Angiogenic activity of human CC chemokine CCL15 in vitro and in vivo

CCL15 is a novel human CC chemokine and exerts its biological activities on immune cells through CCR1 and CCR3. Because a number of chemokines induce angiogenesis and endothelial cells express CCR1 and CCR3, we investigated the angiogenic activity of CCL15. Both CCL15(1-92) and N-terminal truncated CCL15(25-92) stimulate the chemotactic endothelial cell migration and differentiation, but CCL15(25-92) is at least 100-fold more potent than CCL15(1-92). Treatment with pertussis toxin (PTX), with anti-CCR1, or with anti-CCR3 antibody inhibits the CCL15(25-92)-induced endothelial cell migration. CCL15(25-92) also stimulates sprouting of vessels from aortic rings and mediates angiogenesis in the chick chorioallantoic membrane assay. Our findings demonstrate that CCL15(25-92) has in vitro and in vivo angiogenic activity, and suggest roles of the chemokine in angiogenesis.     

The N-terminal domain of CCL21 reconstitutes high affinity binding, G protein activation, and chemotactic activity, to the C-terminal domain of CCL19

CC chemokine receptor 7 (CCR7), which regulates the trafficking of leucocytes to the secondary lymphoid organs, has two endogenous chemokine ligands: CCL19 and CCL21. Although both ligands possess similar affinities for the receptor and similar abilities to promote G protein activation and chemotaxis, they share only 25% sequence identity. Here, we show that substituting N-terminal six amino acids of CCL21 (SDGGAQ) for the corresponding N-terminal domain of CCL19 (GTNDAE) results in a chimeric chemokine that exhibits high affinity binding and G protein activation of CCR7. These data demonstrate that despite dissimilar sequences, the amino terminal hexapeptide of these two chemokines is capable of performing similar roles resulting in receptor activation.     

Renal cytoplasmic proteasome proteinase activities are altered in chronic renal failure

The effect of uremia on renal cortex cytoplasmic proteasomes was examined by comparing proteasomes isolated from 5/6th nephrectomy rats 3-months post-surgery and age-matched control rats with normal renal function. ATP-dependent proteasome activity was reduced 50% in chronic renal failure rats (CRF) 3-months post-surgery compared to age-matched control rats. Trypsin-like (T-like) proteasome activity was decreased 90% compared to 70% for caspase-like activity (PGPHase) and 30% for chymotrypsin-like activity (C-like). ATP-independent proteasome activity was decreased 60% in CRF rats 3-months post-surgery. ATP-independent renal cortex proteasome T-like activity in CRF rats was 4% of age-matched control rats. C-like and PGPHase activities were 60% and 50% of age-matched controls, respectively. Uremia was associated with decreased 26S proteasome beta subunits. CRF rat 26S proteasomes had decreased levels of beta1, beta3, alpha4, and alpha7 abundances. Compared to age-matched control rats with normal renal function, CRF rats had a 25% increase in ubiquitinated cytoplasmic proteins. Decreased renal cytoplasmic proteasome activity may play a role in renal tubule hypertrophy common to renal diseases associated with decreased functioning nephrons.     

Zinc bone loss in chronic renal failure and chronic metabolic acidosis

The effects of chronic metabolic acidosis (CMA) on zinc (Zn) bone content and urinary excretion were examined in the presence of normal or reduced renal function together with some aspects of calcium (Ca) metabolism. Four groups of rats were compared. All were fed a 30% protein and 9 mg Zn/100 g diet. Two were uremic (U): The first developed acidosis (UA), which was suppressed in the other (UNA) by NaHCO₃ supplement. Two other groups had normal renal function: One was normal (CNA), and the other had NH₄Cl in the drinking water and acidosis (CA). Femur total Zn and Ca content was markedly reduced by CMA and was not affected by uremia. Zn urinary excretion was increased by CMA and unaltered by uremia. Ca urinary excretion was markedly reduced in uremic rats, but was enhanced in both acidotic conditions. Urinary Ca and Zn showed a strong correlation in uremic and in control rats. Plasma parathormone and 1,25(OH)₂D₃ were unchanged by CMA. These data are in agreement with a direct primary effect of CMA on bone in releasing buffers. CMA induces bone resorption and a parallel decrease of mineral bone components, such as Ca and Zn, with little or no role of PTH, 1,25(OH)₂D₃ and of uremia itself.     

