Glomerular Matrix Accumulation Research Articles

FOXA1 Suppresses SATB1 Transcription and Inactivates the Wnt/β-Catenin Pathway to Alleviate Diabetic Nephropathy in a Mouse Model.

ObjectiveDiabetic nephropathy (DN) represents the most common diabetic complication that may lead to end-stage renal disease. This work focused on the effect of FOXA1 on the DN development and the molecular mechanism.MethodsA mouse model of DN was induced by high-fat diets and streptozotocin. The concentrations of blood glucose and urinary protein in mice, and the pathological changes in mouse kidney tissues were determined. A podocyte cell line MPC-5 was treated with high glucose (HG) to mimic a DN-like condition in vitro. FOXA1 and SATB1 were overexpressed in HG-treated MPC-5 cells and in DN mice to explore their effects on cell proliferation and apoptosis, and on pathological changes in mouse kidney tissues. The binding relationship between FOXA1 and STAB1 was predicted and validated. Activation of the Wnt/β-catenin pathway was detected.ResultsFOXA1 was poorly expressed in the kidney tissues of DN mice. Overexpression of FOXA1 reduced the concentrations of fasting blood glucose and 24-h urinary protein in mice. It also suppressed the accumulation of glomerular mesangial matrix and hyperplasia of glomerular basement membrane, and reduced collagen deposition and interstitial fibrosis in mouse kidney. Also, FOXA1 reduced HG-induced apoptosis of MPC-5 cells. FOXA1 bound to the promoter region of SATB1 for transcription suppression. Overexpression of SATB1 activated the Wnt/β-catenin pathway and blocked the protective roles of FOXA1 in DN mice and in HG-treated MPC-5 cells.ConclusionThis study demonstrated that FOXA1 transcriptionally suppresses SATB1 expression and inactivates the Wnt/β-catenin signaling pathway, thereby inhibiting podocyte apoptosis and DN progression.

Clitoria ternatea L. extract prevents kidney damage by suppressing the Ang II/Nox4/oxidative stress cascade in l-NAME-induced hypertension model of rats

Clitoria ternatia L. (CT) has been reported to have anti-inflammatory and antioxidant effects. This study investigated the effect of CT aqueous flower extract on blood pressure and renal alterations in Nω-nitro-l-arginine methyl ester hydrochloride (l-NAME)-induced hypertensive rats. Male Sprague Dawley rats received l-NAME in drinking water and were treated with CT flower extract or lisinopril. CT aqueous flower extract and lisinopril alleviated l-NAME-induced hypertension (p < 0.05). Glomerular extracellular matrix accumulation, renal fibrosis, and increased serum creatinine levels were observed in l-NAME-induced hypertensive rats and attenuated by CT flower extract or lisinopril co-treatment (p < 0.05). High levels of plasma angiotensin II (Ang II) and upregulated nicotinamide adenine dinucleotide phosphate oxidase 4 (Nox4) protein expression in the kidneys induced by l-NAME were alleviated by CT flower extract or lisinopril co-treatment (p < 0.05). Furthermore, CT flower extract and lisinopril treatment reduced lipid peroxidation and elevated plasma and kidney malondialdehyde levels in l-NAME-induced hypertensive rats (p < 0.05). In conclusion, CT flower extract prevented l-NAME-induced renal injury and dysfunction in rats. The possible mechanism may be related to the suppression of Ang II-mediated Nox4 expression and the oxidative stress cascade in rats.

