The efficacy of percutaneous transluminal angioplasty and arteriovenous fistula reconstruction for immature arteriovenous fistula.

BACKGROUND: To access the efficacy of percutaneous transluminal angioplasty and arteriovenous fistula reconstruction for immature arteriovenous fistula, compare the long-term patency and post-operative complications between them. MATERIALS AND METHODS: The medical records and Hemodialysis record sheets from 44 patients between May 2020 and January 2022 who underwent percutaneous transluminal angioplasty or arteriovenous fistula reconstruction treatment for immature autogenous arteriovenous fistula (AVF) were retrospectively reviewed. The patients were divided into two groups according to the type of surgery they received, including 25 patients in the PTA group and 19 patients in the AVF reconstruction group. Clinical outcomes were included, such as the primary and secondary patency rates following the procedure, maturation time, peak systolic velocity (PSV) of brachial artery, maximum pump-controlled blood flow at initial dialysis, and post-operative complications rates in the two groups. RESULTS: Technical and clinical success was achieved in 100% of the 44 cases. For patients who underwent percutaneous transluminal angioplasty, the primary patency rate at 3, 6, and 9 months was 84.0%, 68.0%, 60.0%, and the secondary patency rate was 92.0%, 84.0%, 80.0%, respectively. And for patients who underwent arteriovenous fistula reconstruction, the primary patency rate at 3, 6, and 9 months was 89.5%, 73.7%, 68.4%, and the secondary patency rate was 100.0%, 94.7%, 94.7%, respectively. There were no significant differences between the two groups in terms of patency rates (p > .050). In patients whose maturation was successful, the average maturation time of fistula after the PTA procedure was 19.36 ± 13.94 days, and 58.63 ± 18.95 days for the reconstruction procedure (p < .010). The PSV of brachial artery before and after the procedure was 87.64 ± 23.87 cm/s and 153.20 ± 21.69 cm/s in PTA group, for reconstruction group, the number was 86.26 ± 20.59 cm/s and 151.26 ± 29.94 cm/s, respectively. No statistically significant differences (p > .050). The maximum pump-controlled blood flow at initial dialysis was 232.60 ± 16.72 ml/min in PTA group, which was significantly higher than 197.11 ± 10.45 ml/min in reconstruction group (p < .010). Subcutaneous hematoma, restenosis, thrombus formation, and pseudoaneurysm were major complications in PTA group. Restenosis, thrombus formation, and pseudoaneurysm were major complications in reconstruction group, with no statistically significant differences between the two groups (p > .050). CONCLUSION: When immature AVFs require reconstruction surgery, the patency outcomes are comparable to AVFs that undergo successful management by PTA. While, when AVFs are successfully managed by PTA, they have significantly less maturation times and higher maximum pump-controlled blood flow rates at initial dialysis AVF use.

Effect and mechanism of TFEB on pyroptosis in HK-2 cells induced by high glucose.

As a newly discovered way of cell death, pyroptosis has been gradually discovered in acute and chronic kidney disease. Existing studies have shown that reactive oxygen species (ROS) can induce pyroptosis and release a large number of inflammatory mediators, resulting in kidney damage. As a transcription factor, transcription factor EB(TFEB) can regulate mitochondrial energy metabolism, reduce the production of ROS, and reduce the inflammatory damage of vascular endothelial cells. In a high-glucose environment, whether TFEB can regulate oxidative stress in HK-2 cells, thereby reducing pyroptosis, has not yet been studied. This study found that in HK-2 cells, with the prolongation of high concentration glucose stimulation, the expression level of TFEB showed a trend of first increasing and then decreasing; and nuclear translocation of TFEB expression occurred within 24 h. In high-glucose environment, the expression of pyroptosis-related proteins gradually increased over time, while the expression of anti-oxidative stress proteins superoxide dismutase2(SOD2)and NAD(P)H: quinone oxidoreductase 1(NQO1) showed a trend of first increasing and then decreasing. After TFEB was transfected with overexpression plasmid, the expression levels of SOD2 and NQO1 increased significantly, and the expression of pyroptosis-related proteins decreased. Observed under a confocal microscope after Mitosox red staining, the expression of ROS in the TFEB overexpression group decreased. After down-regulating the expression of TFEB, the expression of ROS increased. The research results suggested that in HK-2 cells in the high glucose environment, TFEB may affect the pyroptosis by regulating the expression of antioxidant enzymes SOD2 and NQO1, which provides a new therapeutic idea for the treatment of diabetic nephropathy.

