Aspirin attenuates podocyte injury in diabetic rats through overriding cyclooxygenase-2-mediated dysregulation of LDL receptor pathway.

AIM: This study aimed to investigate the effects of aspirin on podocyte injury and its underlying mechanisms in diabetic nephropathy (DN). METHODS: Eight-week-old male Sprague-Dawley rats were divided into three groups: non-diabetic rats (Control), streptozotocin-induced diabetic rats (DM), and diabetic rats treated with aspirin (DM + Aspirin) for 12 weeks. Intracellular lipid accumulation was evaluated by Oil Red O staining and quantitative free cholesterol assays. Podocyte injury and the levels of COX-2, inflammatory cytokines, and low-density lipoprotein receptor (LDLr) pathway-related proteins were evaluated by electron microscopy, immunohistochemical staining, and Western blotting, respectively. RESULTS: Lipid levels and urinary albumin-creatinine ratios were higher in the DM rats than in the Control rats. Periodic acid-Schiff staining showed glomerular hypertrophy and mild mesangial area widening in the DM rats. Electron microscopy showed that the podocyte foot processes were significantly flattened or absent in the DM rats. The protein expression levels of WT-1 and nephrin in the podocytes of DM rats were reduced. Interestingly, lipid accumulation in the kidneys of DM rats was significantly increased due to increased protein expression levels of LDLr, sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP), SREBP-2, cyclooxygenase-2 (COX-2), and inflammatory cytokines. Confocal immunofluorescent staining showed that COX-2 and WT-1 were co-expressed. Furthermore, COX-2 protein expression levels were positively correlated with LDLr protein expression levels. However, when COX-2 expression was inhibited by aspirin, these changes in the DM rats were significantly attenuated. CONCLUSION: Aspirin attenuates podocyte injury in DN, which may be through COX-2-mediated dysregulation of LDLr pathway.

Platelet Microparticles Mediate Glomerular Endothelial Injury in Early Diabetic Nephropathy.

BACKGROUND: Glomerular endothelium dysfunction, which plays a crucial role in the pathogenesis of early diabetic nephropathy, might be caused by circulating metabolic abnormalities. Platelet microparticles, extracellular vesicles released from activated platelets, have recently emerged as a novel regulator of vascular dysfunction. METHODS: We studied the effects of platelet microparticles on glomerular endothelial injury in early diabetic nephropathy in rats with streptozotocin-induced diabetes and primary rat glomerular endothelial cells. Isolated platelet microparticles were measured by flow cytometry. RESULTS: Plasma platelet microparticles were significantly increased in diabetic rats, an effect inhibited in aspirin-treated animals. In cultured glomerular endothelial cells, platelet microparticles induced production of reactive oxygen species, decreased nitric oxide levels, inhibited activities of endothelial nitric oxide synthase and SOD, increased permeability of the glomerular endothelium barrier, and reduced thickness of the endothelial surface layer. Conversely, inhibition of platelet microparticles in vivo by aspirin improved glomerular endothelial injury. Further analysis showed that platelet microparticles activated the mammalian target of rapamycin complex 1 (mTORC1) pathway in glomerular endothelial cells; inhibition of the mTORC1 pathway by rapamycin or raptor siRNA significantly protected against microparticle-induced glomerular endothelial injury in vivo and in vitro. Moreover, platelet microparticle-derived chemokine ligand 7 (CXCL7) contributed to glomerular endothelial injury, and antagonizing CXCL7 using CXCL7-neutralizing antibody or blocking CXCL7 receptors with a competitive inhibitor of CXCR1 and CXCR2 dramatically attenuated such injury. CONCLUSIONS: These findings demonstrate a pathogenic role of platelet microparticles in glomerular endothelium dysfunction, and suggest a potential therapeutic target, CXCL7, for treatment of early diabetic nephropathy.

CRH/CRHR1 mediates prenatal synthetic glucocorticoid programming of depression-like behavior across 2 generations.

