YKL-40 (chitinase-3-like protein 1) serum levels are associated with abdominal aortic calcification in hemodialysis patients.

BACKGROUND: Vascular calcification (VC) is highly prevalent and predicts cardiovascular mortality in dialysis patients. The mechanisms are still unclear. Inflammation is a well-known inducer of VC. YKL-40 has been suggested as a novel biomarker of inflammation and has been demonstrated to be associated with cardiovascular mortality in hemodialysis patients. This study aims to evaluate the relationship between serum YKL-40 and VC in hemodialysis (HD) patients. METHODS: A total of 109 HD patients and 31 healthy controls were enrolled in the study from September 2014 to December 2014. We evaluated the abdominal aortic calcification (AAC) score by plain X-ray films of the abdomen and measured serum YKL-40 concentrations using enzyme-linked immunosorbent assay. We also examined the relationship between YKL-40 levels and AAC scores in HD patients. RESULTS: Serum YKL-40 levels in HD patients were significantly higher than those in healthy controls [199.8 (144.8, 288.7) vs. 71.9 (52.8, 89.3) ng/ml; P < 0.001]. There was a tendency that YKL-40 levels in diabetic hemodialysis patients were higher than those in nondiabetic patients [217.8 (155.3, 335.8) vs. 192.9 (135.9, 274.4) ng/ml; P = 0.093]. A significant positive correlation was found between serum YKL-40 level and AAC score in these patients (r = 0.410, P = 0.003). Multiple regression analysis showed that Ln(YKL-40) was independently associated with AAC score in HD patients (P = 0.044). CONCLUSION: This study showed high serum YKL-40 concentrations in chronic HD patients and that YKL-40 was independently associated with increased AAC in hemodialysis patients.

Single-cell RNA sequencing confirms IgG transcription and limited diversity of VHDJH rearrangements in proximal tubular epithelial cells.

Increasing evidence has confirmed that immunoglobulins (Igs) can be expressed in non-B cells. Our previous work demonstrated that mesangial cells and podocytes express IgA and IgG, respectively. The aim of this work was to reveal whether proximal tubular epithelial cells (PTECs) express Igs. High-throughput single-cell RNA sequencing (scRNA-seq) detected Igs in a small number of PTECs, and then we combined nested PCR with Sanger sequencing to detect the transcripts and characterize the repertoires of Igs in PTECs. We sorted PTECs from the normal renal cortex of two patients with renal cancer by FACS and further confirmed their identify by LRP2 gene expression. Only the transcripts of the IgG heavy chain were successfully amplified in 91/111 single PTECs. We cloned and sequenced 469 VHDJH transcripts from 91 single PTECs and found that PTEC-derived IgG exhibited classic VHDJH rearrangements with nucleotide additions at the junctions and somatic hypermutations. Compared with B cell-derived IgG, PTEC-derived IgG displayed less diversity of VHDJH rearrangements, predominant VH1-24/DH2-15/JH4 sequences, biased VH1 usage, centralized VH gene segment location at the 3' end of the genome and non-Gaussian distribution of the CDR3 length. These results demonstrate that PTECs can express a distinct IgG repertoire that may have implications for their role in the renal tubular epithelial-mesenchymal transition.

Urotensin II Induces Mice Skeletal Muscle Atrophy Associated with Enhanced Autophagy and Inhibited Irisin Precursor (Fibronectin Type III Domain Containing 5) Expression in Chronic Renal Failure.