Effects of transcription factor EB on oxidative stress and apoptosis induced by high glucose in podocytes.

The aim of the present study was to investigate the effects of transcription factor EB (TFEB) overexpression on oxidative stress, mitochondrial function and apoptosis in podocytes induced with high glucose. High glucose­induced time­dependent changes in TFEB expression were identified and nuclear translocation of TFEB was observed in podocytes. Overexpression of TFEB markedly reduced high glucose­induced oxidative stress in podocytes, and increased the expression of superoxide dismutase 2 and heme oxygenase 1 antioxidant enzymes. It was further observed that TFEB overexpression could partially restore the expression of peroxisome proliferator­activated receptor­Î³ coactivator­1α, transcription factor A, mitochondrial, and cytochrome c oxidase subunit 4, thereby enhancing mitochondrial biosynthesis. Furthermore, overexpression of TFEB reduced mitochondrial swelling and fragmentation, restored mitochondrial membrane potential, and contributed to the restoration of mitochondrial function. By overexpressing TFEB, it was revealed that TFEB increased the ratios of phosphorylated (p)­Akt/Akt and p­Bad/Bad, and the expression of downstream Bcl­xl, and reduced the ratio of Bax/Bcl­2 and the expression of cleaved­caspase­3 compared with high glucose­treatment. Furthermore, when the Akt phosphorylation inhibitor Ly294002 was added, the improvement by TFEB to high glucose­induced apoptosis was significantly reduced. These findings suggest that overexpressing TFEB could reduce the production of reactive oxygen species in podocytes in a high glucose environment, relieve oxidative stress, promote mitochondrial biogenesis and renewal functions, and reduce high glucose­induced podocyte apoptosis by activating the Akt/Bad pathway.

Protective effects of pyrroloquinoline quinine against oxidative stress-induced cellular senescence and inflammation in human renal tubular epithelial cells via Keap1/Nrf2 signaling pathway.

Oxidative stress-induced cellular senescence and inflammation are important biological events in diabetic nephropathy (DN). Our recent studies have found that pyrroloquinoline quinine (PQQ) has protective effects against HG-induced oxidative stress damage and apoptosis in HK-2 cells. Nevertheless, whether PPQ has the effect of anti-inflammation and anti-senescence in HK-2 cells remains unclear. Here, we showed that low-dose PPQ treatment (100 nM) downregulates the expression of P16, P21, IL-1ß, TNF-α and NF-κB in HG cultured HK-2 cells. A low dose of PPQ also upregulated the protein expression of SOD2, CAT and inhibited the generation of ROS. We also indicated that PPQ affected the activity of Keap1/Nrf2 pathway, increased the nuclear accumulation of Nrf2 and the downstream pathway protein expression of Keap1/Nrf2 signaling pathway (HO-1, NQO-1, GST and GPx-3). When ML385 was added to inhibit the activity of Keap1/Nrf2 signaling pathway, the effects of PPQ on anti-oxidative stress, anti-inflammation and anti-senescence in HK-2 cells under HG condition were weakened. In conclusion, our results suggest that PPQ could modulate HG-induced inflammation and senescence in HK-2 cells via the inhibition of ROS generation and achieves the protective effects through Keap1/Nrf2 pathway and upregulating the expression of its target protein.

Pyridoxamine Treatment of HK-2 Human Proximal Tubular Epithelial Cells Reduces Oxidative Stress and the Inhibition of Autophagy Induced by High Glucose Levels.

BACKGROUND Diabetic nephropathy is a predominant cause of renal failure, which is an important chronic complication of diabetes. Pyridoxamine (PM) has been reported to protect renal tubular epithelial cells against oxidative damage and delay or inhibit the development and generation of glucose-induced renal insufficiency at the early stage of disease. In this study, we attempted to explore the protection mechanism of PM on human proximal tubular epithelial cells (HK-2 cells) induced by high glucose. MATERIAL AND METHODS HK-2 cells were cultivated by high glucose medium in the absence or presence of PM. Cell Counting Kit-8 was used to investigate the most appropriate drug concentration of PM by detecting the cell viability of HK-2 cells. The expression of autophagy-related protein Beclin-1, LC-3II, and p62 was measured by western blot analysis, reverse transcription­quantitative polymerase chain reaction (RT­qPCR), and immunofluorescence. The expression and localization of Beclin-1 and p62 were also detected via immunofluorescence. The intracellular reactive oxygen species generation was detected using the reactive oxygen species assay kit. The effects of PM on antioxidant defenses were evaluated with glutathione peroxidase (GPx), manganese superoxide dismutase (MnSOD) activity, and glutathione/glutathione disulfide (GSH/GSSG) ratio. RESULTS High glucose levels were able to upregulate the expression of oxidative stress associated protein and inhibit autophagy­associated changes verified by western blotting, RT­qPCR and immunofluorescence. Administration of PM reversed the high glucose­induced low-expressed Beclin-1 and LC-3II, and overexpressed p62 and intracellular reactive oxygen species levels. Furthermore, non-enzymatic antioxidant defenses and enzymatic antioxidant defenses were turned on by the application of PM. CONCLUSIONS Treatment with PM could reverse high glucose-induced inhibition of autophagy and oxidative stress.

