Mesangial Matrix Accumulation Research Articles

Low molecular weight fucoidan alleviates diabetic nephropathy by binding fibronectin and inhibiting ECM-receptor interaction in human renal mesangial cells

Diabetic nephropathy (DN) is the most common cause of end-stage renal disease (ESRD). Currently, approximately 20–40% of individuals with diabetes are diagnosed with DN. Mesangial cells (MCs) are critical for maintaining and regulating glomerular filtration, and the abnormal proliferation of MCs causes the accumulation of mesangial extracellular matrix (ECM), further promoting glomerular dysfunction and renal diseases. Low molecular weight fucoidan (LMWF) extracted from Saccharina japonica could alleviate DN, but the mechanism was not analysed. Based on the ability of LMWF to ameliorate the human renal mesangial cell (HRMC) injury caused by advanced glycation end products (AGEs), we identified fibronectin (FN) as the most obviously impacted protein in the ECM-receptor interaction by proteomic analysis. The co-localization of LMWF and FN indicated direct interaction between them, and surface plasmon resonance (SPR) analysis confirmed the specific binding with a KD of 453.7 μmol L−1. Positively charged protamine sulfate (PS) promoted the combination of LMWF and HRMCs and further enhanced the effect of LMWF on HRMC injury. Our results indicated that LMWF alleviates the HRMC injury caused by AGEs via binding FN and inhibiting the ECM-receptor interaction pathway. These results provide a foundation for the in-depth analysis of the mechanism of polysaccharide functions.

Dietary nobiletin suppresses TGF-β1-Src-caveolin-1 dependent signaling involved with high glucose-induced renal mesangial matrix accumulation

Dietary nobiletin suppresses TGF-β1-Src-caveolin-1 dependent signaling involved with high glucose-induced renal mesangial matrix accumulation

The effectiveness of chitosan-mediated silencing of PDGF-B and PDGFR-β in the mesangial proliferative glomerulonephritis therapy

Platelet-derived growth factor-B (PDGF-B) is a growth factor that plays an important role in the progression of mesangial proliferative glomerulonephritis (MsPGN). PDGF-B may contribute to mesangioproliferative changes and is overexpressed in MsPGN. Recently, small interfering RNAs (siRNAs) have been widely used for gene silencing effects in experimental models of renal diseases. Nanoparticle-based therapeutics are preferred for reasons such as increasing therapeutic efficacy and reducing toxic effects caused by high doses. The distribution of nanoparticles to the kidney is a significant advantage in siRNA delivery. The aim of this study was to investigate the efficacy of chitosan/siRNA nanoplexes in silencing of PDGF-B and PDGFR-β genes in kidney and to decrease mesangial cell proliferation and matrix accumulation in MsPGN model induced by anti-Thy-1.1 antibody. The therapeutic effects of chitosan/siPDGF-B + siPDGFR-β nanoplexes in glomerulonephritic rats were studied by molecular, biochemical, and histopathologic evaluations. Chitosan/siPDGF-B + siPDGFR-β nanoplexes markedly reduced PDGF-B and PDGFR-β mRNA and protein expressions in experimental MsPGN model. Histopathologic examination results showed that the silencing of PDGF-B and its receptor PDGFR-β led to reduction in mesangial cell proliferation and matrix accumulation. The use of chitosan/siPDGF-B + siPDGFR-β nanoplexes for silencing the PDGF-B pathway in MsPGN can be considered as a new effective therapeutic strategy.

Chronic exposure to arsenic and high fat diet induces sex-dependent pathogenic effects on the kidney

Both obesity and arsenic exposure are global public health problems that are associated with increased risk of renal disease. The effect of whole-life exposure to environmentally relevant levels of arsenic within dietary high fat diet on renal pathogenesis were examined. In this study, C57BL/6 J mice were parentally exposed to 100 ppb arsenic before conception. After weaning, both male and female offspring were maintained on 100 ppb arsenic and fed either a normal (LFD) or high fat diet (HFD). At 10 and 24 weeks of age, the offspring were sacrificed and kidneys collected. Exposure to arsenic led to an increase body-weight in LFD diet-fed female but not male mice. This response was not observed in HFD-fed female mice; however male mice showed significant increases in body weight in both As- and non-treated animals. Histological analysis shows that arsenic exposure significantly increases HFD-induced glomerular area expansion, mesangial matrix accumulation and fibrosis compared to LFD control animals. HFD alone increases renal inflammation and fibrosis; reflected by increases in IL-1β, ICAM-1 and fibronectin levels. Arsenic exposure significantly increases HFD-induced inflammatory and oxidative stress responses. In general, male mice have more severe responses than female mice to HFD or arsenic treatment. These results demonstrate that arsenic exposure causes sex-dependent alterations in HFD-induced kidney damage.