Pregnant women at risk of preterm labor usually receive synthetic glucocorticoids (sGCs) to promote fetal lung development. Emerging evidence indicates that antenatal sGC increases the risk of affective disorders in offspring. Data from animal studies show that such disorders can be transmitted to the second generation. However, the molecular mechanisms underlying the intergenerational effects of prenatal sGC remain largely unknown. Here we show that prenatal dexamethasone (Dex) administration in late pregnancy induced depression-like behavior in first-generation (F1) offspring, which could be transmitted to second-generation (F2) offspring with maternal dependence. Moreover, corticotropin-releasing hormone (CRH) and CRH receptor type 1 (CRHR1) expression in the hippocampus was increased in F1 Dex offspring and F2 offspring from F1 Dex female rats. Administration of a CRHR1 antagonist to newborn F1 Dex offspring alleviated depression-like behavior in these rats at adult. Furthermore, we demonstrated that increased CRHR1 expression in F1 and F2 offspring was associated with hypomethylation of CpG islands in Crhr1 promoter. Our results revealed that prenatal sGC exposure could program Crh and Crhr1 gene expression in hippocampus across 2 generations, thereby leading to depression-like behavior. Our study indicates that prenatal sGC can cause epigenetic instability, which increases the risk of disease development in the offspring's later life.-Xu, Y.-J., Sheng, H., Wu, T.-W., Bao, Q.-Y., Zheng, Y., Zhang, Y.-M., Gong, Y.-X., Lu, J.-Q., You, Z.-D., Xia, Y., Ni, X. CRH/CRHR1 mediates prenatal synthetic glucocorticoid programming of depression-like behavior across 2 generations.

Activation of the CXCL16/CXCR6 Pathway by Inflammation Contributes to Atherosclerosis in Patients with End-stage Renal Disease.

Background: Chronic inflammation plays a critical role in the progression of atherosclerosis (AS). This study aimed to determine the effects of the CXC chemokine ligand 16 (CXCL16)/CXC chemokine receptor 6 (CXCR6) pathway on cholesterol accumulation in the radial arteries of end-stage renal disease (ESRD) patients with concomitant microinflammation and to further investigate the potential effects of the purinergic receptor P2X ligand-gated ion channel 7 (P2X7R). Methods: Forty-three ESRD patients were divided into the control group (n=17) and the inflamed group (n=26) based on plasma C-reactive protein (CRP) levels. Biochemical indexes and lipid profiles of the patients were determined. Surgically removed tissues from the radial arteries of patients receiving arteriovenostomy were used for preliminary evaluation of AS. Haematoxylin-eosin (HE) and Filipin staining were performed to assess foam cell formation. CXCL16/CXCR6 pathway-related protein expression, P2X7R protein expression and the expression of monocyte chemotactic protein-1 (MCP-1), tumour necrosis factor-α (TNF-α), and CD68 were detected by immunohistochemical and immunofluorescence staining. Results: Inflammation increased both MCP-1 and TNF-α expression and macrophage infiltration in radial arteries. Additionally, foam cell formation significantly increased in the radial arteries of the inflamed group compared to that of the controls. Further analysis showed that protein expression of CXCL16, CXCR6, disintegrin and metalloproteinase-10 (ADAM10) in the radial arteries of the inflamed group was significantly increased. Furthermore, CXCL16 expression was positively correlated with P2X7R expression in the radial arteries of ESRD patients. Conclusions: Inflammation contributed to foam cell formation in the radial arteries of ESRD patients via activation of the CXCL16/CXCR6 pathway, which may be regulated by P2X7R.

[Effect of Cordyceps sinensis powder on renal oxidative stress and mitochondria functions in 5/6 nephrectomized rats].