BACKGROUND/AIMS: Skeletal muscle atrophy is one of the main manifestations of protein energy wasting. We hypothesized that urotensin II (UII) can lead to skeletal muscle atrophy through upregulating autophagy and affecting Irisin precursor fibronectin type III domain containing 5 (FNDC5) expressions. METHODS: Three animal models (the sham operation, wild-type C57BL/6 mice with 5/6 nephrectomy, UII receptor (UT) gene knockout (UTKO) mice with 5/6 nephrectomy) were designed. Skeletal muscle weight, cross-sectional area (CSA) along with UII, FNDC5, LC3, and p62 expression were investigated. C2C12 cells were differentiated for up to 4 days into myotubes. These cells were then exposed to different UII concentrations (10-5 to 10-7 M) for 6-12 h and analyzed for the expressions of autophagic markers. These cells were also exposed to the same predetermined UII concentrations for 48-72 h and analyzed for the FNDC5 expression. Myotube diameter was measured. RESULTS: Upregulation of UII expression in skeletal muscle tissue was accompanied by reduced muscle weight and skeletal muscle CSA in the 2 posterior limbs, upregulated autophagy markers expression, and downregulated FNDC5 expression in 5/6 nephrectomy mice. The decrease of skeletal muscle weight, skeletal muscle CSA, downregulation of FNDC5 expression, and the upregulation of autophagy markers were inhibited in UTKO with 5/6 nephrectomy mice. Our in vitrostudy showed that UII could directly decrease myotube diameter, induce autophagy markers upregulation, and inhibit expression of FNDC5. When UII receptor gene was interfered by UT-specific siRNA, UII induced autophagy markers upregulation and FNDC5 downregulation were inhibited. CONCLUSION: We are the first to verify UII induces mice skeletal muscle atrophy associated with enhanced skeletal muscle autophagy and inhibited FNDC5 expression in chronic renal failure.

Lower Serum Irisin Levels Are Associated with Increased Vascular Calcification in Hemodialysis Patients.

BACKGROUND/AIMS: Vascular calcification, which involves an active cellular transformation of vascular smooth muscle cells into bone forming cells, is prevalent and predicts mortality in dialysis patients. Its mechanisms are complex and unclear. We presume that irisin, a newly identified myokine also may play roles in vascular calcification in hemodialysis patients. This study aims to evaluate serum irisin levels and establish their relation to vascular calcification and other parameters in hemodialysis patients. METHODS: A total of 150 patients on maintenance hemodialysis treatment and 38 age- and sex-matched healthy controls were enrolled in this cross-sectional study. Serum irisin concentrations were measured by ELISA. Vascular calcification was evaluated by abdominal aortic calcification scores. RESULTS: Serum irisin concentrations were significantly lower in hemodialysis patients than in controls [52.8 (22.0, 100.0) vs. 460.8 (434.8, 483.4) ng/ml, P<0.01]. In addition, irisin was negatively correlated with the parathyroid hormone level (P=0.01). The HD patients with vascular calcification showed significantly lower serum irisin concentrations [39.0 (21.7, 86.2) vs.79.0 (39.5, 130.2) ng/mL, P<0.01]. Compared with the group without vascular calcification multivariate logistic regression analyses revealed that serum irisin, HD vintage and age were significant independent determinant factors for vascular calcification in HD patients. CONCLUSION: Our results are the first to provide a clinical evidence of the association between serum irisin and vascular calcification in HD patients. Lower irisin levels, long-term hemodialysis and old ages are independent risk factors in HD patients.

Expression of immunoglobulin G in human podocytes, and its role in cell viability and adhesion.

Podocyte injury occurs during the initiation and development of numerous forms of glomerular disease, and antibodies targeting podocytes have become a biomarker for diagnosis and monitoring treatment response. Accumulating evidence has suggested that immunoglobulin (Ig) is expressed in non­B lineage cells, including epithelial cancer cells, myeloid cells and several types of normal cells. The main aim of the present study was to ascertain the expression of IgG in human podocytes and to determine its potential role in cellular bioactivity. The present study detected positive staining for IgG heavy chain (Igγ) and its subtype γ4, and the light chains κ and λ in the cytoplasm or on the membrane by immunofluorescence. In addition, positive bands were detected for Igγ, γ1, γ3, γ4, κ and λ in the lysates of a podocyte cell line by western blotting. Mass spectrometry confirmed IgG1 as an intact tetramer in the culture supernatant. Constant region transcripts of Igγ, γ1, γ3, γ4, κ and λ were identified by reverse transcription­polymerase chain reaction, and DNA sequencing of these transcripts revealed 96­99% similarity with Ig mRNAs in the National Center for Biotechnology Information database. Compared with the diverse gene rearrangements from B cell-derived Ig, podocyte­derived Ig exhibited conservative V(D)J patterns in the variable regions of Igγ and κ chains. Furthermore, the present study investigated the mechanism underlying IgG production in these cells by examining the expression of recombination activating gene (RAG)1, RAG2 and activation­induced cytidine deaminase. The expression levels of these proteins suggested that podocyte­derived Ig and traditional Ig may be generated in a similar manner. Furthermore, small interfering RNA­mediated downregulation of IgG expression reduced podocyte viability and adhesive capabilities. These findings suggested that IgG is expressed in podocytes and that this expression may be associated with podocyte function. Due to its potential biological and clinical significance, this phenomenon warrants further investigation.