Resveratrol Reduces Oxidative Stress and Apoptosis in Podocytes via Sir2-Related Enzymes, Sirtuins1 (SIRT1)/Peroxisome Proliferator-Activated Receptor γ Co-Activator 1α (PGC-1α) Axis.

BACKGROUND PGC-1α can be activated by deacetylation reactions catalyzed by SIRT1. Resveratrol is currently known as a potent activator of SIRT1. However, it is unknown whether the renal-protective effect of resveratrol is further related to activation of the podocyte SIRT1/PGC-1α pathway. MATERIAL AND METHODS High glucose was used to stimulate mouse podocytes. Resveratrol and PGC-1α siRNA transfection were used to perform co-intervention treatments. The protein and mRNA expression levels of SIRT1, PGC-1α, NRF1, and TFAM were detect by immunofluorescence, Western blot analysis, and qRT-PCR in the podocytes, respectively. DCHF-DA and MitoSOX™ staining were used to monitor the total ROS and mitochondrial ROS levels, respectively. The specific activities of complexes I and III were measured using Complex I and III Assay Kits. Mitochondrial membrane potential and cell apoptosis were measured using JC-1 staining and Annexin V-FITC/PI double-staining, respectively. RESULTS We found that high-glucose stimulation results in time-dependent decreases in the expression of SIRT1, PGC-1α, and its downstream genes NRF1 and mitochondrial transcription factor A (TFAM) for mouse podocytes, and increases ROS levels in cells and mitochondria. Moreover, the expression of nephrin was downregulated and the cell apoptotic rate was increased. Resveratrol treatment can improve abnormalities caused by high-glucose stimulation. In addition, it can also reduce the release of mitochondrial cytochrome C and DIABLO proteins to the cytoplasm and increase respiratory chain complex I and III activity and mitochondrial membrane potential. CONCLUSIONS Resveratrol can reduce the oxidative damage and apoptosis of podocytes induced by high-glucose stimulation via SIRT1/PGC-1α-mediated mitochondrial protection.

Resveratrol ameliorates podocyte damage in diabetic mice via SIRT1/PGC-1α mediated attenuation of mitochondrial oxidative stress.

Excessive generation of mitochondrial reactive oxygen species (ROS) is considered to be initiating event in the development of diabetic nephropathy (DN). Mitochondrial biosynthesis mediated by coactivator PGC-1α and its downstream transcription factors NRF1 and TFAM may be a key target in maintaining mitochondrial function. Resveratrol (RESV), a natural polyphenolic antioxidant, is a potent SIRT1 agonist. In this study we established diabetes mouse and podocyte exposed to high glucose as in vivo and in vitro models to investigate the efficacy and mechanism of RESV on renoprotection. We found that RESV alleviated proteinuria of diabetic mice, decreased malondialdehyde content while increased Mn-SOD activity in renal cortex, inhibited the apoptosis of glomerular podocytes and renal tubular epithelial cells, ameliorated pathological manifestations, and restored the expression of SIRT1 and PGC-1α in renal tissues of DN mice. In podocytes exposed to high glucose, RESV inhibited excessive ROS production and apoptosis. In addition, RESV decreased mitochondrial ROS production, improved respiratory chain complex I and III activity, elevated mitochondrial membrane potential, and inhibited the release of Cyto C and Diablo in the mitochondria into the cytoplasm. Taken together, our findings suggest that RESV ameliorates podocyte damage in diabetic mice via SIRT1/PGC-1α mediated attenuation of mitochondrial oxidative stress.

Pyrroloquinoline quinine protects HK-2 cells against high glucose-induced oxidative stress and apoptosis through Sirt3 and PI3K/Akt/FoxO3a signaling pathway.