Noninvasive assessment of radiation-induced renal injury in mice

Purpose The kidney is a radiosensitive late-responding normal tissue. Injury is characterized by radiation nephropathy and decline of glomerular filtration rate (GFR). The current study aimed to compare two rapid and cost-effective methodologies of assessing GFR against more conventional biomarker measurements. Methods C57BL/6 mice were treated with bilateral focal X-irradiation (1x14Gy or 5x6Gy). Functional measurements of kidney injury were assessed 20 weeks post-treatment. GFR was estimated using a transcutaneous measurement of fluorescein-isothiocyanate conjugated (FITC)-sinistrin renal excretion and also dynamic contrast-enhanced CT imaging with a contrast agent (ISOVUE-300 Iopamidol). Results Hematoxylin and eosin (H&E) and Periodic acid-Schiff staining identified comparable radiation-induced glomerular atrophy and mesangial matrix accumulation after both radiation schedules, respectively, although the fractionated regimen resulted in less diffuse tubulointerstitial fibrosis. Albumin-to-creatinine ratios (ACR) increased after irradiation (1x14Gy: 100.4 ± 12.2 µg/mg; 6x5Gy: 80.4 ± 3.02 µg/mg) and were double that of nontreated controls (44.9 ± 3.64 µg/mg). GFR defined by both techniques was negatively correlated with BUN, mesangial expansion score, and serum creatinine. The FITC-sinistrin transcutaneous method was more rapid and can be used to assess GFR in conscious animals, dynamic contrast-enhanced CT imaging technique was equally safe and effective. Conclusion This study demonstrated that GFR measured by dynamic contrast-enhanced CT imaging is safe and effective compared to transcutaneous methodology to estimate kidney function.

Hesperidin inhibits L-NAME-induced vascular and renal alterations in rats by suppressing the renin-angiotensin system, transforming growth factor-β1, and oxidative stress.

The protective effect of hesperidin on vascular and renal alterations and possible underlying mechanisms involved in Nω -nitro-L-arginine methyl ester hydrochloride (L-NAME)-induced hypertensive rats were investigated in this study. Male Sprague-Dawley rats were administered L-NAME (40mg/kg/day), L-NAME plus hesperidin (30mg/kg/day), and L-NAME plus captopril (2.5mg/kg/day) for 5weeks. Hesperidin and captopril significantly prevented L-NAME-induced hypertension, vascular and renal dysfunction, intrarenal artery remodelling, glomerular extracellular matrix accumulation, and renal fibrosis. The preventive treatment with hesperidin and captopril also significantly decreased serum angiotensin-converting enzyme activity and plasma transforming growth factor-β1 (TGF-β1) levels and downregulated angiotensin II receptor type I and TGF-β1 protein expression in the kidneys. In addition, decreased malondialdehyde levels and increased superoxide dismutase activity in the plasma and kidney were observed after co-treatment with hesperidin or captopril. These findings suggest that hesperidin inhibits L-NAME-induced vascular and renal alterations in rats. The possible mechanism may be related to the suppression of the activation of the renin-angiotensin system and expression of TGF-β1, and reduction of oxidative stress.

Nobiletin ameliorates high-fat diet-induced vascular and renal changes by reducing inflammation with modulating AdipoR1 and TGF-β1 expression in rats

AimsWe investigate the effect of nobiletin on vascular and renal alterations and possible mechanisms involved in high-fat diet (HFD)-fed rats. Main methodsMale Sprague-Dawley rats were fed a HFD with fructose 15% in drinking water for 16 weeks. HFD-fed rats were treated with nobiletin (20 or 40 mg/kg/day) or vehicle for the last 4 weeks. Key findingsHFD-fed rats treated with nobiletin was significantly reduced obesity, hypertension, dyslipidemia and hyperinsulinemia. Nobiletin improved vascular endothelial function, restored creatinine clearance, and reduced plasma urea and creatinine levels, as well as urinary protein excretion. Nobiletin markedly alleviated vascular medial cross-sectional area (CSA) and collagen deposition, glomerular extracellular matrix (ECM) accumulation, and renal fibrosis. Nobiletin significantly elevated plasma adiponectin levels, together with upregulated adiponectin receptor 1 (AdipoR1) and suppressed transforming growth factor-β1 (TGF-β1) expression in kidney. In addition, an increase of plasma tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) was significantly attenuated after nobiletin treatment. SignificanceOur results suggest that nobiletin attenuates HFD-induced vascular and renal alterations in rats, which is possibly related to the modulation of AdipoR1 and TGF-β1expression, and suppression of inflammation.