Expression of immunoglobulin A in human mesangial cells and its effects on cell apoptosis and adhesion.

IgA nephropathy (IgAN) is characterized by predominant IgA deposition in the glomerular mesangium. It has been considered that the deposited IgA is synthesized by B cells, although recent reports have suggested the implication of other cell types. Therefore, the present study investigated whether glomerular mesangial cells could produce IgA by themselves. Semi­quantitative reverse transcription-polymerase chain reaction, and immunostaining analysis revealed that the IgA protein and gene transcripts were expressed in primary human renal mesangial cells (HRMCs). Furthermore, the IgA heavy chain (α1 and α2) and the light chain (κ and λ) were localized in the cytoplasm or were located on the cell membranes of human mesangial cells (HMCs). Mass spectrometry results indicated that Ig α1 and Ig α2 were secreted in the culture media of HMCs. The transcripts of Ig α, Ig κ and Ig λ constant regions were detected. The predominant rearrangement pattern of the variable region of Ig κ, was Vκ3­20*01/Jκ1*01 in HMCs and Vκ1­12*01/Jκ4*01 in HRMCs. In addition, knockdown of Ig α1 expression by small interfering RNA (siRNA) inhibited cell adhesion and promoted apoptosis. Our findings demonstrate that HMCs can express IgA, and that this expression is associated with cell functions, which may contribute to the deposition of IgA in patients with IgAN.

Irisin is Associated with Urotensin II and Protein Energy Wasting in Hemodialysis Patients.

AIMS/INTRODUCTION: Irisin is a newly identified myokine which can promote energy expenditure. Urotensin II (UII) is identified as the most potent mammalian vasoconstrictor to date. Previous studies showed that UII can aggravate insulin resistance while irisin alleviate insulin resistance. Through this study, it is our aim to elucidate if UII can induce insulin resistance and also have an association with the irisin level in hemodialysis (HD) patients. MATERIALS AND METHODS: One hundred and twenty-eight patients on maintenance hemodialysis treatment and forty healthy subjects were enrolled in this study. Blood irisin concentrations and UII concentrations were measured by ELISA and RIA respectively. The body composition was analyzed by bioelectrical impedance. RESULTS: The serum irisin levels and UII levels were both significantly lower in HD patients in comparison to that of the healthy subjects. The serum irisin levels were lower in HD patients with protein energy wasting than those of the patients without protein energy wasting. The independent determinants of circulating Ln (irisin) (the natural logarithm of irisin) were UII lean body mass and patients with protein energy wasting. CONCLUSIONS: Our results are the first to provide the clinical evidence of the association among irisin, UII, and protein energy wasting. Our results hint that UII and protein energy wasting might inhibit the release or synthesis of irisin from skeletal muscles in HD patients.

Increased urinary angiotensinogen precedes the onset of albuminuria in normotensive type 2 diabetic patients.

It was previously reported that intrarenal renin angiotensin system (RAS) plays a pivotal role in the onset and progression of diabetic nephropathy (DN). Urinary angiotensinogen (UAGT) was employed as a special index of the intrarenal RAS status and enhanced significantly at a very early stage of chronic kidney disease and type 1 diabetes. On the basis of these findings, the present study was performed to test the hypothesis that UAGT levels are increase even before the development of DN in type 2 diabetic patients without hypertension. 102 patients with type 2 diabetes mellitus (T2DM) and 18 healthy volunteers were studied cross-sectionally. Clinical data were collected and morning spot urine samples were obtained from all participants. UAGT levels were detected by an enzyme-linked immunosorbent assay (ELISA). As a result, UAGT to creatinine ratio (UAGT/Cr) was significantly enhanced in T2DM patients before the appearance of urinary albumin (UALB) and further increased to a greater degree in albuminuric patients. UAGT/Cr levels were positively correlated with Log (UALB to creatinine ratio) and diastolic blood pressure, but negatively correlated with estimated glomerular filtration rate. These data indicate that elevated UAGT levels precede the onset of albuminuria in normotensive T2DM patients. UAGT might potentially serve as an early marker to determine intrarenal RAS activity and predict progressive kidney disease in T2DM patients without hypertension.

