scholarly journals Role of the USF1 transcription factor in diabetic kidney disease

2011 ◽  
Vol 301 (2) ◽  
pp. F271-F279 ◽  
Author(s):  
Amber P. Sanchez ◽  
JingHong Zhao ◽  
Young You ◽  
Anne-Emilie Declèves ◽  
Maggie Diamond-Stanic ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Kidney Disease ◽  
High Glucose ◽  
Diabetic Kidney Disease ◽  
Nuclear Accumulation ◽  
Diabetic Mice ◽  
Mesangial Matrix ◽  
Diabetic Kidney ◽  
Ampk Activity ◽  
Tgf Β1

The predominant transcription factors regulating key genes in diabetic kidney disease have not been established. The transcription factor upstream stimulatory factor 1 (USF1) is an important regulator of glucose-mediated transforming growth factor (TGF)-β1 expression in mesangial cells; however, its role in the development of diabetic kidney disease has not been evaluated. In the present study, wild-type (WT; USF1 +/+), heterozygous (USF1 +/−), and homozygous (USF1 −/−) knockout mice were intercrossed with Akita mice (Ins2/Akita) to induce type 1 diabetes. Mice were studied up to 36 wk of age. The degree of hyperglycemia and kidney hypertrophy were similar in all groups of diabetic mice; however, the USF1 −/− diabetic mice had significantly less albuminuria and mesangial matrix expansion than the WT diabetic mice. TGF-β1 and renin gene expression and protein were substantially increased in the WT diabetic mice but not in USF1 −/− diabetic mice. The underlying pathway by which USF1 is regulated by high glucose was investigated in mesangial cell culture. High glucose inhibited AMP-activated protein kinase (AMPK) activity and increased USF1 nuclear translocation. Activation of AMPK with AICAR stimulated AMPK activity and reduced nuclear accumulation of USF1. We thus conclude that USF1 is a critical transcription factor regulating diabetic kidney disease and plays a critical role in albuminuria, mesangial matrix accumulation, and TGF-β1 and renin stimulation in diabetic kidney disease. AMPK activity may play a key role in high glucose-induced regulation of USF1.

scholarly journals S-Nitrosylation of RhoGAP Myosin9A Is Altered in Advanced Diabetic Kidney Disease

2021 ◽  
Vol 8 ◽  
Author(s):  
Qi Li ◽  
Delma Veron ◽  
Alda Tufro
Keyword(s):  
Kidney Disease ◽  
High Glucose ◽  
Diabetic Kidney Disease ◽  
Diabetic Mice ◽  
Post Translational Modification ◽  
No Donor ◽  
Diabetic Kidney ◽  
Rhoa Activity

The molecular pathogenesis of diabetic kidney disease progression is complex and remains unresolved. Rho-GAP MYO9A was recently identified as a novel podocyte protein and a candidate gene for monogenic FSGS. Myo9A involvement in diabetic kidney disease has been suggested. Here, we examined the effect of diabetic milieu on Myo9A expression in vivo and in vitro. We determined that Myo9A undergoes S-nitrosylation, a post-translational modification dependent on nitric oxide (NO) availability. Diabetic mice with nodular glomerulosclerosis and severe proteinuria associated with doxycycline-induced, podocyte-specific VEGF164 gain-of-function showed markedly decreased glomerular Myo9A expression and S-nitrosylation, as compared to uninduced diabetic mice. Immortalized mouse podocytes exposed to high glucose revealed decreased Myo9A expression, assessed by qPCR, immunoblot and immunocytochemistry, and reduced Myo9A S-nitrosylation (SNO-Myo9A), assessed by proximity link assay and biotin switch test, functionally resulting in abnormal podocyte migration. These defects were abrogated by exposure to a NO donor and were not due to hyperosmolarity. Our data demonstrate that high-glucose induced decrease of both Myo9A expression and SNO-Myo9A is regulated by NO availability. We detected S-nitrosylation of Myo9A interacting proteins RhoA and actin, which was also altered by high glucose and NO dependent. RhoA activity inversely related to SNO-RhoA. Collectively, data suggest that dysregulation of SNO-Myo9A, SNO-RhoA and SNO-actin may contribute to the pathogenesis of advanced diabetic kidney disease and may be amenable to therapeutic targeting.

scholarly journals AMP-activated protein kinase (AMPK) activation inhibits nuclear translocation of Smad4 in mesangial cells and diabetic kidneys