The caveolin-1 regulated protein follistatin protects against diabetic kidney disease

Glomerular matrix protein accumulation, mediated largely by mesangial cells, is central to the pathogenesis of diabetic kidney disease. Our previous studies showed that the membrane microdomains caveolae and their marker protein caveolin-1 regulate matrix protein synthesis in mesangial cells in response to diabetogenic stimuli, and that caveolin-1 knockout mice are protected against diabetic kidney disease. In a screen to identify the molecular mechanism underlying this protection, we also established that secreted antifibrotic glycoprotein follistatin is significantly upregulated by caveolin-1 deletion. Follistatin potently neutralizes activins, members of the transforming growth factor-β superfamily. A role for activins in diabetic kidney disease has not yet been established. Therefore, invitro, we confirmed the regulation of follistatin by caveolin-1 in primary mesangial cells and showed that follistatin controls both basal and glucose-induced matrix production through activin inhibition. Invivo, we found activin A upregulation by immunohistochemistry in both mouse and human diabetic kidney disease. Importantly, administration of follistatin to type 1 diabetic Akita mice attenuated early diabetic kidney disease, characterized by albuminuria, hyperfiltration, basement membrane thickening, loss of endothelial glycocalyx and podocyte nephrin, and glomerular matrix accumulation. Thus, activin A is an important mediator of high glucose-induced profibrotic responses in mesangial cells, and follistatin may be a potential novel therapy for the prevention of diabetic kidney disease.

The Role of SMPDL3b in Regulating Radiation Nephropathy

Radiation nephropathy (RN) is functionally characterized by an initial acute phase (proteinuria) followed by a chronic decline of glomerular filtration rate leading to renal failure. RN is irreversible and affects the quality of life of both short- and long-term cancer survivors. The molecular mechanisms responsible for RN remain largely unknown. The current study investigates the role of sphingolipids in radiation-induced podocytopathy and RN. Sphingomyelin phosphodiesterase acid-like 3B (SMPDL3b) expression was assessed in cultured podocytes after X-irradiation (1-8 Gy) by real-time PCR and Western blotting. Changes in podocyte morphology and radiation-induced DNA damage repair were assessed in wild-type, SMPDL3b-KO and SMPDL3b-over-expressing podocytes. Wild-type C57BL/6 male and female mice (10–14 weeks) were X-irradiated using CT image-guidance (SD 14 Gy, fractionated 24x2 Gy; and 4 Gy SD + 6 mg/kg cisplatin). 50 mg/kg Rituximab (or IgG) was administered IP 30 min before irradiation to protect SMPDL3b. Functional kidney parameters (proteinuria and albumin/creatinine urine ratio, along with serum BUN [blood urea nitrogen], creatinine and hematocrit) were analyzed 10–40 weeks post treatment. Kidney histology, podocyte number and SMPDL3b levels were assessed by immunohistochemistry and Western blotting. Following irradiation, SMPDL3b expression was significantly reduced in vitro and in vivo . In irradiated kidneys, podocyte number decreased significantly and H&E kidney sections demonstrated a multifocal increase in the number of pericytes, tubular atrophy and glomerular damage. Transmission electron microscopy indicated radiation-mediated changes to glomerular base membrane thickness and podocyte foot process effacement. Periodic Acid-Schiff (PAS) staining demonstrated increases in glomerular mesangial matrix accumulation post-irradiation, along with diffuse intertubular fibrosis as determined by Sirius red staining, especially in the renal cortex. Rituximab improved kidney functional parameters, vascular structure, normalization of pericyte coverage and suppressed the development of fibrosis and tubular damage post irradiation. SMPDL3b expression was identified as a molecular determinant of podocyte injury after single dose and fractionated X-irradiation. Rituximab pretreatment protected mice against radiation-induced nephrotoxicity, which may have therapeutic implications for radiation-induced injuries in cancer patients.

Regulation of profibrotic responses by ADAM17 activation in high glucose requires its C-terminus and FAK.