慢性肾衰竭大鼠模型的建立

目的 建立两种慢性肾衰竭大鼠模型,观察瘦素蛋白在大鼠组织、器官中的表达.方法 建立两种慢性肾衰竭CRF动物模型:(1)大鼠肾大部分切除诱发肾衰(Platt法).(2)腺嘌呤诱发大鼠慢性肾衰竭的动物模型(Yokozawa法).分别测定血清中血尿素氮(BUN),血肌酐(Scr)Ca2+、P5+等含量.取肾脏组织,HE染色,行免疫荧光,检测瘦素蛋白在两种慢性肾衰竭大鼠模型中的表达情况.结果 模型组大鼠血清中血尿素氮(BUN),血肌酐(Scr)等含量明显升高,免疫荧光检测显示两种模型大鼠肾脏组织瘦素蛋白的表达.结论 成功建立两种慢性肾衰竭CRF动物模型,显示不同模型组织部位的瘦素蛋白的表达.为进一步探讨瘦素蛋白在动物体内的生物学作用提供实验基础.     

Ang Ⅱ拮抗剂对慢性肾衰大鼠肾血流量和肾内氧耗的影响

目的观察血管紧张素II(AngⅡ)拮抗剂对5/6(ablation/infarction,A/I)肾切除诱导慢性肾衰竭(CRF)大鼠肾功能、肾血流量及肾内氧耗的影响。方法制备5/6(A/I)肾切除诱导慢性肾衰大鼠模型,设正常组(A组,n=14只),模型组(B组,n=14只),AngⅡ拮抗剂治疗组(氯沙坦钾联合福辛普利钠)(C组,n=14只)。给予相应干预,疗程60 d。分别测量尾动脉收缩压(SBP)、舒张压(DBP),检测大鼠尾静脉血清肌酐(Scr)、尿素氮(BUN)、血红蛋白(Hb),计算内生肌酐清除率(Ccr)。干预60 d后,检测肾血流量(RBF)、腹主动脉和肾静脉血气(AABG and RVBG),左肾静脉压(RVpO2),计算残余肾内氧耗(QO2/TNa)及观察残肾组织病理变化。结果 (1)造模后与A组比较,B、C两组的Scr、BUN和尾动脉SBP、DBP显著增加(P0.01),Ccr、Hb显著降低(P0.01),提示造模成功。(2)干预后与B组比较,C组的Scr、尾动脉SBP、DBP、QO2/TNa明显下降(P0.01),BUN降低(P0.05),Hb、Ccr、RVpO2显著升高(P0.01),RBF升高(P0.05)。(3)残肾组织病理形态学变化显示,C组的肾组织病理变化明显减轻,优于B组。结论 AngⅡ拮抗剂可以增加慢性肾衰大鼠肾血流量,降低肾内氧耗,改善肾功能及减轻肾组织病理变化,其肾脏保护作用机制可能与其调节细胞能量代谢,改善肾内氧耗有关。     

Clinical grade production and characterization of a fusion protein comprised of the chemokine CCL2-ligand genetically fused to a mutated and truncated form of the Shiga A1 subunit

First generation chemokine ligand-Shiga A1 (SA1) fusion proteins (leukocyte population modulators, LPMs) were previously only obtained in small quantities due to the ribosomal inactivating protein properties of the SA1 moiety which inhibits protein synthesis in host cells. We therefore employed 4-aminopyrazolo[3,4-d]-pyrimidine, an inhibitor of Shiga A1, to allow the growth of these cells prior to induction and during the expression phase post-induction with IPTG. Scale-up allowed the production of gram quantities of clinical grade material of the lead candidate, OPL–CCL2–LPM. A manufacturing cell bank was established and used to produce OPL–CCL2–LPM in a fed-batch fermentation process. Induction of the expression of OPL–CCL2–LPM led to the production of 22.47 mg/L per OD₆₀₀ unit. The LPM was purified from inclusion bodies using solubilization, renaturation, refolding and chromatography steps. The identity and purity of the OPL–CCL2–LPM was determined using several analytical techniques. The product retained the ability of the SA1 moiety to inhibit protein synthesis as measured in a rabbit reticulocyte lysate cell-free protein synthesis assay and was cytotoxic to target cells. Binding studies established that the protein exerts its effects via CCR2, the cognate receptor for CCL2. Clinical trials in inflammatory nephropathies are planned.     