2015 ◽  
Vol 308 (10) ◽  
pp. F1167-F1177 ◽  
Author(s):  
Jinghong Zhao ◽  
Satoshi Miyamoto ◽  
Young-Hyun You ◽  
Kumar Sharma
Keyword(s):  
Kidney Disease ◽  
Diabetic Kidney Disease ◽  
Nuclear Translocation ◽  
Mesangial Cells ◽  
Nuclear Accumulation ◽  
Ampk Activation ◽  
Mesangial Matrix ◽  
Diabetic Kidney ◽  
Amp Activated Protein Kinase ◽  
Stimulation Of

Diabetic nephropathy is characterized by diffuse mesangial matrix expansion and is largely dependent on the TGF-β/Smad signaling pathway. Smad4 is required for TGF-β signaling; however, its regulation has not been well characterized in diabetic kidney disease. Here, we report that high glucose is sufficient to stimulate nuclear translocation of Smad4 in mesangial cells and that stimulation of the major energy sensor AMP-activated protein kinase (AMPK) has a potent effect to block Smad4 nuclear translocation. Activation of AMPK by 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) inhibited high glucose-induced and TGF-β stimulation of nuclear Smad4. To identify which of the catalytic α-subunits may be involved, small interfering (si) RNA-based inhibition of AMPK α1- or α2-subunit was employed. Inhibition of either subunit reduced overall AMPK activity and contributed to Smad4 nuclear accumulation. In an animal model of early diabetic kidney disease, induction of diabetes was found to markedly stimulate Smad4 protein levels and enhance nuclear accumulation. AMPK activation with AICAR completely prevented the upregulation of Smad4 and reduced mesangial matrix accumulation. We conclude that stimulation of Smad4 in cell culture and in in vivo models of early diabetic kidney disease is dependent on AMPK.

scholarly journals KCa3.1 Mediates Dysregulation of Mitochondrial Quality Control in Diabetic Kidney Disease

2021 ◽  
Vol 9 ◽  
Author(s):  
Chunling Huang ◽  
Hao Yi ◽  
Ying Shi ◽  
Qinghua Cao ◽  
Yin Shi ◽  
...  
Keyword(s):  
Quality Control ◽  
Kidney Disease ◽  
Diabetic Kidney Disease ◽  
Diabetic Mice ◽  
Mitochondrial Quality Control ◽  
Diabetic Kidney ◽  
Hk2 Cells ◽  
Tgf Β1

Mitochondrial dysfunction is implicated in the pathogenesis of diabetic kidney disease. Mitochondrial quality control is primarily mediated by mitochondrial turnover and repair through mitochondrial fission/fusion and mitophagy. We have previously shown that blockade of the calcium-activated potassium channel KCa3.1 ameliorates diabetic renal fibrosis. However, the mechanistic link between KCa3.1 and mitochondrial quality control in diabetic kidney disease is not yet known. Transforming growth factor β1 (TGF-β1) plays a central role in diabetic kidney disease. Recent studies indicate an emerging role of TGF-β1 in the regulation of mitochondrial function. However, the molecular mechanism mediating mitochondrial quality control in response to TGF-β1 remains limited. In this study, mitochondrial function was assessed in TGF-β1-exposed renal proximal tubular epithelial cells (HK2 cells) transfected with scrambled siRNA or KCa3.1 siRNA. In vivo, diabetes was induced in KCa3.1+/+ and KCa3.1−/− mice by low-dose streptozotocin (STZ) injection. Mitochondrial fission/fusion-related proteins and mitophagy markers, as well as BCL2 interacting protein 3 (BNIP3) (a mitophagy regulator) were examined in HK2 cells and diabetic mice kidneys. The in vitro results showed that TGF-β1 significantly inhibited mitochondrial ATP production rate and increased mitochondrial ROS (mtROS) production when compared to control, which was normalized by KCa3.1 gene silencing. Increased fission and suppressed fusion were found in both TGF-β1-treated HK2 cells and diabetic mice, which were reversed by KCa3.1 deficiency. Furthermore, our results showed that mitophagy was inhibited in both in vitro and in vivo models of diabetic kidney disease. KCa3.1 deficiency restored abnormal mitophagy by inhibiting BNIP3 expression in TGF-β1-induced HK2 cells as well as in the diabetic mice. Collectively, these results indicate that KCa3.1 mediates the dysregulation of mitochondrial quality control in diabetic kidney disease.

scholarly journals Wogonin protects glomerular podocytes by targeting Bcl-2-mediated autophagy and apoptosis in diabetic kidney disease