Glomerular matrix accumulation is the hallmark of diabetic nephropathy. The metalloprotease ADAM17 mediates high glucose (HG)-induced matrix production by kidney mesangial cells through release of ligands for the epidermal growth factor receptor. Here, we study the mechanism by which HG activates ADAM17. We find that the C-terminus is essential for ADAM17 activation and the profibrotic response to HG. In the C-terminus, Src-mediated Y702 phosphorylation and PI3K-MEK-Erk-mediated T735 phosphorylation are crucial for ADAM17 activation, both are also required for the HG-induced increase in cell surface mature ADAM17. The non-receptor tyrosine kinase FAK is a central mediator of these processes. These data not only support a crucial role for the C-terminus in ADAM17 activation and downstream profibrotic responses to HG, but also highlight FAK as a potential alternative therapeutic target for diabetic nephropathy.

IL-6 receptor blockade ameliorates diabetic nephropathy via inhibiting inflammasome in mice

Background and ObjectiveInterleukin 6 (IL-6) has been identified as a key mediator in inflammation, immune responses and glucose metabolism. In this study, we assessed the effects of an IL-6 receptor antibody on diabetic nephropathy in a mouse model of type 2 diabetes mellitus. MethodsTwelve week old male db/db mice were treated with Tocilizumab (an IL-6 receptor antibody), normal IgG1 control antibody, insulin or normal saline for 12 weeks. Renal injury, inflammation and insulin resistance were assessed. ResultsDb/db mice treated with Tocilizumab exhibited reduced proteinuria and glomerular mesangial matrix accumulation compared to db/db + IgG controls. Additionally, Tocilizumab suppressed inflammatory response, oxidative stress and the IL-6 signaling pathway in the diabetic kidneys. It is noteworthy that blockade of IL-6 receptor blunted the activation of NLRP3 inflammasome partly through inhibition of IL-17A. Furthermore, insulin resistance assessed by glucose tolerance test, was ameliorated by Tocilizumab treatment. ConclusionsThe protective effects of an IL-6 receptor blockade against diabetic renal injury may be due to decreased insulin resistance and inhibition of the inflammasome.

Protective effects of IFN-γ on the kidney of type- 2 diabetic KKAy mice.

Development of novel therapeutic strategies that specifically target diabetic kidney disease (DKD) is urgently needed. Male KKAy mice were divided randomly into three equal groups - KK, KI, and KF; Male C57BL/6 mice were the control group. All KKAy mice were fed a high-fat diet. From the 16th week, the KI group was given IFN-γ, and the KF group was assigned to be treated with fludarabine. C57BL/6 mice were always fed a normal mouse diet. Every 4 weeks, body weight, random blood sugar, urine albumin and urea of all mice were measured. At the 20th week, all mice were killed, renal tissue was obtained to observe the pathological manifestations and extract proteins, and transforming growth factor- beta1 (TGF-β1), collagen IV and Janus kinase 2/signal transducers and activators of transcription 1 (JAK2/STAT1) pathway proteins were measured by western blot. The present study showed that all KKAy mice appeared obese and hyperglycaemic from 12 weeks old and exhibited an increased urine albumin-to-creatinine ratio (ACR) from 16 weeks old. At the 20th week, compared to the KK group, the KI group showed lower ACR, more overexpression of P-STAT1 and less expression of TGF-β1 and collagen IV proteins in renal tissue. The KI group mice showed less accumulation of glomerular mesangial matrix than those in the KK group. Our results indicate that IFN-γ might activate STAT1 to suppress the overexpression of TGF-β1 and collagen IV proteins and attenuate the excessive accumulation of mesangial matrix under DKD conditions in KKAy mice.