Irisin levels are associated with urotensin II levels in diabetic patients.

AIMS/INTRODUCTION: Irisin is a newly identified myokine that can promote energy expenditure. Previous studies showed that circulating urotensin II (UII) levels were increased in diabetes, and UII could inhibit the glucose transport in skeletal muscle in diabetic mice and aggravated insulin resistance. We presumed that irisin levels are associated with UII in diabetic patients. MATERIALS AND METHODS: A total of 71 patients with type 2 diabetes and 40 healthy subjects were recruited. Blood and urinary irisin concentrations were measured by using enzyme-linked immunosorbent assay, and UII concentrations were measured by bioelectrical impedance analysis. Every participant's body composition was analyzed by bioelectrical impedance. RESULTS: The serum irisin levels were significantly lower in diabetic patients than that of controls, whereas serum UII levels were significantly higher in diabetic patients than that in that of controls. Serum irisin levels were negatively associated with circulating UII, hemoglobin A1c and the natural logarithm transformation of urinary albumin excretion, whereas serum irisin was positively associated with estimated glomerular filtration rate, and low-density lipoprotein cholesterol and urinary irisin were positively associated with urinary UII. Furthermore, circulating irisin is positively associated with muscle mass, whereas circulating UII is negatively associated with muscle mass in diabetic patients. Hemoglobin A1c and circulating UII are independent determinants of circulating irisin by multiple regression analysis. CONCLUSIONS: The present results provide the clinical evidence of an association between irisin and UII in diabetic patients. Hemoglobin A1c and circulating UII are independent determinants of circulating irisin. Our results hint that UII and high glucose might inhibit the release of irisin from skeletal muscle in diabetic patients.

Urinary angiotensin converting enzyme 2 increases in patients with type 2 diabetic mellitus.

BACKGROUND/AIMS: Angiotensin converting enzyme 2 (ACE2) is highly expressed in the kidney and recognized to be renoprotective by degrading Angiotensin II to Angiotensin (1-7) in diabetic nephropathy. However, little is known about the role of urinary ACE2 (UACE2) in diabetes. The present study was performed to evaluate UACE2 levels in type 2 diabetic patients with various degrees of albuminuria and its associations with metabolic parameters. The effect of RAS inhibitors on UACE2 excretion was also assessed. METHODS: A total of 132 type 2 diabetic patients with different degrees of albuminuria and 34 healthy volunteers were studied. UACE2 levels and activity were measured. RESULTS: Compared to healthy controls, UACE2 to creatinine (UACE2/Cr) levels were significantly increased in both albuminuric and non-albuminuric diabetic patients. UACE2/Cr levels were much higher in hypertensive diabetic patients compared with their normotensive counterparts and treatment with RAS inhibitors markedly attenuated the augmentation. Furthermore, UACE2/Cr was positively correlated with fasting blood glucose, hemoglobin A1C (HbA1C), triglyceride, and total cholesterol. In multiple regression analysis, UACE2/Cr was independently predicted by HbA1C and RAS inhibitors treatment. CONCLUSIONS: UACE2 increased in type 2 diabetic patients with various degrees of albuminuria and RAS inhibitors suppresses UACE2 excretion. UACE2 might potentially function as a marker for monitoring the metabolic status and therapeutic response of RAS inhibitors in diabetes.

[Up-regulation of intrarenal renin-angiotensin system contributes to renal damage in high-salt induced hypertension rats].