Effect of Acacia gum on blood pressure in rats with adenine-induced chronic renal failure

Chronic renal failure (CRF) occurring naturally in patients or induced by subtotal nephrectomy in rats induces several alterations in the cardiovascular system (CVS). However, the effect of chemically induced CRF in rats on the CVS is less well known. We induced CRF in rats by feeding adenine (0.75%, w/w, four weeks) and investigated the effect of the ensuing CRF on the systolic and diastolic blood pressure (BP) and heart rate (HR). Further, we investigated the effect of giving acacia gum (AG, 10%, w/v) in the drinking water concomitantly with adenine on the above parameters. AG has been previously shown to ameliorate the severity of CRF in humans and rats. We confirmed here that adenine-induced CRF significantly increased the plasma concentrations of urea and creatinine, and reduced creatinine clearance. Additionally, it significantly increased both systolic and diastolic BP, with no significant effect on HR. Both of these actions were significantly mitigated by AG treatment. The antihypertensive angiotenisn-converting enzyme inhibitor lisinopril (10 mg/kg) was given by gavage to rats concomitantly with adenine, significantly reduced the rise in blood pressure induced by adenine. In conclusion, adenine-induced CRF in rats significantly increased BP, and this was significantly mitigated by administration of AG. Possible mechanisms of these changes and the protective effect of AG will be investigated.     

Accumulation of plasma N-methyl-2-pyridone-5-carboxamide in patients with chronic renal failure

Intracellular catabolism of NAD in mammalian cells occurs mainly via reaction catalyzed by poly(ADP-ribose) polymerase (PARP) with the release of nicotinamide, which is then metabolized predominantly to N-methyl-2-pyridone-5-carboxamide (2PY). PARP could be activated by binding to broken DNA and is known to be involved in DNA repair mechanisms, cell stress response and regulation of apoptosis. 2PY may accumulate under disease conditions resulting in accelerated DNA damage and retention of catabolic products. Our hypothesis was that chronic renal failure would lead to elevation of 2PY and potentially to inhibition of PARP and related physiological mechanisms. In the present study we: (a) compared plasma 2PY concentration in healthy subjects and in patients with chronic renal failure (CRF); (b) evaluated the relationship between plasma 2PY concentration and the severity of CRF; (c) evaluated the effect of hemodialysis treatment and kidney transplantation on 2PY concentration.We found that the plasma 2PY concentration in healthy subjects is 0.83 ± 0.18 M but it could increase up to 40 M in patients with CRF. A significant correlation was found in CRF between plasma 2PY and creatinine concentration. A single hemodialysis treatment was associated with significant reduction of plasma 2PY concentration after the hemodialysis, but it increased rapidly 48 h after the end of treatment. Successful kidney transplantation was associated with return of 2PY concentration to the normal range.In conclusion, our results indicated significant production of 2PY in humans. In healthy subjects 2PY is cleared from the plasma by excretion in the urine. Altered excretion by the kidney leads to increase in plasma concentration of 2PY. It is possible that 2PY may play a significant role in the development of uremic toxemia, especially as an inhibitor of poly(ADP-ribose)polymerase.     

肾康注射液联合血液透析治疗慢性肾功能衰竭的临床效果及对肾功能、营养指标的影响

目的 探讨肾康注射液联合血液透析治疗对慢性肾功能衰竭患者的肾功能、营养指标及临床疗效的影响.方法 选取2018年3月～2019年2月我院肾内科收治的116例慢性肾功能衰竭患者作为研究对象,随机分为对照组和观察组各58例.对照组给予单纯血液透析治疗,观察组给予肾康注射液联合血液透析治疗,治疗16周后观察两组患者的肾功能及...     

CpG-ODNs induces up-regulated expression of chemokine CCL9 in mouse macrophages and microglia

Unmethylated CpG oligodeoxynucleotides (CpG-ODNs) interact with Toll-like receptor (TLR) 9 to activate macrophage/microglia in central nervous system (CNS). Here, we investigated the potential involvement of the chemokine CCL9 and its receptor CCR1 in the effects of CpG-ODNs on macrophage/microglial cells. CpG-ODNs enhanced the expression of TLR9 mRNA of RAW264.7 macrophage and BV2 microglia cells time dependently. The expression of CCL9 of macrophages/microglia showed different responsiveness upon stimulation with a variety of CpG-ODN sequences. The CpG-ODNs-mediated induction of CCL9 was TLR9/MyD88 dependent and associated with activation of stress kinases, particularly ERK, p38 MAPK and PI3K. The expression of CCR1 was also significantly increased by CpG-ODNs that increased CCL9 expression. These results reveal the potential involvement of CCL9 and CCR1 in regulation of macrophage and microglial cells by CpG-ODNs and may help improving our understanding about the role of the chemokine/chemokine receptor pairs in macrophage/microglia under physiologic and pathologic conditions.     