Abstract 904: Podocyte-specific SMPDl3b modulates radiation-induced renal dysfunction

Abstract Background: The underlying mechanisms responsible for the renal failure and proteinuria in radiation nephropathy remain largely unknown. Radiotherapy with or without chemotherapy may result in radiation-induced kidney injury in pelvic malignancies such as gynecologic cancers, lymphomas, gastrointestinal cancers, sarcomas of the upper abdomen and during total body irradiation. The current study investigates the role of sphingolipids in radiation-induced podocytopathy using a murine model. The molecular and functional effects of kidney irradiation were evaluated after single-dose exposures. Material/Methods: In cell culture, SMPDL3b expression post radiation (8Gy) was determined by real-time PCR (RT-PCR) and Western blotting. Morphological changes and DNA damage were detected post radiation using immunofluorescence microscopy. Wild-type C57BL/6 male and female mice (age 10-14 weeks) were irradiated with a single dose of X-ray (14 Gy) using an image guided small animal arc radiation treatment system (iSMAART). Rituximab/IgG was administered (50 mg/kg, intraperitoneal injection) 30 min before the single dose of irradiation. Functional kidney parameters, kidney histology, and gene expression were analyzed at 20, 30 and 40 weeks after irradiation. Results: Following irradiation, SMPDL3b expression at protein level was significantly reduced in vitro and in vivo. However, no significant changes were observed at the transcriptional level. Podocyte number also decreased significantly post radiation in vivo. iSMAART dynamic contrast enhanced (DCE) imaging data analysis showed reduced glomerular filtration rate post radiation. In agreement with the functional data, hematoxylin and eosin staining of kidney sections showed a multifocal increase in the number of pericytes, tubular atrophy, and glomerular damage. Periodic Acid-Schiff (PAS) staining showed an increase in glomerular mesangial matrix accumulation post radiation. Sirius red staining showed diffuse intertubular fibrosis, especially in the renal cortex post radiation. These histological changes were paralleled by the change in serum Creatinine, urine albumin. Rituximab pretreatment to mice, improved kidney functional parameters, vascular structure, normalization of pericyte coverage, suppress the development of fibrosis and tubular damage post irradiation. Conclusion: This study shows that rituximab pretreatment protects mice against radiation-induced nephrotoxicity, which may have therapeutic implications for radiation-induced injuries in cancer patients. Citation Format: Anis Ahmad, Alla Mitrofanova, Saba Ansari, Thirupandiyur Udayakumar, Jacek Bielawski, Alan Pollack, Alesia Fornoni, Brian Marples, Youssef Zeidan. Podocyte-specific SMPDl3b modulates radiation-induced renal dysfunction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 904. doi:10.1158/1538-7445.AM2017-904

Parathyroid hormone inhibits TGF-β/Smad signaling and extracellular matrix proteins upregulation in rat mesangial cells

Accumulation of glomerular matrix is a hallmark of diabetic nephropathy. TGF-β1 is a major cytokine mediating the production of various extracellular matrix (ECM) proteins. The aim of this study is to elucidate the effect of parathyroid hormone (PTH) on TGF-β1 and high glucose-induced upregulation of ECM proteins in primary mesangial cells from Sprague-Dawley rat. The results showed that PTH pretreatment prevented TGF-β1 and high glucose-induced Smad2/3 phosphorylation and consequent upregulation of fibronectin and type IV collagen within 4 h. The inhibitory effect of PTH is due to PTH1R activation, because knocking down PTH 1 receptor (PTH1R) by RNA interference reversed the inhibitory effect of PTH on TGF-β1 and high glucose-induced Smad2/3 phosphorylation and ECM upregulation. Furthermore, it is found that PTH1R associated with TGF-β type II receptor (TβR II) and both receptors internalized into the cytoplasm when mesangial cells were stimulated with PTH alone. The internalization of TβR II might reduce the amount of membrane TβR II, attenuate the sensitivity of mesangial cells to TGF-β1, and therefore inhibit Smad activation and ECM upregulation induced by TGF-β1 and high glucose. Further studies are needed to know whether the endocytic receptors are to be degraded or recycled, and evaluate the role of PTH in TGF-β1 signaling more comprehensively.

IL-17 Expression in the Time Course of Acute Anti-Thy1 Glomerulonephritis.