OBJECTIVE: To test the hypothesis that in a high-salt induced hypertension in normal rats, whether the changes of intrarenal renin-agiotensin system (RAS) play a critical role in renal damage and could be reflected by urinary angiotensinogen (AGT). METHODS: In the study, 27 normotensive male Wistar-Kyoto rats were divided into control group [0.3% (mass faction) NaCl in chow, n=9, NS], high-salt diet group [8% (mass faction) NaCl in chow, n=9, HS] and high-salt diet with Losartan group [8% (mass faction) NaCl in chow and 20 mg/(kg×d) Losartan in gavages, n=9, HS+L)], and were fed for six weeks. The blood pressure was monitored and urine samples were collected every 2 weeks. AGTs in plasma, kidney and urine were measured by ELISA kits. The renal cortex expression of mRNA and protein of AGT were measured by Real-time PCR and immunohistochemistry (IHC). The renin activity and ANG II were measured by radioimmunoassay (RIA) kits. RESULTS: Compared with NS, the systolic blood pressure (SBP) [(156 ± 2) mmHg vs. (133 ± 3) mmHg, P<0.05] increased significantly at the end of the 2nd week, and the urinary protein [(14.07 ± 2.84) mg/24 h vs. (7.62 ± 3.02) mg/24 h, P<0.05] increased significantly at the end of the 6th week in HS. Compared with HS, there was no significant difference in SBP (P>0.05) but the proteinuria [(9.69 ± 2.73) mg/24 h vs. (14.07 ± 2.84) mg/24 h, P<0.01] decreased significantly in HS+L. Compared with NS, there was no significant difference in the plasma renin activity, angiotensinogen and ANG II level in HS (P>0.05), but the renal cortex renin content [(8.72 ± 1.98) ng/(mL × h) vs. (4.37 ± 1.26) ng/(mL × h), P<0.05], AGT formation [(4.02 ± 0.60) ng/mg vs. (2.59 ± 0.42) ng/mg, P<0.01], ANG II level [(313.8 ± 48.76) pmol/L vs. (188.9 ± 46.95) pmol/L, P<0.05] were increased significantly in HS, and the urinary AGT and ANG II excretion rates increased significantly (P<0.05). Compared with HS, the plasma renin activity, angiotensinogen and ANG II level were significantly increased (P<0.05), but the renal cortex renin content, AGT formation, ANG II level significantly decreased (P<0.05), and the urinary AGT and ANG II excretion rates decreased significantly in HS+L (P<0.05). The urinary AGT excretion rates were positively correlated with the AGT level in the renal cortex (P<0.05). CONCLUSION: Up-regulation of intarenal RAS may contribute to renal damage in high-salt induced hypertension rats. Urinary AGT may reflect the status of intrarenal RAS.

Up-regulation of intrarenal renin-agiotensin system contributes to renal damage in high-salt induced hypertension rats.

BACKGROUND/AIMS: To investigate the change of intrarenal renin-agiotensin system (RAS) and its role in high-salt induced hypertension. METHODS: Wistar rats were divided into normal-salt (NS), high-salt diet (HS) and high-salt diet with Losartan group (HS+L), for 6 weeks. Systolic blood pressure (SBP) was monitored. Blood and urine samples were collected every 2 weeks. Angiotensinogen (AGT) was measured by ELISA. AGT mRNA and protein were measured by real-time PCR and immunohistochemistry. Renin activity and angiotensin II (Ang II) were measured by radioimmunoassay. RESULTS: HS versus NS group, SBP increased from 2(nd) week (P<0.05), urinary protein increased at 6(th) week (P<0.05). Although plasma renin, AGT and Ang II had no significant changes (P>0.05), renal cortex renin, AGT, and Ang II increased significantly in HS (P<0.05). In HS+L, Losartan failed to reduce SBP (P>0.05) but abolished the increase of proteinuria (P<0.01), renal cortex renin, AGT, Ang II and urinary AGT reduced (P<0.05) while plasma renin, AGT and Ang II enhanced (P<0.05) when compared with HS. Urinary AGT was positively correlated with renal AGT (r=0.592, P <0.01) and Ang II (r=0.726, P <0.01). CONCLUSION: Inappropriate response of the renal RAS to a high salt diet may contribute to hypertension and renal damage, and urinary AGT could reflect intrarenal RAS activity.

Effects of stocking rate on the variability of peak standing crop in a desert steppe of Eurasia grassland.

