with Chronic Kidney Disease.

Nucleic acid oxidation plays an important role in the pathophysiology progress of a variety of diseases. 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dGsn) and 8-oxo-7,8-dihydroguanosine (8-oxo-Gsn), which originate from DNA and RNA oxidation, were the most widely used indicators for oxidative stress. The study investigated the relation between 8-oxo-dGsn, 8-oxo-Gsn, and CKD. 146 patients with CKD were divided into five disease stages, and their fasting blood and morning urine were collected. The levels of 8-oxo-dGsn and 8-oxo-Gsn in plasma and urine were quantified by LC-MS/MS. The ratio of urinary 8-oxo-Gsn to creatinine increased from stages 1 to 4 corresponding to the increased severity of CKD, but it decreased in stage 5. And plasma 8-oxo-Gsn gradually increased with the decline of renal function. In particular, the increased ratio of plasma and urine 8-oxo-Gsn in stage 5 exceeded the concentration of creatinine. This trend was similar to the estimated glomerular filtration rate (eGFR), which indicates that 8-oxo-Gsn could be an appropriate indicator for renal function. Our finding indicates that as the disease progresses, RNA oxidation is increased. The significant increase in the ratio of plasma and urinary 8-oxo-Gsn is a novel evaluation index of end-stage renal disease.

Levels of 8-oxo-dGsn and 8-oxo-Gsn in random urine are consistent with 24 h urine in healthy subjects and patients with renal disease.

Oxidatively generated damage to nucleic acids may play an important role in the pathophysiological processes of a variety of diseases. 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dGsn) and 8-oxo-7,8-dihydroguanosine (8-oxo-Gsn) are oxidatively generated products of DNA and RNA, respectively. Our previous studies have suggested that the amounts of 8-oxo-dGsn and 8-oxo-Gsn in urine were considerably higher than other body fluid or tissue. The aim of this study was to investigate whether 8-oxo-dGsn and 8-oxo-Gsn levels in random urine samples are consistent with those in 24 h urine samples in healthy subjects and patients with renal disease. A total of 16 healthy subjects and 104 renal disease patients were enrolled in this study, and their random and 24 h urine samples were collected. The levels of urinary 8-oxo-dGsn and 8-oxo-Gsn were quantified by LC-MS/MS and corrected by creatinine. Regardless of healthy subjects or renal disease patients, the levels of oxidised nucleosides in random urine samples were consistent with 24 h urine samples. Regardless of the age bracket, there is no significant difference between random samples and 24 h urine samples. In conclusion, 8-oxo-dGsn and 8-oxo-Gsn levels in random urine samples could replace those in 24 h urine samples, and were considered as the representative of the level of systemic oxidative stress for the whole day.