2021 ◽  
Author(s):  
Xue-qi Liu ◽  
Ling Jiang ◽  
Yuan-yuan Li ◽  
Yue-bo Huang ◽  
Xue-ru Hu ◽  
...  
Keyword(s):  
Kidney Disease ◽  
High Glucose ◽  
Diabetic Kidney Disease ◽  
Cell Damage ◽  
Microvascular Complications ◽  
Therapeutic Drug ◽  
Protective Effects ◽  
Diabetic Mice ◽  
Mice Model ◽  
Diabetic Kidney

AbstractDiabetic kidney disease (DKD) is one of the microvascular complications of diabetes mellitus and a major cause of end-stage renal disease with limited treatment options. Wogonin is a flavonoid derived from the root of Scutellaria baicalensis Georgi, which has shown a potent renoprotective effect. But the mechanisms of action in DKD are not fully elucidated. In this study, we investigated the effects of wogonin on glomerular podocytes in DKD using mouse podocyte clone 5 (MPC5) cells and diabetic mice model. MPC5 cells were treated with high glucose (30 mM). We showed that wogonin (4, 8, 16 μM) dose-dependently alleviated high glucose (HG)-induced MPC5 cell damage, accompanied by increased expression of WT-1, nephrin, and podocin proteins, and decreased expression of TNF-α, MCP-1, IL-1β as well as phosphorylated p65. Furthermore, wogonin treatment significantly inhibited HG-induced apoptosis in MPC5 cells. Wogonin reversed HG-suppressed autophagy in MPC5 cells, evidenced by increased ATG7, LC3-II, and Beclin-1 protein, and decreased p62 protein. We demonstrated that wogonin directly bound to Bcl-2 in MPC5 cells. In HG-treated MPC5 cells, knockdown of Bcl-2 abolished the beneficial effects of wogonin, whereas overexpression of Bcl-2 mimicked the protective effects of wogonin. Interestingly, we found that the expression of Bcl-2 was significantly decreased in biopsy renal tissue of diabetic nephropathy patients. In vivo experiments were conducted in STZ-induced diabetic mice, which were administered wogonin (10, 20, 40 mg · kg−1 · d−1, i.g.) every other day for 12 weeks. We showed that wogonin administration significantly alleviated albuminuria, histopathological lesions, and p65 NF-κB-mediated renal inflammatory response. Wogonin administration dose-dependently inhibited podocyte apoptosis and promoted podocyte autophagy in STZ-induced diabetic mice. This study for the first time demonstrates a novel action of wogonin in mitigating glomerulopathy and podocytes injury by regulating Bcl-2-mediated crosstalk between autophagy and apoptosis. Wogonin may be a potential therapeutic drug against DKD.

scholarly journals Early Renoprotective Effect of Ruxolitinib in a Rat Model of Diabetic Nephropathy

2021 ◽  
Vol 14 (7) ◽  
pp. 608
Author(s):  
Mohamed M. El-Kady ◽  
Reham A. Naggar ◽  
Maha Guimei ◽  
Iman M. Talaat ◽  
Olfat G. Shaker ◽  
...  
Keyword(s):  
Diabetes Mellitus ◽  
Kidney Disease ◽  
Rat Model ◽  
Diabetic Kidney Disease ◽  
Tubulointerstitial Fibrosis ◽  
Favorable Effect ◽  
Glomerular Sclerosis ◽  
Diabetic Kidney ◽  
Renoprotective Effect ◽  
Tgf Β1

Diabetic kidney disease (DKD) is still one of the unresolved major complications of diabetes mellitus, which leads ultimately to end-stage renal disease in both type 1 and type 2 diabetes patients. Available drugs that suppress the renin–angiotensin system have partially minimized the disease impact. Yet, there is an unmet need for new therapeutic interventions to protect the kidneys of diabetic patients. In DN, glomerular sclerosis and tubulointerstitial fibrosis are mediated through several pathways, of which JAK/STAT is a key one. The current study explored the potential renoprotective effect of the JAK1/JAK2 inhibitor ruxolitinib (at doses of 0.44, 2.2, and 4.4 mg·kg−1) compared to that of enalapril at a dose of 10 mg·kg−1, in a rat model of streptozotocin-induced diabetes mellitus over 8 weeks. The effect of ruxolitinib was assessed by determining urinary albumin/creatinine ratio, serum level of cystatin, and levels of TGF-β1, NF-κB, and TNF-α in renal tissue homogenates by biochemical assays, the glomerular sclerosis and tubulointerstitial fibrosis scores by histological analysis, and fibronectin, TGF-β1, and Vimentin levels by immunohistochemical staining with the respective antibodies. Our results revealed a significant early favorable effect of a two-week ruxolitinib treatment on the renal function, supported by a decline in the proinflammatory biomarkers of DKD. This pre-clinical study suggests that the renoprotective effect of ruxolitinib in the long term should be investigated in animals, as this drug may prove to be a potential option for the treatment of diabetic kidney disease.