High glucose(HG)-induced oxidative stress and apoptosis in renal tubular epithelial cells play an important role in the pathogenesis of diabetic nephropathy. Pyrroloquinoline quinine (PQQ), a new B vitamin, has been demonstrated to be important in antioxidant and anti-apoptotic effects. However, its effect on HK-2 cells and the potential mechanism are rarely investigated. In this study, we investigated that PPQ had protective effects against HG-induced oxidative stress damage and apoptosis in vitro model of diabetic nephropathy. PPQ at 10, 100, 500, 1000 and 10000 nM could protect HK-2 cell from HG-induced inhibition. The protective effects of PQQ were associated with increasing the level of antioxidants(SOD2, CAT), inhibition of reactive oxygen species(ROS) production, and dependent modulation of Bcl-2 family proteins. PPQ significantly upregulated the protein and mRNA expression of Sirtuin3(Sirt3) in HG-induced HK-2 cells. PPQ also reduced apoptosis in HG-induced HK-2 cells by the PI3K/Akt/FoxO3a signal pathway. As down-regulated sirt3 or inhibitory the activity of PI3K/Akt/FoxO3a pathway, the protective effects of PPQ were weakened. In conclusion, our data suggest that PPQ achieves the protective effects through PI3K/Akt/FoxO3a pathway and dependent modulation of Sirt3.

Genistein and Myd88 Activate Autophagy in High Glucose-Induced Renal Podocytes In Vitro.

BACKGROUND Renal podocyte damage plays a crucial role in the development of diabetic nephropathy. Genistein is derived from a leguminous plant, and MyD88 and TRIF are adaptor molecules in the Toll-like receptor (TLR) signaling pathway, which may play a role in autophagy. In this study, we utilized an in vitro high glucose (HG)-treated podocyte model to investigate the effects and underlying mechanisms of Genistein and MyD88 or TRIF siRNA induced autophagy and renal protection. MATERIAL AND METHODS An immortalized mouse podocyte cell line was treated with HG, Genistein, chloroquine, and/or transfected with specific Myd88 and TRIF siRNAs. The formation of autophagosomes and related autophagic vacuoles were monitored by transmission electron microscopy. The expression of autophagy-related factors and podocyte structure and functional markers, including LC3, p62, p-mTOR, synaptopodin, and nephrin, were measured by Western blot, and LC3 and p-mTOR expression were also assessed by immunofluorescence. RESULTS We showed that HG transiently (after 6-h exposure) induced expression of the autophagy activation marker LC3-II in podocytes. Genistein treatment induced autophagy in both normal and HG-treated podocytes through inactivating mTOR signaling. Moreover, Genistein protected podocytes against chloroquine in HG-cultured conditions in vitro by maintaining the level of autophagy-related proteins. In addition, MyD88 siRNA downregulated expression of autophagy-related proteins, whereas Genistein treatment reversed these effects. CONCLUSIONS This study demonstrated that Genistein-induced autophagy could be a potential treatment strategy for glomerular diseases.

Salidroside Reduces High-Glucose-Induced Podocyte Apoptosis and Oxidative Stress via Upregulating Heme Oxygenase-1 (HO-1) Expression.

BACKGROUND Hyperglycemia is one of the most dangerous factors causing diabetic nephropathy. Salidroside is considered to have the effects of reducing oxidative stress damage and improving cell viability. This study was performed to investigate whether and how salidroside reduces high-glucose (HG)-induced apoptosis in mouse podocytes. MATERIAL AND METHODS We examined whether salidroside could decrease HG-induced podocyte oxidative stress and podocyte apoptosis in vitro. The potential signaling pathways were also investigated. Podocytes (immortalized mouse epithelial cells) were treated with normal glucose (5.5 mM) as control or HG (30 mM), and then exposed to salidroside treatment. RESULTS HG enhanced the generation of intracellular reactive oxygen species (ROS) and apoptosis in podocytes. Salidroside reduced HG-induced apoptosis-related consequences via promoting HO-1 expression. Salidroside increased the expression level of phosphorylated Akt (p-Akt) and phosphorylated ILK (p-ILK), p-JNK, and p-ERK and localization of Nrf-2. JNK inhibitor and ILK inhibitor decreased HO-1 expression to different degrees. Moreover, specific siRNAs of ILK, Nrf-2, and HO-1, and inhibitors of HO-1 and ILK significantly increased ROS generation and Caspase9/3 expression in the presence of salidroside and HG. CONCLUSIONS The results suggest that salidroside reduces HG-induced ROS generation and apoptosis and improves podocytes viability by upregulating HO-1 expression. ILK/Akt, JNK, ERK1/2, p38 MAPK, and Nrf-2 are involved in salidroside-decreased podocyte apoptosis in HG condition.