BackgroundInterleukin-17 (IL-17) is a new pro-inflammatory cytokine involved in immune response and inflammatory disease. The main source of IL-17 is a subset of CD4+ T-helper cells, but is also secreted by non-immune cells. The present study analyzes expression of IL-17 in the time course of acute anti-thy1 glomerulonephritis and the role of IL-17 as a potential link between inflammation and fibrosis.MethodsAnti-thy1 glomerulonephritis was induced into male Wistar rats by OX-7 antibody injection. After that, samples were taken on days 1, 5, 10 (matrix expansion phase), 15 and 20 (resolution phase). PBS-injected animals served as controls. Proteinuria and histological matrixes score served as the main markers for disease severity. In in vitro experiments, NRK-52E cells were used. For cytokine expressions, mRNA and protein levels were analyzed by utilizing RT-PCR, in situ hybridization and immunofluorescence.ResultsHighest IL-17 mRNA-expression (6.50-fold vs. con; p<0.05) was found on day 5 after induction of anti-thy1 glomerulonephritis along the maximum levels of proteinuria (113 ± 13 mg/d; p<0.001), histological glomerular-matrix accumulation (82%; p<0.001) and TGF-β1 (2.2-fold; p<0.05), IL-6 mRNA expression (36-fold; p<0.05). IL-17 protein expression co-localized with the endothelial cell marker PECAM in immunofluorescence. In NRK-52E cells, co-administration of TGF-β1 and IL-6 synergistically up-regulated IL-17 mRNA 4986-fold (p<0.001).ConclusionsThe pro-inflammatory cytokine IL-17 is up-regulated in endothelial cells during the time course of acute anti-thy1 glomerulonephritis. In vitro, NRK-52E cells secrete IL-17 under pro-fibrotic and pro-inflammatory conditions.

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-p67phox, 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.

Heparan sulfate 6-O-endosulfatases, Sulf1 and Sulf2, regulate glomerular integrity by modulating growth factor signaling.

Glomerular integrity and functions are maintained by growth factor signaling. Heparan sulfate, the major component of glomerular extracellular matrixes, modulates growth factor signaling, but its roles in glomerular homeostasis are unknown. We investigated the roles of heparan sulfate 6-O-endosulfatases, sulfatase (Sulf)1 and Sulf2, in glomerular homeostasis. Both Sulf1 and Sulf2 were expressed in the glomeruli of wild-type (WT) mice. Sulf1 and Sulf2 double-knockout (DKO) mice showed glomerular hypercellularity, matrix accumulation, mesangiolysis, and glomerular basement membrane irregularity. Platelet-derived growth factor (PDGF)-B and PDGF receptor-β were upregulated in Sulf1 and Sulf2 DKO mice compared with WT mice. Glomeruli from Sulf1 and Sulf2 DKO mice in vitro stimulated by either PDGF-B, VEGF, or transforming growth factor-β similarly showed reduction of phospho-Akt, phospho-Erk1/2, and phospho-Smad2/3, respectively. Since glomerular lesions in Sulf1 and Sulf2 DKO mice were reminiscent of diabetic nephropathy, we examined the effects of Sulf1 and Sulf2 gene disruption in streptozotocin-induced diabetes. Diabetic WT mice showed an upregulation of glomerular Sulf1 and Sulf2 mRNA by in situ hybridization. Diabetic DKO mice showed significant increases in albuminuria and serum creatinine and an acceleration of glomerular pathology without glomerular hypertrophy; those were associated with a reduction of glomerular phospho-Akt. In conclusion, Sulf1 and Sulf2 play indispensable roles to maintain glomerular integrity and protective roles in diabetic nephropathy, probably by growth factor modulation.

Podocyte-specific Nox4 deletion affords renoprotection in a mouse model of diabetic nephropathy.