Effects of a new amino acid supplement on blood AA pools in patients with chronic renal failure

Summary We designed a new formula for AA supplement in order to correct blood pools of AA in chronic renal failure (CRF). This supplement was given to 5 patients with CRF and its effectiveness was tested during long term (12–24 weeks) administration. The patients had previously been on a diet providing 0.6 g of protein and 34–36 kcal/kg/day. The diet was then modified to one providing the same caloric content but only 0.3 g/kg high biological value protein per day with the addition of the AA supplement (0.3 g/kg). The new diet corrected most of the abnormalities in blood AA pools. After 1 month of treatment Val, Leu, Thr, Ser and Tyr levels rose and became normal throughout the study. Ratios Tyr/Phe, Ser/Gly and Val/Gly also improved. During the treatment no side effect or toxicity was observed, and serum albumin, transferrin and nutritional anthropometric parameters persisted to be normal. It is concluded that this specially designed AA supplement added to a hypoproteic diet is an acceptable regimen which can quite completely correct the imbalance in blood AA pools in CRF.     

IL-15 alters expression and function of the chemokine receptor CX3CR1 in human NK cells

The chemokine receptor CX3CR1 is thought to regulate inflammation in part by modulating NK cell adhesion, migration, and killing in response to its ligand CX3CL1 (fractalkine). Recent reports indicate that IL-15, which is essential for development and survival of NK cells, may negatively regulate CX3CR1 expression, however, the effects of the cytokine on human NK cell CX3CR1 expression and function have not been fully delineated. Here, we demonstrate that short term culture in IL-15 decreases surface expression of CX3CR1 on cultured CD56+ cells from human blood resulting in diminished chemotaxis and calcium flux in response to CX3CL1. Cells cultured long term in IL-15 (more than five days) completely lost surface expression as well as mRNA and protein for CX3CR1. The effect was specific since mRNA for CCR5 was increased and mRNA for CXCR4 was unchanged in these cells by IL-15. Thus, exogenous IL-15 is a negative regulator of CX3CR1 expression and function in human CD56+ NK cells. The data imply that the use of IL-15 alone to expand NK cells ex vivo for immunotherapy may produce cells impaired in their ability to traffic to sites of inflammation.     

D-Amino acids in chronic renal failure and the effects of dialysis and urinary losses

Summary Total D-amino acids were measured in plasma for 20 non-dialysed patients (creatinine clearance < 12 ml/minute), 20 on CAPD, 20 on haemodialysis and 20 normals. Plasma D-tyrosine and D-phenylalanine were measured in 8 of each group by HPLC. Total D-amino acids, D-tyrosine and D-phenylalanine were significantly greater for patients than normals. D-amino acids and D-tyrosine correlated with creatinine and were decreased during HD. During dialysis, the mean losses for D-tyrosine and D-phenylalanine were similar, about 0.2 mg/CAPD exchange and 3 mg/4 hour haemodialysis (i.e. 2% of the total amino acid, as in plasma). Clearance was unaffected by stereochemical configuration. Urinary losses/24 hour in the non-dialysed patients were 0.35 mg D-tyrosine and 0.25 mg D-phenylalanine. Clearance for D-phenylalanine was greater than for the L-enantiomer. Increases in D-amino acids in renal failure are probably due to depletion of D-amino acid oxidase, but may be enhanced by a D-amino acid rich diet, peptide antibiotics and D-amino acid oxidase inhibiting drugs and metabolites. Possible toxic effects need further investigation.     