Abstract 016: Profound and Sustained Amplification of Circulating ACE2 Activity Does Not Protect Diabetic Mice from Kidney Disease

Hypertension ◽  
2015 ◽  
Vol 66 (suppl_1) ◽  
Author(s):  
Jan Wysocki ◽  
Minghao Ye ◽  
Ahmed M Khattab ◽  
Yashpal Kanwar ◽  
Mark Osborn ◽  
...  
Keyword(s):  
Kidney Disease ◽  
Diabetic Kidney Disease ◽  
Kidney Injury ◽  
Renin Angiotensin System ◽  
Diabetic Mice ◽  
Diabetic Kidney ◽  
Over Expression ◽  
Akita Mice

ACE2 is a monocarboxypeptidase that by converting AngII to Ang1-7 should down-regulate the renin-angiotensin system and therefore provide a means to therapeutically target diabetic kidney disease, a condition where the kidney RAS is overactive. Previous work indicated that soluble human recombinant (r)ACE2 administration for 4 weeks attenuated kidney injury in diabetic Akita mice. Whether such effect of rACE2 can be confirmed and attributed to augmented ACE2 activity is uncertain because chronic use of human rACE2 in mice induces immunogenicity and the development of antibodies that neutralize serum ACE2 activity. To examine the effect of chronic amplification of circulating ACE2 on kidney injury caused by STZ-induced diabetes and to circumvent the immunogenicity arising from xenogeneic ACE2, ACE2 of mouse origin was administered to mice using either daily i.p. injections (1 mg/kg) of mrACE2 for 4 weeks or after 20 weeks of ACE2 mini-circle (MC) (10-30ug/mouse) administration. MC provides a form of gene delivery that is resistant to gene silencing and, in addition, greatly optimizes long-term in vivo overexpression of proteins of interest. ACE2MC resulted in a profound and sustained increase in serum ACE2 activity (2.4±0.3 vs. 497±135 RFU/ul/hr, p<0.01) but kidney ACE2 activity was unchanged (17.4±1.3 vs. 19.0±0.8 RFU/ug prot/hr). mACE2-treated mice injected with STZ developed diabetes similar to sham mice injected with STZ. Systolic BP was not different between non-diabetic mice, sham STZ-mice, and STZ-mice receiving mACE2 by either i.p. mrACE2 or ACE2MC. Urinary albumin was similarly increased in sham STZ-mice and in STZ-mice receiving mACE2. Glomerular mesangial score and glomerular cellularity were both increased to a similar extent in sham STZ-mice and in STZ-mice with mACE2 administration, as compared to non-diabetic controls. In conclusion, profound and long-term augmentation of ACE2 activity confined to the circulation is not sufficient to attenuate glomerular pathology and albuminuria in STZ-induced diabetic kidney disease probably because of lack of kidney delivery of ACE2. Strategies to achieve over-expression of ACE2 at the kidney level are needed to demonstrate a beneficial effect of this enzyme on diabetic kidney disease.

scholarly journals Dapagliflozin Ameliorates Diabetic Kidney Disease via Upregulating Crry and Alleviating Complement Over-activation in db/db Mice

2021 ◽  
Vol 12 ◽  
Author(s):  
Dong-Yuan Chang ◽  
Xiao-Qian Li ◽  
Min Chen ◽  
Ming-Hui Zhao
Keyword(s):  
Kidney Disease ◽  
Protective Effect ◽  
High Glucose ◽  
Diabetic Kidney Disease ◽  
Membrane Attack Complex ◽  
Sglt2 Inhibitors ◽  
Tubulointerstitial Injury ◽  
Diabetic Kidney ◽  
Proximal Tubular Epithelial Cells ◽  
Complement Regulator