512-P: Sexual Differences in Progression of Diabetic Nephropathy in over 26 Type 1 Diabetic Mice

As a transgenic type 1 diabetic model, OVE26 mice develop diabetic nephropathy (DN) with the feature close to advanced DN in human, which makes it a valuable rodent model to study DN. Nevertheless, sexual differences in renal lesions, especially dynamic progression with different ages, remain undefined in OVE26 mice. To explore these issues, male and female OVE26 and wild type nondiabetic FVB mice at 4-, 12-, 24 - and 36-weeks old were used (N=6). Urine albumin-to-creatinine ratio (UACR) was detected to evaluate renal function. Renal histopathology and immunochemical and Western blotting protein expression of inflammation, tubular epithelial-myofibroblast transdifferentiation and fibrosis markers were examined. In FVB mice, blood glucose, triglyceride, UACR and podocytes numbers were constant during aging and comparable in male and female. Age-dependent slightly increase of glomerular mesangial matrix, inflammatory cells infiltration and renal fibrosis were observed in FVB mice. Compared with the age- and sex- matched FVB mice, male OVE26 mice developed hyperglycemia, mesangial matrix accumulation and increased trend of triglyceride at 4-week-old and albuminuria, glomerular enlargement, and upregulations of TGF-β1, α-SMA at 12 weeks old. Significant loss of podocytes, tubular dilation, collagen accumulation, increased trend of macrophage and T lymphocytes infiltrations, and increased protein levels of TNF-α, ICAM and fibronectin in kidney were observed in 24-week old male OVE26 mice. Compared with age-matched male OVE26 mice, female OVE26 mice had significant hypertriglyceridemia, worse renal function and kidney lesions at older age, including higher UACR and sustained loss of podocytes at age of 24 weeks; more inflammatory cells infiltration and renal fibrosis at age of 36 weeks. Our findings suggest the sexual difference in the progression of DN in OVE26 mice to provide evidence for researchers to properly select the age and sex of OVE26 mice in the future type 1 DN study. Disclosure W. Wang: None. L. Cai: None.

Metanephric mesenchyme-derived Foxd1+ mesangial precursor cells alleviate mesangial proliferative glomerulonephritis.

Mesangial proliferative glomerulonephritis (MsPGN) is a glomerular disease characterized by the proliferation of mesangial cells and the accumulation of mesangial matrix. No effective treatment is currently able to stop or reverse the disease process. Here, we isolated metanephric mesenchyme-derived Foxd1+ mesangial precursor cells from E13.5 Foxd1Cre; DTRflox double transgenic embryo mice. The Foxd1+ cells showed the cell-specific expression of high levels of Foxd1 without Six2 and manifested specific cell surface markers of mesenchymal stem cells while retaining their differentiation potential. Next, an anti-Thy1 MsPGN rat model was established, and the Foxd1+ cells were injected into the tail veins 24h later. We found that the Foxd1+ cells could improve the pathological changes to the kidney and significantly reduce proteinuria by inhibiting the sonic hedgehog pathway. Moreover, the Foxd1+ cells could inhibit PDGF-BB-induced activation of mesangial cells by secreting multiple cytokines, including hepatocyte growth factor. Our study uncovered the novel function of Foxd1+ mesangial precursor cells in repairing the damaged mesangium, stabilizing the structure and function of mesangial cells, and restoring their therapeutic effect on the MsPGN. KEY MESSAGES: It is viable to isolate highly purified metanephric mesenchyme-derived Foxd1+ mesangial precursor cells using transgenic mice. Foxd1+ cells could alleviate experimental mesangial proliferative glomerulonephritis and PDGF-induced mesangial cell proliferation through a variety of mechanisms including inhibiting the sonic hedgehog pathway and secreting multiple cytokines. As the progenitor cells of mesangial cells, Foxd1+ cells could stabilize the structure and function of mesangial cells that had undergone pathological changes.