Proper grazing management practices can generate corresponding compensatory effects on plant community production, which may reduce inter-annual variability of productivity in some grassland ecosystems. However, it remains unclear how grazing influences plant community attributes and the variability of standing crop. We examined the effects of sheep grazing at four stocking rate treatments [control, 0 sheep ha(-1) month(-1); light (LG), 0.15 sheep ha(-1 )month(-1); moderate (MG), 0.30 sheep ha(-1) month(-1); and heavy (HG), 0.45 sheep ha(-1) month(-1)] on standing crop at the community level and partitioned by species and functional groups, in the desert steppe of Inner Mongolia, China. The treatments were arranged in a completely randomized block design over a 9-year period. Standing crop was measured every August from 2004 to 2012. Peak standing crop decreased (P < 0.05) with increasing stocking rate; peak standing crop in the HG treatment decreased 40 % compared to the control. May-July precipitation explained at least 76 % of the variation in peak standing crop. MG and HG treatments resulted in a decrease (P < 0.05) in shrubs, semi-shrubs, and perennials forbs, and an increase (P < 0.05) in perennial bunchgrasses compared to the control. The coefficients of variation at plant functional group and species level in the LG and MG treatments were lower (P < 0.05) than in the control and HG treatments. Peak standing crop variability of the control and HG community were greatest, which suggested that LG and MG have greater ecosystem stability.

Yohimbine protects against endotoxin-induced acute lung injury by blockade of alpha 2A adrenergic receptor in rats.

BACKGROUND: Alpha 2A adrenergic receptor (AR) is a subtype of α2 AR belonging to G protein-coupled receptors, and exerts a variety of biological effects. Recent studies have demonstrated that the α2A AR activation was closely related with inflammatory reaction. The present study aimed to investigate the influence of α2A AR antagonist, yohimbine, on the severity of endotoxin-induced acute lung injury in rats. METHODS: A total of 72 male Sprague-Dawley rats were randomly divided into three groups: control group, lipopolysaccharide (LPS) group and LPS + yohimbine group. Rats were intratracheally administrated with normal saline or LPS (300 µg), and the rats in the LPS + yohimbine group were treated with additional yohimbine (2 mg/kg, i.p) soon after LPS administration. Six, 24 and 48 hours after treatment, arterial blood gas analysis was carried out, and optical microscopy was performed to evaluate pathological changes in the lung, and lung injury score was assessed. The count of white blood cells in bronchoalveolar lavage fluid (BALF) was determined. The levels of norepinephrine, tumor necrosis factor (TNF)-α, interleukin (IL)-1ß and IL-6 in BALF were measured with enzyme-linked immunosorbent assay. Immunocytochemistry was performed for the detection of α2A AR on inflammatory cells in BALF. RESULTS: When compared with the control group, the oxygenation index in the LPS group was significantly decreased, and white blood cell count, the lung histopathological scores, levels of norepinephrine and IL-6 as well as α2A AR expression on inflammatory cells in the BALF were dramatically increased at different time points, and the concentrations of TNF-α and IL-1ß were also increased except at 48 hours after LPS administration. The oxygenation index decreased while white blood cell count in BALF and the lung histopathological scores were obviously increased in the LPS + yohimbine group. The level of norepinephrine in BALF was increased at each time interval in the LPS + yohimbine group, and so did the levels of TNF-α, IL-1ß and IL-6 at 6 and 48 hours after LPS administration respectively. When compared with the LPS group, the oxygenation index, white blood cell count, the lung histopathological scores and the level of IL-6 in the LPS + yohimbine group were significantly improved at each time interval, and the concentrations of TNF-α and IL-1ß were also lower at 24 hours of LPS administration (all P < 0.05). Correlation analysis indicated the level of norepinephrine was related to the levels of TNF-α, IL-1ß and IL-6 in the BALF and the lung histopathological scores (r = 0.703, r = 0.595, r = 0.487 and r = 0.688, respectively, P < 0.001) and the intensity scores of immunoreactivity to α2A AR on inflammatory cells were also associated with the levels of TNF-α, IL-1ß and IL-6 as well as the lung histopathologial scores (r = 0.803, r = 0.978, r = 0.716 and r = 0.808, respectively, P < 0.001). CONCLUSIONS: Yohimbine can inhibit TNF-α, IL-1ß and IL-6 overproduction and relieve the severity of pulmonary inflammation induced by endotoxin, which is maybe mediated by blockade of α2A AR on inflammatory cells.