OBJECTIVE: To observe the effect of Cordyceps sinensis (CS) powder on renal oxidative stress and mitochondria functions in 5/6 nephrectomized rats, and to primarily explore its possible mechanisms. METHODS: Totally 30 male Sprague-Dawley rats were divided into the sham-operation group, the model group, and the treatment group by random digit table, 10 in each group. A chronic kidney disease (CKD) rat model was prepared by one step 5/6 nephrectomy. Rats in the treatment group were intragastrically administered with CS powder solution at the daily dose of 2 g/kg, once per day. Equal volume of double distilled water was intragastrically administered to rats in the sham-operation group and the model group. All medication lasted for 12 weeks. The general condition of rats, their body weight, blood pressure, 24 h proteinuria, urinary N-acetyl-ß-D-glucosaminidase (NAG), serum creatinine (SCr) , and blood urea nitrogen (BUN) were assessed before surgery, at week 2, 4, 6, 8, 10, and 10 after surgery. Pathological changes of renal tissues were observed under light microscope. Morphological changes of mitochondria in renal tubular epithelial cells were observed under transmission electron microscope. Activities of antioxidant enzymes including reduced glutathione (GSH), manganese superoxide dismutase (MnSOD), and malondialdehyde (MDA) in fresh renal tissue homogenate were detected. Mitochondria of renal tissues were extracted to detect levels of mitochondrial membrane potential and changes of reactive oxygen species (ROS). And expressions of cytochrome-C (Cyto-C) and prohibitin in both mitochondria and cytoplasm of the renal cortex were also measured by Western blot. RESULTS: (1) Compared with the sham-operation group, body weight was significantly decreased at week 2 (P <0. 01), but blood pressure increased at week 4 (P <0. 05) in the model group. Compared with the model group, body weight was significantly increased at week 12 (P <0. 01), but blood pressure decreased at week 8 (P < 0. 01) in the treatment group. (2) Compared with the sham-operation group, 24 h proteinuria, urinary NAG, blood SCr and BUN significantly increased in the model group (all P <0. 01). Compared with the model group, blood and urinary biochemical indices all significantly decreased in the treatment group (all P <0. 01). (3) Results of pathological renal scoring: Glomerular sclerosis index, scoring for tubulointerstitial fibrosis, degree of tubulointerstitial inflammatory infiltration were all obviously higher in the model group than in the sham-operation group (all P <0. 01). All the aforesaid indices were more obviously improved in the treatment group than in the model group (all P <0. 01). (4) Compared with the sham-operation group, activities of MnSOD and GSH-Px were significantly reduced, but MDA contents obviously increased in the renal cortex of the model group (all P <0. 01). Compared with the model group, activities of MnSOD and GSH-Px obviously increased (P <0. 05, P <0. 01), but MDA contents obviously decreased in the renal cortex of the treatment group (P <0. 01). (5) Compared with the sham-operation group, the mitochondrial membrane potential significantly decreased, but ROS levels significantly increased in the model group (all P <0.01). Compared with the model group, mitochondrial transmembrane potential increased in the treatment group, thereby inhibiting the tendency of increased production of ROS (both P < 0. 01). (6) Results of Western blot showed that, compared with the sham-operation group, expression levels of mitochondrial Cyto-C and Prohibitin were significantly reduced in the renal cortex (P <0. 01), but significantly elevated in the cytoplasm of the model group (P <0. 01). Compared with the model group, each index was obviously improved in the treatment group with statistical difference (P <0. 05, P <0. 01). CONCLUSION: CS powder had renal protection, and its mechanism might partially depend on in- hibition of oxidative stress and protection for mitochondria.

Influence of irbesartan on expression of ILK and its relationship with epithelial-mesenchymal transition in mice with unilateral ureteral obstruction.