Role of Sirtuin3 in high glucose-induced apoptosis in renal tubular epithelial cells.

The apoptosis of renal tubular epithelial cells contributes to the pathogenesis of diabetic nephropathy. High glucose-induced mitochondrial oxidative stress is considered to be an important mediator for renal tubular cell apoptosis. Sirtuin3(Sirt3), a kind of mitochondria-localized nicotinamide adenine dinucleotide(NAD+)-dependent protein deacetylase, has been reported to regulate the generation of ROS in mitochondria through regulating acetylation level and activity of several key mitochondrial enzymes. In this study, we investigated the role of Sirt3 on high glucose-induced apoptosis in HK-2 cells. High glucose decreased the protein and mRNA expression of Sirt3 in a time-dependent manner, along with increased cell apoptosis in HK-2 cells. Furthermore, high glucose-induced oxidative stress and apoptosis were reversed by Sirt3 overexpression or antioxidant treatment. Meanwhile, we also found that overexpression of Sirt3 or antioxidant could regulate the activity of Akt/FoxO signaling pathway associated with cell apoptosis in diabetic nephropathy. In conclusion, our data suggest that Sirt3 overexpression antagonize high glucose-induced apoptosis by controlling ROS accumulation and ROS-sensitive Akt/FoxO signaling pathway in HK-2 cells.

Matrix metalloproteinase 12 modulates high-fat-diet induced glomerular fibrogenesis and inflammation in a mouse model of obesity.

Obesity-induced kidney injury contributes to albuminuria, which is characterized by a progressive decline in renal function leading to glomerulosclerosis and renal fibrosis. Matrix metalloproteinases (MMPs) modulate inflammation and fibrosis by degrading a variety of extracellular matrix and regulating the activities of effector proteins. Abnormal regulation of MMP-12 expression has been implicated in abdominal aortic aneurysm, atherosclerosis, and emphysema, but the underlying mechanisms remain unclear. The present study examined the function of MMP-12 in glomerular fibrogenesis and inflammation using apo E(-/-) or apo E(-/-)MMP-12(-/-) mice and maintained on a high-fat-diet (HFD) for 3, 6, or 9 months. MMP-12 deletion reduced glomerular matrix accumulation, and downregulated the expression of NADPH oxidase 4 and the subunit-p67(phox), indicating the inhibition of renal oxidative stress. In addition, the expression of the inflammation-associated molecule MCP-1 and macrophage marker-CD11b was decreased in glomeruli of apo E(-/-)MMP-12(-/-) mice fed HFD. MMP-12 produced by macrophages infiltrating into glomeruli contributed to the degradation of collagen type IV and fibronectin. Crescent formation due to renal oxidative stress in Bowman's space was a major factor in the development of fibrogenesis and inflammation. These results suggest that regulating MMP-12 activity could be a therapeutic strategy for the treatment of crescentic glomerulonephritis and fibrogenesis.

The Notch pathway mediates the angiotensin II-induced synthesis of extracellular matrix components in podocytes.

The Notch pathway is known to contribute to the development of glomerular disease. Angiotensin II (Ang II), an important member of the renin-angiotensin system, stimulates the accumulation of extracellular matrix components in glomerular disease; however, the exact mechanisms involved remain to be elucidated. In the present study, we aimed to investigate the effects of the Notch pathway on the synthesis of extracellular matrix components in Ang II-stimulated podocytes. Mouse podocytes were stimulated with Ang II (10-6 mol/l). The activation of the Notch pathway was inhibited by a vector carrying short hairpin RNA (shRNA) targeting Notch1 (sh-Notch1) or by γ-secretase inhibitor (GSI). The protein levels of Notch1, Notch intracellular domain 1 (NICD1), hairy and enhancer of split-1 (Hes1), matrix metalloproteinase (MMP)-2, MMP-9, transforming growth factor-ß1 (TGF-ß1), type IV collagen and laminin were determined by western blot analysis. The Notch1, Hes1, MMP-2, MMP-9, TGF-ß1, type IV collagen and laminin mRNA levels were detected by RT-PCR. The MMP-2 and MMP-9 activity was measured using a cell active fluorescence assay kit. The levels of TGF-ß1, type IV collagen and laminin were determined in the culture medium of the podocytes by enzyme-linked immunosorbent assay (ELISA). Our results revealed that Ang II upregulated Notch1, NICD1, Hes1, TGF-ß1, type IV collagen and laminin expression and downregulated MMP-2 and MMP-9 expression in the cultured podocytes. The inhibition of the Notch pathway by sh-Notch1 or GSI increased MMP-2 and MMP-9 expression, decreased the TGF-ß1 level and suppressed type IV collagen and laminin expression. The inhibition of the Notch pathway by sh-Notch1 or GSI also increased MMP-2 and MMP-9 activity, and decreased TGF-ß1 levels, type IV collagen levels and laminin secretion. These findings indicate that the Notch pathway potentially mediates the Ang II-induced synthesis of extracellular matrix components in podocytes through the regulation of MMPs and TGF-ß1.