Changes in podocyte morphology and function are associated with albuminuria and progression of diabetic nephropathy. NADPH oxidase 4 (NOX4) is the main source of reactive oxygen species (ROS) in the kidney and Nox4 is upregulated in podocytes in response to high glucose. We assessed the role of NOX4-derived ROS in podocytes in vivo in a model of diabetic nephropathy using a podocyte-specific NOX4-deficient mouse, with a major focus on the development of albuminuria and ultra-glomerular structural damage. Streptozotocin-induced diabetes-associated changes in renal structure and function were studied in male floxedNox4 and podocyte-specific, NOX4 knockout (podNox4KO) mice. We assessed albuminuria, glomerular extracellular matrix accumulation and glomerulosclerosis, and markers of ROS and inflammation, as well as glomerular basement membrane thickness, effacement of podocytes and expression of the podocyte-specific protein nephrin. Podocyte-specific Nox4 deletion in streptozotocin-induced diabetic mice attenuated albuminuria in association with reduced vascular endothelial growth factor (VEGF) expression and prevention of the diabetes-induced reduction in nephrin expression. In addition, podocyte-specific Nox4 deletion reduced glomerular accumulation of collagen IV and fibronectin, glomerulosclerosis and mesangial expansion, as well as glomerular basement membrane thickness. Furthermore, diabetes-induced increases in renal ROS, glomerular monocyte chemoattractant protein-1 (MCP-1) and protein kinase C alpha (PKC-α) were attenuated in podocyte-specific NOX4-deficient mice. Collectively, this study shows the deleterious effect of Nox4 expression in podocytes by promoting podocytopathy in association with albuminuria and extracellular matrix accumulation in experimental diabetes, emphasising the role of NOX4 as a target for new renoprotective agents.

Epidermal growth factor receptor inhibition with erlotinib ameliorates anti-Thy 1.1-induced experimental glomerulonephritis.

Mesangial proliferative glomerulonephritis is a common glomerular disorder that may lead to end-stage renal disease. Epidermal growth factor (EGF) plays an important role in the regulation of cell growth, proliferation, and differentiation and in the pathology of various renal diseases. Erlotinib is a novel, oral, highly selective tyrosine kinase inhibitor of the EGF receptor. It is clinically used to treat non-small cell lung and pancreatic cancers. Here, we investigated the effect of erlotinib on the progression of mesangioproliferative glomerulonephritis in an experimental model. Mesangial glomerulonephritis was induced with anti-rat Thy-1.1 antibody in male Wistar rats weighing 150-160g. Rats were treated with erlotinib (10mg/kg/day p.o.) or vehicle only (polyethylene glycol). Native Wistar rat kidneys were used as histological controls. Serum creatinine levels were measured at day 7. Kidneys were harvested 7days after antibody administration for histology. Native controls showed no histological signs of glomerular pathology. In the vehicle group, intense glomerular inflammation developed after 7days and prominent mesangial cell proliferation and glomerular matrix accumulation was seen. Erlotinib was well tolerated and there were no adverse effects during the follow-up period. Erlotinib significantly prevented progression of the glomerular inflammatory response and glomerular mesangial cell proliferation as well as matrix accumulation when compared with the vehicle group. Erlotinib also preserved renal function. These results indicate that erlotinib prevents the early events of experimental mesangial proliferative glomerulonephritis. Therefore, inhibition of the EGF receptor with erlotinib could prevent the progression of glomerulonephritis also in clinical nephrology.

Following specific podocyte injury captopril protects against progressive long term renal damage

Background: Angiotensin converting enzyme inhibitors (ACEi) reduce proteinuria and preserve kidney function in proteinuric renal diseases. Their nephroprotective effect exceeds that attributable to lowering of blood pressure alone. This study examines the potential of ACEi to protect from progression of injury after a highly specific injury to podocytes in a mouse model.Methods: We created transgenic (Podo-DTR) mice in which graded specific podocyte injury could be induced by a single injection of diphtheria toxin. Transgenic and wild-type mice were given the ACEi captopril in drinking water, or water alone, commencing 24h after toxin injection. Kidneys were examined histologically at 8 weeks and injury assessed by observers blinded to experimental group.Results: After toxin injection, Podo-DTR mice developed acute proteinuria, and at higher doses transient renal impairment, which subsided within 3 weeks to be followed by a slow glomerular scarring process. Captopril treatment in Podo-DTR line 57 after toxin injection at 5ng/g body weight reduced proteinuria and ameliorated glomerular scarring, matrix accumulation and glomerulosclerosis almost to baseline (toxin: 17%; toxin + ACEi 10%, p<0.04; control 7% glomerular scarring). Podocyte counts were reduced after toxin treatment and showed no recovery irrespective of captopril treatment (7.1 and 7.3 podocytes per glomerular cross section in water and captopril-treated animals compared with 8.2 of wild-type controls, p<0.05).Conclusions: Observations in Podo-DTR mice support the hypothesis that continuing podocyte dysfunction is a key abnormality in proteinuric disease. Our model is ideal for studying strategies to protect the kidney from progressive injury following podocyte depletion. Demonstrable protective effects from captopril occur, despite indiscernible preservation or restoration of podocyte counts, at least after this degree of relatively mild injury.