Ramipril can alleviate the accumulation of renal mesangial matrix in rats with diabetic nephropathy by inhibiting insulin-like growth factor-1.

Purpose To investigate the impact of Ramipril (RAM) on the expressions of insulin-like growth factor-1 (IGF-1) and renal mesangial matrix (RMM) in rats with diabetic nephropathy (DN). Methods The Sprague Dawley rats were divided into normal control (NC) group (n = 12), DN group (n = 11), and DN+RAM group (n = 12). The ratio of renal weight to body weight (RBT), fasting blood glucose (FBG), HbA1c, 24-h urine protein (TPU), blood urea nitrogen (BUN), creatinine (Cr), renal pathological changes, the levels of IGF-1, fibronectin (FN), type IV collagen (Col-IV), and matrix metalloproteinases (MMP)-2 were compared among the groups. Results Compared with NC group, the RBT, FBG, HbA1c, TPU, BUN, Cr, and RMM in DN group were significantly increased (P < 0.05), the IGF-1, FN, and Col-IV were significantly upregulated (P < 0.05), while MMP was significantly downregulated (P < 0.05). Compared with DN group, the indexes except for the FBG and HbA1c in DN+RAM group were significantly improved (P < 0.05), among which IGF-1 exhibited significant positive correlation with TPU(r=0.937), FN(r=0.896) and Col-IV(r=0.871), while significant negative correlation with MMP-2 (r=-0.826) (P<0.05). Conclusion RAM may protect the kidneys by suppressing IGF-1 and mitigating the accumulation of RMM.

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.

EP4 knockdown alleviates glomerulosclerosis through Smad and MAPK pathways in mesangial cells

Prostaglandin E2 has exhibited pleiotropic effects in the regulation of glomerulosclerosis progression through its four receptors. The current study aimed to evaluate the effect of prostaglandin receptor EP4 on mesangial cell proliferation. Invivo, 5/6 nephrectomy was introduced into EP4+/‑ and wild‑type (WT) mice. Clinical parameters were monitored post‑surgery. At 8weeks post‑surgery, glomerular fibrosis‑associated indicators were measured by immunohistochemical staining and trichrome staining. Invitro, mesangial cells in different groups (transfected with green fluorescent protein, AD‑EF4 or AD‑CRE) were exposed to transforming growth factor (TGF)‑β1 for 24h to detect the level of downstream signaling. Corresponding signaling inhibitors were also used to validate the signaling effects. Following surgery, EP4+/‑ mice presented a higher survival rate and normal urine volume compared with the WT group, and serum creatinine level and 24h urine protein were lower in the EP4+/‑ mice. Furthermore, associated profibrotic indicators were identified to have decreased at 8weeks post‑surgery along with less tubule‑interstitial fibrosis. Invivo, the inhibition of extracellular signal‑regulated kinase and P38 phosphorylation alleviated the accumulation of mesangial matrix, and these signals were enhanced when EP4 was overexpressed. EP4 enhancement aggravated imbalanced mesangial cell proliferation stimulated by TGF‑β1 and GS of mice treated with 5/6 nephrectomy through the Smad and mitogen‑activated protein kinase pathways.

Oxidative stress mediated mitochondrial damage plays roles in pathogenesis of diabetic nephropathy rat.