Sodium-glucose cotransporter 2(SGLT2) inhibitors show prominent renal protective effect in diabetic kidney disease (DKD), anti-inflammatory effect being one of its key mechanisms. Over-activation of the complement system, a crucial part of innate immunity, plays an important role in DKD. We aimed to investigate the effect of SGLT2 inhibitors on alleviating complement over-activation in DKD. Db/db mice were randomly divided into two groups, with 7 mice in each group treated with dapagliflozin and vehicle respectively, and 7 mice in m/m mice group. Laboratory and renal pathological parameters were evaluated. Mouse proximal tubular epithelial cells (MPTECs) were cultured and treated with high glucose. Dapagliflozin and dimethyloxallyl glycine (DMOG) were added as conditional treatment. Dapagliflozin-treated db/db mice showed significantly lower urinary albumin than vehicle-treated ones. Besides typical glomerular and tubulointerstitial injury, both C3b and membrane attack complex (MAC) depositions were significantly attenuated in dapagliflozin-treated db/db mice. The expression of complement receptor type 1-related protein y (Crry), a key complement regulator which inhibits complement over-activation, was significantly upregulated by dapagliflozin. Dapagliflozin-mediated Crry upregulation was associated with inhibition of HIF-1α accumulation under high glucose. When HIF-1α expression was stabilized by DMOG, the protective effect of dapagliflozin via upregulating Crry was blocked. In conclusion, dapagliflozin could attenuate complement over-activation in diabetic mice via upregulating Crry, which is associated with the suppression of HIF-1α accumulation in MPTECs.

scholarly journals Effects of ZnT8 on epithelial-to-mesenchymal transition and tubulointerstitial fibrosis in diabetic kidney disease

2020 ◽  
Vol 11 (7) ◽  
Author(s):  
Xiuli Zhang ◽  
Tingwen Guan ◽  
Boxuan Yang ◽  
Harvest F. Gu ◽  
Zhihong Chi
Keyword(s):  
Kidney Disease ◽  
Pancreatic Beta Cells ◽  
Diabetic Kidney Disease ◽  
Epithelial To Mesenchymal Transition ◽  
Gene Transfection ◽  
Tubulointerstitial Fibrosis ◽  
Inflammatory Factors ◽  
Diabetic Kidney ◽  
Mesenchymal Transition ◽  
Tgf Β1

Abstract Zinc transporter 8 (ZnT8) transports zinc ions for crystallization and storage of insulin in pancreatic beta-cells and ZnT8 dysfunction is involved in pathogenesis of diabetes. The current study aimed to investigate whether ZnT8 has effects in pathophysiology of diabetic kidney disease (DKD) by using animal models for diabetes, including STZ-induced diabetic, db/db, ZnT8-KO, ZnT8-KO-STZ and ZnT8-KO-db/db mice. Results demonstrated that urine albumin to creatinine ratio and epithelial-to-mesenchymal transition (EMT) were increased in kidneys of ZnT8-KO-STZ and ZnT8-KO-db/db mice compared with C57BL/6 J and ZnT8-KO mice, while serum TGF-β1, IL-6, and TNF-α levels were elevated in parallel. In kidneys of mice intercrossed between ZnT8-KO and STZ-induced diabetic or db/db mice, these three inflammatory factors, ACR and EMT were also found to be increased compared with C57BL/6J, db/db and ZnT8-KO mice. Furthermore, ZnT8 up-regulation by hZnT8-EGFP reduced the levels of high glucose (HG)-induced EMT and inflammatory factors in normal rat kidney tubular epithelial cell (NRK-52E cells). Expression of phosphorylated Smad2/Smad3 was up-regulated after HG stimulation and further enhanced by ZnT8 siRNA but down-regulated after hZnT8-EGFP gene transfection. The current study thus provides the first evidence that ZnT8 protects against EMT-tubulointerstitial fibrosis though the restrain of TGF-β1/Smads signaling activation in DKD.

scholarly journals MMP-10 is Increased in Early Stage Diabetic Kidney Disease and can be Reduced by Renin-Angiotensin System Blockade

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
José María Mora-Gutiérrez ◽  
José Antonio Rodríguez ◽  
María A. Fernández-Seara ◽  
Josune Orbe ◽  
Francisco Javier Escalada ◽  
...  
Keyword(s):  
Kidney Disease ◽  
Diabetic Kidney Disease ◽  
Early Stage ◽  
Renin Angiotensin System ◽  
Diabetic Patients ◽  
Diabetic Mice ◽  
Angiotensin System ◽  
Diabetic Kidney ◽  
Renin Angiotensin