AIM: Irbesartan, a new antagonist of the type 1 angiotensin II receptor, has been proven to be renal protective in both diabetic and non-diabetic nephropathy, but its exact mechanism is still uncertain. Here we investigated the influence of irbesartan on the expression of the integrin-linked kinase (ILK) and its relationship with epithelial-mesenchymal transition (EMT) in mice with unilateral ureteral obstruction (UUO). METHODS: The mice were randomly divided into 3 groups: sham operation (C, n=20), UUO (n=40), and UUO with irbesartan treatment (UUO+irbesartan, n=40). Irbesartan was given at a dose of 50 mg/kg body weight per day by gavage. The experimental animals in the control group received the same volume of vehicle (0.9% saline solution). The animals were sacrificed at d 1, 3, 7, and 14, respectively, after the surgery. RESULTS: The expression of the ILK at mRNA and protein levels were significantly increased in the UUO group 1 d after the surgery, which was significantly decreased by treatment with irbesartan (P<0.01, respectively). The expression of alpha-smooth muscle actin (alpha-SMA) was significantly increased, while E-cadherin was decreased in mice with UUO at d 3 after the surgery. Treatment with irbesartan significantly abrogated such effects (P<0.01). The immunohistochemistry analysis indicated that the protein expression of the ILK was positively correlated with alpha-SMA, but negatively with E-cadherin. CONCLUSION: These results suggested that irbesartan attenuated renal tubulointerstitial fibrosis in UUO mice, which may be related to the inhibition of ILK expression, subsequently preventing the tubular EMT.

[Role of integrin-linked kinase in renal tubular epithelial-mesenchymal transition of mice with obstructive nephropathy].

OBJECTIVE: To investigate the expression of integrin-linked kinase (ILK) in kidneys of mice with unilateral ureteral obstruction and its relevance with the epithelial-mesenchymal transition. METHODS: Mice were randomly divided into two groups, sham operation (C, n = 20) and unilateral ureteral obstruction (UUO, n = 40). The animals were sacrificed at day 1, 3, 7 and 14 respectively after the surgery. Tubulointerstitial fibrosis (TIF) was graded according to Masson staining. The protein level of ILK was examined by Western blot. Tissue/cytological expression for ILK, alpha-SMA and E-cadherin were investigated by immunohistochemistry. The mRNA levels of ILK, alpha-SMA and E-cadherin were analyzed by quantitative real-time PCR. RESULTS: In the control animals (group C), weak staining for ILK was detected mainly in the podocytes. Significant increase of staining for ILK in the experimental mice (UUO group) was detected from day 1 onward (t = 16.5, P < 0.01), reaching the peak at day 7. The protein expression of E-cadherin was continuously down-regulated from day 3 onward after surgery (t = 21.0, P < 0.01), while expression for alpha-SMA was up-regulated. From day 1 to day 7, the protein expression of ILK was positively correlated with alpha-SMA (R = 0.88, P < 0.01), but negatively correlated with E-cadherin (R = -0.87, P < 0.01). The mRNA expression of ILK and alpha-SMA analyzed by real-time PCR increased from postoperative day 1 and 3 respectively, but the mRNA expression of E-cadherin decreased from day 3 onward. CONCLUSION: Increasing expression of ILK occurs in the early phase of UUO mouse and may play an important role in the process of TIF through mediating the epithelial-mesenchymal transition.

[Observation on the ultrastructure of Pneumocystis carinii].

OBJECTIVE: To study the life cycle and morphology of Pneumocystis carinii by ultrastructural observation. METHODS: Wistar rat model of P. carinii infection was established by subcutaneous injection with dexamethasone. Lung tissue of the infected rats was used for the transmission electron microscopical study. RESULTS: The organisms were mainly present in the lung alveolar cavity, and also in the alveolar septum, pulmonary macrophages and neutrophils. More trophozoites of P. carinii attached to the type I alveolar epithelial cells, and rarely to the type II alveolar epithelial cells. Most of these trophozoites showed pseudopodial evaginations on their pellicles. The nucleus-associated organelle and spindle microtubules were observed in some trophozoites. The precyst phase was in three forms: early, intermediate and late form. Synaptonemal complexes indicating meiotic nuclear divisions and a clump of mitochondria were also observed in the precyst. The pellicle of the cyst has a thickened portion with a pore. There were nucleus with nucleolus, mitochondrion, vesicles, endoplasmic reticulum and numerous ribosomes in the organisms, and tubular expansions on its surface. CONCLUSION: The life cycle of P. carinii consists of trophozoite, precyst and cyst stages. The presence of a single pore in the cyst wall reveals that pore formation may be a mode of excystation for intracystic bodies of P. carinii.