Diabetic nephropathy (DN) is considered as a complication of diabetes and accounts for about 40%. Reactive oxygen species (ROS) level continues to increase in DN. However, whether specific ROS levels can alleviate renal damage by improving mitochondrial function has not been investigated. DN model was established by intraperitoneal injection of streptozotocin (STZ). The rats were divided into normal group, STZ model group, and N-acetylcysteine (NAC) group. Fasting blood glucose was tested to assess the modeling. The renal injury was evaluated by using hematoxylin-eosin (HE) staining and periodate-Schiff staining. Serum creatinine and 24 h urinary protein levels were determined by renal function detection kit. The levels of ROS and malondialdehyde (MDA) were assessed by the kit to evaluate the effects of oxidative stress and NAC in rats. The mitochondrial damage marker Cyto C level was detected by Western blot. Blood glucose, serum creatinine, and urinary protein levels were significantly increased in model group compared with the normal group (p<0.05). Blood glucose levels, serum creatinine, and urinary protein levels were markedly improved after the ROS and MDA levels reduced by NAC. Meanwhile, glomerular hypertrophy, mesangial matrix accumulation, and severe renal injury were observed in the model group. NAC administration markedly improved glomerular morphology and mesangial matrix aggregation. Cyto C expression in the model group increased significantly compared with normal control group (p<0.05), while NAC effectively inhibited Cyto C levels. Oxidative stress-mediated mitochondrial damage is an important part of the pathogenesis of DN. Inhibition of ROS production can be a potential target for DN treatment.

Liquiritigenin attenuates high glucose-induced mesangial matrix accumulation, oxidative stress, and inflammation by suppression of the NF-κB and NLRP3 inflammasome pathways

Oxidative stress, inflammation, and hyperglycemia are considered to play crucial roles in the pathogenesis and progression of diabetic nephropathy (DN). Liquiritigenin, one of the flavonoid compounds, has been shown to possess anti-inflammatory, anti-hyperlipidemic, and anti-oxidative properties. Our study aimed to explore the effects of liquiritigenin on high glucose (HG)-induced extracellular matrix (ECM) accumulation, oxidative stress and inflammatory response and delineate the underlying mechanism. In our study, glomerular mesangial cells (HBZY-1) were co-treated with various doses of liquiritigenin and HG. We found that HG, but not normal glucose or mannitol, promoted the proliferation of HBZY-1 cells, which was suppressed by liquiritigenin. Liquiritigenin inhibited HG-induced ECM accumulation in HBZY-1 cells by reducing the expressions and production of collagen IV (Col IV) and fibronectin (FN). Moreover, liquiritigenin attenuated HG-induced oxidative stress, as evidenced by the decreased MDA content and NADPH oxidase 4 (NOX4) expression, and the increased SOD activity in HBZY-1 cells. Liquiritigenin suppressed HG-induced inflammatory response, as demonstrated by the reduced expressions and secretion of interleukin (IL)-6 and IL-1β in HBZY-1 cells. Furthermore, we found that liquiritigenin inhibited HG-induced activation the nuclear factor-kappa B (NF-κB) and nod-like receptor protein 3 (NLRP3) inflammasome pathways. In conclusion, these results demonstrated that liquiritigenin attenuated HG-induced ECM accumulation, oxidative stress, and inflammation by suppression of the NF-κB and NLRP3 inflammasome pathways, suggesting that liquiritigenin might be a promising therapeutic agent for preventing the development of DN.

Potential benefits and harms of NADPH oxidase type 4 in the kidneys and cardiovascular system.

The main function of NADPH oxidases is to catalyse the formation of reactive oxygen species (ROS). NADPH oxidase 4 (NOX4) is expressed at high levels in kidney tubular cells, and at lower levels in endothelial cells, cardiomyocytes and other cell types under physiological conditions. NOX4 is constitutively active producing hydrogen peroxide (H2O2) as the prevalent ROS detected, whereas other NOX isoforms present in the renal and cardiovascular systems (i.e. NOX1, NOX2 and NOX5) generate superoxide radical anions as main products. Pharmacological inhibition of NOX4 has received enormous attention for its potential therapeutic benefit in fibrotic disease and nephropathologies. Ongoing clinical trials are testing this approach in humans. Diabetes elevates NOX4 expression in podocytes and mesangial cells, which was shown to damage glomeruli leading to podocyte loss, mesangial cell hypertrophy and matrix accumulation. Consequently, NOX4 represents an interesting therapeutic target in diabetic nephropathy. On the contrary, experiments using NOX4-deficient mice have shown that NOX4 is cytoprotective in tubular cells, cardiomyocytes, endothelial cells and vascular smooth muscle cells, and has a metabolism-regulating role when these cells are subjected to injury. Mice with systemic NOX4 deletion are more susceptible to acute and chronic tubular injury, heart failure and atherosclerosis. Overall, the current literature suggests a detrimental role of increased NOX4 expression in mesangial cells and podocytes during diabetic nephropathy, but a cytoprotective role of this enzyme in other cellular types where it is expressed endogenously. We review here the recent evidence on the role of NOX4 in the kidneys and cardiovascular system. With the emergence of pharmacological NOX4 inhibitors in clinical trials, caution should be taken in identifying potential side effects in patients prone to acute kidney injury and cardiovascular disease.

