ABSTRACT
Objective:To investigate the injury effect of hyperoxali acid on human arterial endothelial cells (HAECs) and its mechanism.Methods:HAECs were divided into intervention group and control group according to whether oxalic acid was used for intervention. The cells in the intervention group were stimulated with 30, 100, 200 and 300 μmol/L oxalic for different time. The effect of oxalic acid on the proliferation of HAECs was detected by MTT colorimetry. The change of cell cycle was analyzed by flow cytometry. The content of intracellular calcium was detected by fluorescence detection technology. The protein and mRNA expressions of cell cycle and anion transporter-related proteins were detected by Western blotting and fluorescence quantitative PCR. Besides, JAK2/STAT3 signaling pathway-related proteins were measured by Western blotting.Results:MTT colorimetry results showed that the intervention groups with high concentration of oxalic acid (100, 200, 300 μmol/L) could significantly inhibit the proliferation of HAECs, which was significantly different from the control group (all P<0.05). Fluorescence detection showed that the contents of intracellular calcium of HAECs in the intervention groups with high concentration of oxalic acid (100, 200, 300 μmol/L) were significantly higher than those in the control group after 48 hours ( P<0.05, P<0.001, P<0.001, respectively). Flow cytometry showed that the proportion of S phase of cells in the 200 μmol/L oxalic acid intervention group was significantly higher than that in the control group ( P<0.05). The results of Western blotting and PCR showed that the relative protein and mRNA expressions of anion transporter-related proteins slc26a1, slc26a5, slc26a11 in the intervention groups were higher than those in the control group (all P<0.05). Western blotting showed that the expression of p-JAK2 and p-STAT3 in the intervention groups after 24 hours were significantly higher than those in control group (all P<0.05). Conclusions:Hyperoxalic acid may enter HAECs through transporters slc26a1, slc26a5 and slc26a11 to inhibit cell proliferation and increase the intracellular calcium concentration. The mechanism may be through the activation of JAK2/STAT3 signaling pathway. Therefore, oxalic acid may be one of the uremic toxins leading to atherosclerosis.
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Objective:To explore the role and mechanism of C3a-C3a receptor (C3aR) in the progression of autosomal dominant polycystic kidney disease (ADPKD).Methods:Renal tissues of ADPKD patients and PKD1 knockout mice were collected. Then the expression of C3a-C3aR, Ki67 and F4/80 in renal tissues was observed. Macrophages were stimulated with lipopolysaccharide (LPS) and interleukin 4 respectively. The expression of C3aR, TNF-α, typing markers and related signal pathway proteins was detected in each group. PKD1 knockout mice were treated with C3aR inhibitor SB290157 (1 mg/kg). Renal pathology, cyst-related indicators and renal function were observed. Results:The expression of C3a and C3aR in ADPKD was up-regulated (both P<0.05); C3aR and F4/80 were co-located in the kidney of polycystic kidney disease (PKD) mice, indicating that C3aR was mainly expressed on membrane of macrophages. In vitro, the expression of C3aR was up-regulated in M1 macrophages ( P<0.05). After the stimulation of C3a, the expression of iNOS, TNF-α and IL-6 mRNA in M1 macrophages were up-regulated (all P<0.05), as well as the secretion of TNF-α, indicating that C3a not only affected the expression of inflammatory factors of M1 macrophages, but also affected the inflammatory microenvironment. In addition, C3a significantly activated Akt in M1 macrophages ( P<0.05). Compared with the control group, the treatment group showed a decrease in C3a-C3aR as well as serum BUN, Scr, cyst index, and two kidneys weight/body weight (2KW/BW) (all P<0.05), and ADPKD related pathway protein expression such as p-ERK and p-P65 was significantly down-regulated (all P<0.05). Conclusions:The increased C3a in polycystic kidney tissue causes infiltration and activation of macrophages through C3aR, and then promotes ADPKD progression. The mechanism may be mediated by Akt activation and increased TNF-α production. C3aR antagonist is a potential research direction in the treatment of ADPKD.
ABSTRACT
Objective To screen Oxalobacter formigenes (OxF) from fresh feces of healthy adults,and study its effect on the the prevention of calcium oxalate kidney stones.Methods OxF was screened and cultured from fresh feces of healthy adults.The rat model of calcium oxalate stone was established by esophageal gavage of 0.8％ of ethylene glycol.Rats were divided into a control group and four groups of rats with ethylene glycol-induced calcium oxalate kidney stones according to random number table.Three groups were treated with 106 CFU,107 CFU,108 CFU viable OxF every day,respectively,for 4 weeks.The blood and 24-hour urine samples were collected to detect the serum creatinine,urea nitrogen,serum and urine calcium,phosphorus,magnesium and urine oxalate every week.At the end of the 4th week,the rats were sacrificed and the kidney tissues were stained with HE and Yasue.The deposition and content of calcium oxalate crystals were observed under a light microscope.Results The bacteria strain isolated from fresh feces of healthy adults was 100％ as same as the known ATCC35274 bacteria strain,which means the strain screened is OxF.Among the 5 groups,there were no significant differences in body weight,Scr,BUN,serum calcium,blood magnesium,blood phosphorus,urinary magnesium and urinary phosphorus.The 24-hour urinary calcium excretion in the model group was significantly lower than that of the control group (P ＜ 0.05).After intervention with OxF solution,the 24-hour urinary calcium excretion in the 108 CFU OxF group was significantly higher than that in the model group (P ＜ 0.05),while there was no significant difference between the other intervention groups and the model.The oxalic acid excretion of 106 CFU OxF group and 107 CFU OxF group was lower than that of the model,but the difference did not reach statistical significance (P＞ 0.05).The 24 h oxalic acid excretion in the 108 CFU OxF group was significantly lower than that of the model at the end of first week (P ＜ 0.05),and continued to decrease for the next 3 weeks.After 4 weeks of intervention,no crystal formation was observed in the control group under the deflection microscope,but a large amount of calcium oxalate crystals were formed in the renal cortex and renal medulla.The crystals were piled up and connected to each other.Yasue staining coincided with the calcium oxalate crystal in the same part of the kidneys.Compared with the model,there was no significant change in the score of calcium oxalate crystal in the kidneys of 106 CFU OxF group and 107 CFU OxF group,while the score of calcium oxalate crystal in the kidneys of 108 CFU OxF group was significantly lower (P ＜ 0.05).Conclusions OxF are successively screened from healthy adults.Daily administration of 108 CFU OxF can safely and effectively reduce the urinary oxalic acid excretion,prevent the formation of calcium oxalate crystals and inhibit the formation of stones in kidneys of rats.