Following specific podocyte injury captopril protects against progressive long term renal damage.

Angiotensin converting enzyme inhibitors (ACEi) reduce proteinuria and preserve kidney function in proteinuric renal diseases. Their nephroprotective effect exceeds that attributable to lowering of blood pressure alone. This study examines the potential of ACEi to protect from progression of injury after a highly specific injury to podocytes in a mouse model. We created transgenic (Podo-DTR) mice in which graded specific podocyte injury could be induced by a single injection of diphtheria toxin. Transgenic and wild-type mice were given the ACEi captopril in drinking water, or water alone, commencing 24h after toxin injection. Kidneys were examined histologically at 8 weeks and injury assessed by observers blinded to experimental group. After toxin injection, Podo-DTR mice developed acute proteinuria, and at higher doses transient renal impairment, which subsided within 3 weeks to be followed by a slow glomerular scarring process. Captopril treatment in Podo-DTR line 57 after toxin injection at 5ng/g body weight reduced proteinuria and ameliorated glomerular scarring, matrix accumulation and glomerulosclerosis almost to baseline (toxin: 17%; toxin + ACEi 10%, p<0.04; control 7% glomerular scarring). Podocyte counts were reduced after toxin treatment and showed no recovery irrespective of captopril treatment (7.1 and 7.3 podocytes per glomerular cross section in water and captopril-treated animals compared with 8.2 of wild-type controls, p<0.05). Observations in Podo-DTR mice support the hypothesis that continuing podocyte dysfunction is a key abnormality in proteinuric disease. Our model is ideal for studying strategies to protect the kidney from progressive injury following podocyte depletion. Demonstrable protective effects from captopril occur, despite indiscernible preservation or restoration of podocyte counts, at least after this degree of relatively mild injury.

Relaxin Inhibits High Glucose-Induced Matrix Accumulation in Human Mesangial Cells by Interfering with TGF-β1 Production and Mesangial Cells Phenotypic Transition.

Diabetic nephropathy (DN) is the leading cause of end-stage renal disease (ESRD). DN is characterized by glomerular extracellular matrix accumulation, mesangial expansion, basement membrane thickening, and renal interstitial fibrosis. To date, mounting evidence has shown that H2 relaxin possesses powerful antifibrosis properties; however, the mechanisms of H2 relaxin on diabetic nephropathy remain unknown. Here, we aimed to explore whether H2 relaxin can reduce production of extracellular matrix (ECM) secreted by human mesangial cells (HMC). HMC were exposed to 5.5 mM glucose (NG) or 30 mM glucose (HG) with or without H2 relaxin. Fibronectin (FN) and collagen type IV levels in the culture supernatants were examined by solid-phase enzyme-linked immunoadsorbent assay (ELISA). Western blot was used to detect the expression of α-smooth muscle actin (α-SMA) protein. Quantitative polymerase chain reaction (qPCR) method was employed to analyze transforming growth factor (TGF)-β1 mRNA expression. Compared with the normal glucose group, the levels of fibronectin and collagen type were markedly increased after being cultured in high glucose medium. Compared with the high glucose group, remarkable decreases of fibronectin, collagen type IV, α-smooth muscle actin, and TGF-β1 mRNA expression were observed in the H2 relaxin-treated group. The mechanism by which H2 relaxin reduced high glucose-induced overproduction of ECM may be associated with inhibition of TGF-β1 mRNA expression and mesangial cells' phenotypic transition. H2 relaxin is a potentially effective modality for the treatment of DN.