Benazepril affects integrin-linked kinase and smooth muscle α-actin expression in diabetic rat glomerulus and cultured mesangial cells.

BACKGROUND: Diabetic nephropathy (DN) is the leading cause of chronic kidney disease and is associated with excessive cardiovascular morbidity and mortality. The angiotensin converting enzyme inhibitor (ACEI) benazepril has been shown to slow the progression of chronic renal disease and have beneficial effects in patients with a combination of chronic renal disease and cardiovascular disease. Transforming growth factor-ß(1) (TGF-ß(1)) plays a central role in the pathogenesis and progression of DN. Integrin-linked kinase (ILK) can modulate TGF-ß(1)-induced glomerular mesangial cell (GMC) injury, which is a prominent characteristic of renal pathology in kidney diseases. As an integrin cytoplasmic-binding protein, ILK regulates fibronectin (FN) matrix deposition and the actin cytoskeleton. Smooth muscle α-actin (α-SMA) is involved in progressive renal dysfunction in both human and experimental renal disease. METHODS: To explore the mechanisms of benazepril's reno-protective effects, we examined the expression of TGF-ß(1), ILK, and α-SMA in GMC exposed to high glucose (HG) and in the kidneys of streptozotocin (STZ)-induced diabetic rats using real-time quantitative RT-PCR and western blot analysis. To elucidate the mechanism(s) of the effect of benazepril on GMC cellular processes, we assessed the effect of benazepril on Angiotensin II (Ang II) signalling pathways using western blot analysis. RESULTS: The expression of TGF-ß(1), ILK, and α-SMA increased significantly in the diabetic group compared with the control group. Benazepril treatment inhibited the expression of these genes in DN but failed to rescue the same levels in the control group. Similar results were found in GMC treated with HG or benazepril. Ang II increased ERK and Akt phosphorylation in the HG group, and benazepril could not completely block these responses, suggesting that other molecules might be involved in the progression of DN. Our findings suggest that benazepril decreases ILK and α-SMA expression, at least in part, by affecting the interactions between Ang II and TGF-ß(1). CONCLUSIONS: The findings described here support the hypothesis that the HG milieu of diabetes increases TGF-ß(1) secretion, which increases the synthesis of ILK and α-SMA that are involved in the progression of DN. This might be an important mechanism of the benazepril renal-protective function in the pathogenesis of DN.

Gremlin aggravates hyperglycemia-induced podocyte injury by a TGFß/smad dependent signaling pathway.

Gremlin is a bone morphogenic protein (BMP) antagonist and is elevated in diabetic kidney tissues. In the early course of diabetic nephropathy (DN), podocyte are injured. We studied the protein and gene expression of gremlin in mice podocytes cultured in hyperglycemia ambient. The role of gremlin on podocyte injury and the likely signaling pathways involved were determined. Expression of gremlin was visualized by confocal microscopy. Recombinant mouse gremlin and small interfering RNA (siRNA) targeting to gremlin1 identified the role played by gremlin on podocytes. Study of canonical (smad2/3) and non-canonical (p38MAPK and JNK1/2) transforming growth factor beta (TGFß)/smad mediated signaling revealed the putative signaling mechanisms involved. Smad2/3 siRNA and TGFß receptor inhibition (SB431542) were used to probe canonical TGFß/smad signaling in gremlin-induced podocyte injury. Apoptosis of podocytes was measured by TUNEL assay. Gremlin expression was enhanced in high glucose cultured mouse podocytes, and was localized predominantly in the cytoplasm and negligibly on the cell membrane. Not only expression of nephrin and synaptopodin were decreased on treatment with gremlin, but also synaptopodin rearrangement and nephrin relocalization were evident. Knockdown gremlin1 or smad2/3 by siRNA, and inhibition of TGFßR (SB431542) attenuated podocyte injury. Inhibition of canonical TGF-ß signal blocked the injury of gremlin on podocytes. In conclusion, gremlin was clearly elevated in high glucose cultured mouse podocytes, and likely employed endogenous canonical TGFß1/Smad signaling to induce podocyte injury. Knockdown gremlin1 by siRNA may be clinically useful in the attenuation of podocyte injury.