Fmoc-based synthesis of the human CC chemokine CCL14/HCC-1 by SPPS and native chemical ligation

Summary The CC chemokine CCL14/HCC-1(9–74), a 66-residue polypeptide containing two disulfide bonds, was recently discovered from a human hemofiltrate peptide library as a high-affinity ligand of the chemokine receptors CCR1 and CCR5. It has been shown to inhibit HIV infection by blocking CCR5. Using Fmoc methodology, we, report the chemical synthesis of CCL14/HCC-1 by conventional stepwise solid-phase peptide synthesis (SPPS) and, alternatively, native chemical ligation. To optimize SPPS of CCL14/HCC-1, difficult sequence regions were identified by mass spectrometry, in order to obtain a crude tetrathiol precursor suitable for oxidative disulfide formation. For synthesis of CCL14/HCC-1 by native chemical ligation, the peptide was divided into two segments, CCL14/HCC-1(9–39) and CCL14/HCC-1(40–74), the latter containing a cysteine residue at the amino-terminus. The synthesis of the thioester segment was carried out comparing a thiol linker with a sulfonamide safety-catch linker. While the use of the thiol linker led to very low overall yields of the desired thioester, the sulfonamide linker was efficient in obtaining the 31-residue thioester of CCL14/HCC-1(9–39), suggesting a superior suitability of this linker in generating larger thioesters using Fmoc chemistry. The thioester of CCL14/HCC-1 was subsequently ligated with the cysteinyl segment to the full-length chemokine. Disulfides were introduced in the presence of the redox buffer cysteine/cystine. The products of both SPPS and native chemical ligation were identical. The use of a sulfonamide safety-catch linker enables the Fmoc synthesis of larger peptide thioesters, and is thus useful to generate arrays of larger polypeptides.     

Serum metabonomics study of chronic renal failure by ultra performance liquid chromatography coupled with Q-TOF mass spectrometry

A metabonomics technique based on ultra-performance liquid chromatography (UPLC) coupled with Q-TOF mass spectrometry was employed to investigate the sera from 32 patients with chronic renal failure (CRF) without renal replacement therapy and 30 healthy volunteers in order to find potential disease biomarkers and reveal its pathophysiological changes. After data acquisition Waters MarkerLynx software was used to report retention time and m/z pairs for each metabolite peak, these data were exported to an excel table, then handled by using multivariate analysis and the statistical analysis in the SIMCA-P and the SPSS softwares to obtain potential biomarkers which were further identified by MS/MS. Seven potential biomarkers, creatinine, tryptophan, phenylalanine, kynurenine and three lysophosphatidylcholines, were identified. The results suggest that CRF can lead to the increase of reservation of creatinine in the body, and the abnormal metabolism of the two essential amino acids and lysophosphatidylcholines. It has indicated that metabonomics will be a powerful tool in the clinic research.     

Oxidative stress in patients with cardiovascular disease and chronic renal failure

Abstract

Oxidative response regulates many physiological response in human health, but if not properly regulated it could also lead to a number of deleterious effects. The importance of oxidative stress injury depends on the molecular target, the severity of the stress, and the mechanism by which the oxidative stress is imposed: it has been implicated in several diseases including cancer, neurodegenerative diseases, malaria, rheumatoid arthritis and cardiovascular and kidney disease. Most of the common diseases, such as hypertension, atherosclerosis, heart failure, and renal dysfunction, are associated with vascular functional and structural alterations including endothelial dysfunction, altered contractility, and vascular remodeling. Common to these processes is increased bioavailability of reactive oxygen species (ROS), decreased nitric oxide (NO) levels, and reduced antioxidant capacity. Oxidative processes are up-regulated also in patients with chronic renal failure (CRF) and seem to be a cause of elevated risk of morbidity and mortality in these patients.

In this review, we highlight the role of oxidative stress in cardiovascular and renal disease.     

Investigation of albumin properties in patients with chronic renal failure

The aim of this study was the investigation of HSA properties and its structural changes after modification induced in vivo among patients with CRF who underwent haemodialysis. Application of different fluorescent dyes allowed the investigation of different regions of albumin molecule using ANS, bis-ANS, piren, piren maleimide and fluorescein isothiocyanate. As markers of oxidative modification, the total protein thiol, carbonyls, glycosylated plasma proteins and hydroperoxide were estimated in plasma. Additionally, this study investigated plasma viscosity and total antioxidant capacity (TAC) of the plasma. Results show that haemodialysis provoked significant changes in conformational properties of plasma albumin, which resulted in the loss of its biological functions. These findings suggest that oxidative stress and glycation of proteins in plasma are developed during haemodialysis. The results depict that one of the features of uraemia is the presence of signs of oxidative stress before haemodialysis. Nevertheless, oxidative stress and glycation of proteins in plasma are exacerbated during haemodialysis and are a complex process.