Deletion of pro-angiogenic factor vasohibin-2 ameliorates glomerular alterations in a mouse diabetic nephropathy model

Angiogenesis has been implicated in glomerular alterations in the early stage of diabetic nephropathy. We previously reported the renoprotective effects of vasohibin-1 (VASH1), which is a novel angiogenesis inhibitor derived from endothelial cells, on diabetic nephropathy progression. Vasohibin-2 (VASH2) was originally identified as a VASH1 homolog and possesses pro-angiogenic activity in contrast to VASH1. In addition, VASH2 was recently shown to promote epithelial-to-mesenchymal transition via enhanced transforming growth factor (TGF)-β signaling in cancer cells. Herein, we investigated the pathogenic roles of VASH2 in diabetic nephropathy using VAHS2-deficient mice. The type 1 diabetes model was induced by intraperitoneal injections of streptozotocin in VASH2 homozygous knockout (VASH2LacZ/LacZ) or wild-type mice. These mice were euthanized 16 weeks after inducing hyperglycemia. Increased urine albumin excretion and creatinine clearance observed in diabetic wild-type mice were significantly prevented in diabetic VASH2-deficient mice. Accordingly, diabetes-induced increase in glomerular volume and reduction in glomerular slit-diaphragm density were significantly improved in VASH2 knockout mice. Increased glomerular endothelial area was also suppressed in VASH2-deficient mice, in association with inhibition of enhanced vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2), but not VEGF level. Furthermore, glomerular accumulation of mesangial matrix, including type IV collagen, and increased expression of TGF-β were improved in diabetic VASH2 knockout mice compared with diabetic wild-type mice. Based on the immunofluorescence findings, endogenous VASH2 localization in glomeruli was consistent with mesangial cells. Human mesangial cells (HMCs) were cultured under high glucose condition in in vitro experiments. Transfection of VASH2 small interfering RNA (siRNA) into the HMCs resulted in the suppression of type IV collagen production induced by high glucose compared with control siRNA. These results indicate that VASH2 may be involved in diabetes-induced glomerular alterations, particularly impaired filtration barrier and mesangial expansion. Therefore, VASH2 is likely to represent a promising 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

EP4 inhibition attenuates the development of diabetic and non-diabetic experimental kidney disease

The therapeutic targeting of prostanoid subtype receptors may slow the development of chronic kidney disease (CKD) through mechanisms that are distinct from those of upstream COX inhibition. Here, employing multiple experimental models of CKD, we studied the effects of inhibition of the EP4 receptor, one of four receptor subtypes for the prostanoid prostaglandin E2. In streptozotocin-diabetic endothelial nitric oxide synthase knockout mice, EP4 inhibition attenuated the development of albuminuria, whereas the COX inhibitor indomethacin did not. In Type 2 diabetic db/db mice, EP4 inhibition lowered albuminuria to a level comparable with that of the ACE inhibitor captopril. However, unlike captopril, EP4 inhibition had no effect on blood pressure or hyperfiltration although it did attenuate mesangial matrix accumulation. Indicating a glucose-independent mechanism of action, EP4 inhibition also attenuated proteinuria development and glomerular scarring in non-diabetic rats subjected to surgical renal mass ablation. Finally, in vitro, EP4 inhibition prevented transforming growth factor-ß1 induced dedifferentiation of glomerular podocytes. In rodent models of diabetic and non-diabetic CKD, EP4 inhibition attenuated renal injury through mechanisms that were distinct from either broadspectrum COX inhibition or “standard of care” renin angiotensin system blockade. EP4 inhibition may represent a viable repurposing opportunity for the treatment of CKD.

Angiopoietins and diabetic nephropathy.