Gremlin induces cell proliferation and extra cellular matrix accumulation in mouse mesangial cells exposed to high glucose via the ERK1/2 pathway.

BACKGROUND: Gremlin, a bone morphogenetic protein antagonist, plays an important role in the pathogenesis of diabetic nephropathy (DN). However, the specific molecular mechanism underlying Gremlin's involvement in DN has not been fully elucidated. In the present study, we investigated the role of Gremlin on cell proliferation and accumulation of extracellular matrix (ECM) in mouse mesangial cells (MMCs), and explored the relationship between Gremlin and the ERK1/2 pathway. METHODS: To determine expression of Gremlin in MMCs after high glucose (HG) exposure, Gremlin mRNA and protein expression were evaluated using real-time polymerase chain reaction and western blot analysis, respectively. To determine the role of Gremlin on cell proliferation and accumulation of ECM, western blot analysis was used to assess expression of pERK1/2, transforming growth factor-ß1 (TGF-ß1) and connective tissue growth factor (CTGF). Cell proliferation was examined by bromodeoxyuridine (BrdU) ELISA, and accumulation of collagen IV was measured using a radioimmunoassay. This enabled the relationship between Gremlin and ERK1/2 pathway activation to be investigated. RESULTS: HG exposure induced expression of Gremlin, which peaked 12 h after HG exposure. HG exposure alone or transfection of normal-glucose (NG) exposed MMCs with Gremlin plasmid (NG + P) increased cell proliferation. Transfection with Gremlin plasmid into MMCs previously exposed to HG (HG + P) significantly increased this HG-induced phenomenon. HG and NG + P conditions up-regulated protein levels of TGF-ß1, CTGF and collagen IV accumulation, while HG + P significantly increased levels of these further. Inhibition of Gremlin with Gremlin siRNA plasmid reversed the HG-induced phenomena. These data indicate that Gremlin can induce cell proliferation and accumulation of ECM in MMCs. HG also induced the activation of the ERK1/2 pathway, which peaked 24 h after HG exposure. HG and NG + P conditions induced overexpression of pERK1/2, whilst HG + P significantly induced levels further. Inhibition of Gremlin by Gremlin siRNA plasmid reversed the HG-induced phenomena. This indicates Gremlin can induce activation of the ERK1/2 pathway in MMCs. CONCLUSION: Culture of MMCs in the presence of HG up-regulates expression of Gremlin. Gremlin induces cell proliferation and accumulation of ECM in MMCs. and enhances activation of the ERK1/2 pathway.

Delivery of megsin siRNA plasmid reveals therapeutic potential against diabetic nephropathy by down-regulating p27(kip1) level.

BACKGROUND: Diabetic nephropathy is a complex disease with poor outcomes, and our current treatment measures are limited. It is urgent to search for novel therapeutic targets. Recently, a mesangium-predominant gene, megsin, has emerged as a participant in mesangial cell proliferation and/or mesangial matrix expansion. This study investigated the effect of megsin down-regulation on the progression of diabetic nephropathy. METHODS: Streptozotocin (STZ)-induced diabetic CD-1 mice after uninephrectomy received a pBAsi mU6 Neo megsin siRNA plasmid for 12 weeks and were compared with age-matched nondiabetic mice. In vitro mouse mesangial cells were transfected with pBAsi mU6 Neo megsin siRNA plasmid using Lipofectamine 2000 reagent and further cultured in DMEM containing high glucose for up to 48 hours. All of the cells were collected for protein extraction and the supernatant for type IV collagen measurement. The expression of megsin, matrix metalloproteinase-2 (MMP-2), tissue inhibitor of metalloproteases-2 (TIMP-2) and p27(Kip1) was determined by Western blotting. RESULTS: The megsin siRNA plasmid alleviated proteinuria and glomerular type IV collagen accumulation 12 weeks after the STZ injection, down-regulated renal cell proliferation and normalized the imbalance between MMP-2 and TIMP-2. Also, in vitro experiments showed that the glomerular mesangial cellular proliferation and type IV collagen production induced by high glucose were relieved after the transfection of megsin siRNA plasmid. The level of p27(kip1) was down-regulated in transfected mesangial cells significantly. CONCLUSIONS: The study suggests that the down-regulation of megsin might exert beneficial effects on the diabetic kidney partly through down-regulation of p27(kip1) level and that megsin may serve as a novel therapeutic target in the management of diabetic nephropathy.