AbstractMatrix metalloproteinases have been implicated in diabetic microvascular complications. However, little is known about the pathophysiological links between MMP-10 and the renin-angiotensin system (RAS) in diabetic kidney disease (DKD). We tested the hypothesis that MMP-10 may be up-regulated in early stage DKD, and could be down-regulated by angiotensin II receptor blockade (telmisartan). Serum MMP-10 and TIMP-1 levels were measured in 268 type 2 diabetic subjects and 111 controls. Furthermore, histological and molecular analyses were performed to evaluate the renal expression of Mmp10 and Timp1 in a murine model of early type 2 DKD (db/db) after telmisartan treatment. MMP-10 (473 ± 274 pg/ml vs. 332 ± 151; p = 0.02) and TIMP-1 (573 ± 296 ng/ml vs. 375 ± 317; p < 0.001) levels were significantly increased in diabetic patients as compared to controls. An early increase in MMP-10 and TIMP-1 was observed and a further progressive elevation was found as DKD progressed to end-stage renal disease. Diabetic mice had 4-fold greater glomerular Mmp10 expression and significant albuminuria compared to wild-type, which was prevented by telmisartan. MMP-10 and TIMP-1 are increased from the early stages of type 2 diabetes. Prevention of MMP-10 upregulation observed in diabetic mice could be another protective mechanism of RAS blockade in DKD.

P0996THE INVOLVEMENT OF PRO-INFLAMMATORY IL-1 CYTOKINES IN TUBULAR INJURY IN DIABETIC KIDNEY DISEASE

2020 ◽  
Vol 35 (Supplement_3) ◽  
Author(s):  
Vladislav Slobodsky ◽  
Adi Litmanovich ◽  
Kamal Hassan ◽  
Khaled Khazim
Keyword(s):  
Kidney Disease ◽  
Protein Expression ◽  
Western Blotting ◽  
High Glucose ◽  
Diabetic Kidney Disease ◽  
Diabetic Patients ◽  
Mrna Levels ◽  
Diabetic Kidney ◽  
Tubular Cells ◽  
Fibronectin Expression

Abstract Background and Aims Pro-inflammatory cytokines are one of several factors which contribute to the progression of diabetic kidney disease (DKD), a condition characterized by chronic kidney inflammation which results in the tubulointerstitial fibrosis which contributes to the progression of DKD. Interleukin 1 (IL-1) two main agonists IL-1α and IL-1β activate a pro-inflammatory cascade in response to different inflammatory stimuli, including hyperglycemia. It was previously shown that a deficiency of NLRP3 which is required for the conversion of IL-1 to its active state, protects mice from the development and progression of DKD. We hypothesize that the chronic hyperglycemia in diabetic patients triggers the activation and release of IL1α and/or IL-1β from renal tubular cells and that this activation leads to the tissue fibrosis. We aim to assess Il-1 and fibronectin expression in an immortalized proximal tubule epithelial cell line from normal adult human kidney (HK-2). In addition, we evaluate the influence of Anakinra™, a pharmaceutical inhibitor of the Il-1 receptor, currently indicated mainly for rheumatoid diseases, on the levels of fibronectin expression in this model. Methods HK-2 cells were cultured and treated with either physiological glucose concentration (5.5mM), high glucose (30mM) or 30mM mannitol as osmotic control for 24 hours to evaluate their effects on Il-1 expression and fibronectin expression. mRNA levels of IL-1α, IL-1β and fibronectin were assessed in q-PCR, and protein expression levels were quantified by western blotting. Immunofluorescence was used to visually demonstrate the presence of IL-1α and IL-1β upon stimulation. Finally, Anakinra™ was added to the tissue cultures in a range of physiologic prescribed concentrations and its effect on cell fibrosis was assessed by the measurement of fibronectin expression 24 hours later by western blotting. Results mRNA and protein expression of IL-1α but mostly IL-1β was elevated in HK-2 cells under hyperglycemic conditions but not in physiological glucose environment or under high osmotic conditions. Fibronectin levels were elevated in the high glucose treated cells compared with control. Finally, Anakinra™ was found to attenuate fibronectin expression under high glucose conditions, compared with the untreated cells. Conclusion Proinflammatory IL-1α and IL-1β cytokines are expressed by HK-2 cells upon stimulation with glucose and result in the fibrosis on the cells measured by the production of fibronectin. The addition of Anakinra™, an IL-1 receptor blocker, to the cell culture attenuate the expression of fibronectin by the tubular cells. Our research is the first to describe a causation between hyperglycemia, IL-1 elevated levels and fibrosis in HK-2 cells, as demonstrated by the beneficial effect of Anakinra™ on lowering fibronectin expression.

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