Diabetic nephropathy is the main cause of end-stage renal failure in the Western world. In diabetes, metabolic and haemodynamic perturbations disrupt the integrity of the glomerular filtration barrier, leading to ultrastructural alterations of the glomeruli, including podocyte foot process fusion and detachment, glomerular basement membrane thickening, reduced endothelial cell glycocalyx, and mesangial extracellular matrix accumulation and glomerulosclerosis, ultimately leading to albuminuria and end-stage renal disease. Many vascular growth factors, such as angiopoietins, are implicated in glomerular biology. In normal physiology angiopoietins regulate the function of the glomerular filtration barrier. When they are dysregulated, however, as they are in diabetes, they drive the cellular mechanisms that mediate diabetic glomerular pathology. Modulation of angiopoietins expression and signalling has been proposed as a tool to correct the cellular mechanisms involved in the pathophysiology of diabetic microvascular disease, such as retinopathy in humans. Future work might evaluate whether this novel therapeutic approach should be extended to diabetic kidney disease.

Ultrastructural changes of kidney in diabetic rats.

To demonstrate histopathological changes in diabetic kidneys of the rat on ultrastructural level with focusing on glomerular cellular environment. Male Wistar rats were administered streptozotocin and after 8 weeks, their kidneys were studied using qualitative transmission electron microscope analysis. In diabetic kidneys, the presence of irregularly thickened glomerular membrana basalis (GBM), often up to six times as compared to healthy kidney GBM, mainly around peripheral capillary loops, was striking. We also demonstrated lamination and split appearance of GBM, with presence of delicate fibrillar structure, which was absent within normal GBM. Accumulation of mesangial matrix was found only occasionally. Model may be useful to better understand the mechanisms that play role in the progression of DM-induced renal disease and to comprehend the morphological changes, which this process leaves within the tissue. The clear association between ultra-structure and functional markers may not be necessary (Fig. 4, Ref. 27).

Identification of cysteinylated transthyretin, a predictive biomarker of treatment response to partially hydrolyzed guar gum in type 2 diabetes rats, by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry.

Recent evidence has indicated that total fiber intake is inversely related to type 2 diabetes risk. The present study aimed to investigate the effects of chronic administration of partially hydrolyzed guar gum (PHGG), a water-soluble dietary fiber, on the occurrence of diabetes and its complications, fatty liver and nephropathy. We also identified predictive serum biomarkers of treatment response to PHGG by mass spectroscopy-based proteomic analysis using Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a good model of human non-insulin-dependent diabetes mellitus. In this study, at 5 weeks of age, OLETF rats and control strain Long-Evans Tokushima Otsuka (LETO) rats were fed a control diet or a high-fiber diet (5% PHGG) for 57 weeks. Body weight, food intake, oral glucose tolerance test, plasma insulin levels, and urine glucose and protein levels were regularly measured. Oral glucose tolerance tests (OGTT) and storage of serum in a deep freezer were conducted at the beginning of the experiment and every 4 weeks after overnight fasting during the experiments. PHGG treatment affected neither meal patterns nor the body weight of OLETF and LETO rats. Repeated measure analysis of variance revealed significant differences in fasting plasma glucose and plasma glucose at 2 h after OGTT between control OLETF (OLETF-C) rats and OLETF rats treated with PHGG (OLETF-F). The glucose response determined by the area under the curve of OGTT was significantly greater in OLETF-C rats than that in OLETF-F rats at 25 weeks of age. HOMA-IR, an index of insulin resistance, increased at 25 weeks of age in OLETF-C rats, while this increase was significantly inhibited in OLETF-F rats. At 62 weeks of age, PHGG treatment significantly improved hepatic steatosis as well as renal mesangial matrix accumulation in OLETF rats. To identify the risk marker for diabetes mellitus by SELDI-TOF MS, we collected sera from 21-week-old individuals. Among the 12 specific peaks that were risk marker candidates for diabetes mellitus, the m/z 13,720 peak was identified as that of cysteinylated transthyretin by sequencing of four tryptic peptides using tandem mass spectrometry and peak distribution around the m/z 13,720 peak in the SELDI-TOF spectra. In conclusion, we found that chronic treatment with PHGG improved insulin resistance, delayed the onset of diabetes, and inhibited the development of diabetic complications, as well as identified cysteinylated transthyretin as a predictive biomarker of treatment response to PHGG in OLETF rats.