Effects of dermatan sulfate derivatives on platelet surface P-selectin expression and protein C activity in blood of inflammatory bowel disease patients.

AIM: To investigate the effect of dermatan sulfate (DS) derivatives on platelet surface P-selectin expression and blood activated protein C (APC) activity in patients with inflammatory bowel disease (IBD), and to clarity the anti-inflammatory mechanism of DS derivatives. METHODS: Dermatan sulfate (DS) was sulfated with chlorosulfonic acid to prepare polysulfated dermatan sulfate (PSDS). The major disaccharides of DS and PSDS were determined by 1H nuclear magnetic resonance spectroscopy (1H-NMR) and 13C-NMR. Both DS and PSDS were depolymerized with hydrogen peroxide. The fragments were separated by gel filtration chromatography. The effects of DS derivatives on P-selectin expression were assayed by ELISA method, and blood APC activity was assayed by the synthetic chromogenic substrate method. RESULTS: The major disaccharides of DS and PSDS were IdoA-1-3-GalNAc-4-SO3 and IdoA-2SO3-1-3-GalNAc4, 6-diSO3, respectively. Compared with the adenosine diphosphate stimulated group and IBD control group, DS and its derivatives all had significant inhibitory effects on P-selectin expression (P<0.01), but there was no difference between DS-derived oligosaccharides (DSOSs) and PSDS-derived oligosaccharides (PSDSOSs). The experiments on APC activity showed that DS and its derivatives all enhanced APC activity. The most active DSOS was the one with a relative molecular weight (Mr) of 4,825, which enhanced the APC activity from 106.5+/-11.5% to 181.8+/-22.3% (P<0.01). With the decrease of Mr, the activity of DSOSs decreased gradually. The effect of PSDS on APC activity enhancement was more significant than that of DS, and the APC activity was raised to 205.2+/-22.1% (P<0.01). All the PSDSOSs were more active than DSOSs on the basis of comparable Mr. With the decrease of Mr, the activity of PSDSOSs increased gradually, and the most active PSDSOS was PSDSOS3 with Mr of 2,749, which enhanced the APC activity to 331.2+/-27.8% (P<0.01), then the activity of PSDSOSs decreased gradually. CONCLUSION: DS and its derivatives can significantly inhibit P-selectin expression on platelet surface, but the effect has no correlation with DS molecular mass and sulfation. The effect of DS or its derivatives on APC activity at molecular level involves complex mechanisms that depend on the molecular mass, the degree of sulfation, and the heterogeneous composition of DS. On the same molecular size, the higher the degree of DS sulfation, the more significant the effect on enhancing APC activity.

Inhibitory effect of heparin-derived oligosaccharides on secretion of interleukin-4 and interleukin-5 from human peripheral blood T lymphocytes.

AIM: To investigate the inhibitory effect of heparin-derived oligosaccharides (Oligs) on secretion of interleukin-4 (IL-4) and interleukin-5 (IL-5) from human peripheral blood T lymphocytes (PBTLs). METHODS: Oligs were prepared by three different heparin depolymerization methods and separated by gel filtration chromatography. PBTLs from ten adult patients with allergic eosinophilic gastroenteritis were treated with phytahematoagglutinin (PHA) and Oligs. The supernatants from the cell culture of PBTLs were harvested and subjected to the determination of IL-4 and IL-5 contents by ELISA method. RESULTS: At the concentration of 5 microg/mL, Oligs with different Mr had different effects on the secretion of IL-4 and IL-5. The tetrasaccharide with Mr of 1,142, produced by depolymerizing heparin with hydrogen peroxide, had the strongest inhibitory effect on the secretion of IL-4. It decreased the IL-4 content from 375.6+/-39.2 ng/L (PHA group) to 12.5+/-5.7 ng/L (P<0.01). The hexasaccharide with Mr of 1,806, produced by depolymerizing heparin with beta-elimination method, had the strongest inhibitory effect on the secretion of IL-5. It decreased the IL-5 content from 289.2+/-33.4 ng/L (PHA group) to 22.0+/-5.2 ng/L (P<0.01). CONCLUSION: The inhibitory activity of Oligs on the secretion of IL-4 and IL-5 from human PBTLs closely depends on their molecular structure, and there may be an essential structure to act as an inhibitor. The most effective inhibitors of IL-4 and IL-5 secretion are tetrasaccharides and hexasaccharides, respectively.