Glomerular Mesangial Matrix Expansion Research Articles

β-cryptoxanthin suppresses oxidative stress via activation of the Nrf2/HO-1 signaling pathway in diabetic kidney disease.

This study aims to explore the role and investigate mechanisms of β-Cryptoxanthin (BCX) in high glucose (HG)-induced podocyte injury and renal dysfunction. In this study, db/db mice were orally treated with BCX. Blood glucose, body weight, urinary albumin creatinine ratio (ACR) were recorded to evaluate the mice renal function. The H&E, PAS staining, and transmission electron microscopy (TEM) were utilized to examine the effect of BCX on the morphological changes of glomeruli in db/db mice. In addition, reactive oxygen species (ROS) content, mitochondrial membrane potential (MMP) level, ATP level, and SA-β-gal staining were used to assess the podocyte oxidative damage, mitochondrial dysfunction and senescence. Furthermore, the effects of BCX on Nrf2/HO-1 signaling pathway were evaluated in vivo and in vitro through Western blotting, immunohistochemistry and immunofluorescence analysis. In vivo, BCX reversed glomerular mesangial matrix expansion and reduced proteinuria in db/db mice, as well as decreased glomerular oxidative stress and kidney aging. Similarly, in vitro study showed that BCX effectively alleviated the oxidative stress, mitochondrial dysfunction, and senescence induced by HG in podocytes. Furthermore, we identified that the antioxidative effects of BCX are associated with the activation of Nrf2/HO-1 signaling pathway, and that Nrf2 knockdown partially abrogated the protective effects of BCX in vitro. Our study demonstrated for the first time that BCX alleviates podocyte injury in DKD by promoting Nrf2/HO-1 signaling pathways. BCX may be a potential candidate compound for preventing Diabetic kidney disease (DKD).

SGLT2i and GLP1-RA exert additive cardiorenal protection with a RAS blocker in uninephrectomized db/db mice.

Diabetic Kidney Disease (DKD) is the main cause of end-stage renal disease in the developed world. The current treatment of the DKD with renin-angiotensin system (RAS) blockade does not totally halt the progression to end stage kidney disease. Currently, several drugs have shown to delay DKD progression such as sodium-glucose co-transporter-2 inhibitors (SGLT2i) and glucagon-like-1 receptor agonists (GLP-1RA). We hypothesized that by combining several drugs that prevent DKD progression on top of RAS blockade a synergistic effect would be achieved in terms of cardiorenal protection. In the present study, we analysed if the combination of a RAS blocker (ramipril) with a SGLT2i (empagliflozin) and/or GLP-1RA (semaglutide) in a type 2 diabetic mouse model could have add-on effects in kidney and heart protection. Male and female uninephrectomized type 2 diabetic db/db mice were treated with empagliflozin and/or semaglutide on top of ramipril during 8weeks. During the study body weight, water and food intake were weekly monitored, glycaemia biweekly and albuminuria and glomerular filtration rate (GFR) before and after the treatment. At the end of the experiment, kidney and heart were isolated for histological and gene expression studies as well as for intrarenal RAS state assessment. Semaglutide combined with ramipril and/or empagliflozin significantly decreased albuminuria but only when combined with both compounds, semaglutide further decreased blood glucose, glomerular hyperfiltration in male mice and glomerular mesangial matrix expansion. In kidney, only the triple treatment with empagliflozin, semaglutide and ramipril reduced the expression of the proinflammatory and profibrotic genes ccl2 and TGFß1. In addition, the combination of empagliflozin and semaglutide on top of RAS blockade was superior in decreasing cardiomyocyte hypertrophy and heart fibrosis in db/db mice. Our results suggest that the combination of SGLT2i with GLP-1RA is superior in cardiorenal protection in DKD than the drugs administered alone on top of RAS blockade.

Repurposing Niclosamide to Modulate Renal RNA-Binding Protein HuR for the Treatment of Diabetic Nephropathy in db/db Mice.

Hu antigen R (HuR) plays a key role in regulating genes critical to the pathogenesis of diabetic nephropathy (DN). This study investigates the therapeutic potential of niclosamide (NCS) as an HuR inhibitor in DN. Uninephrectomized mice were assigned to four groups: normal control; untreated db/db mice terminated at 14 and 22 weeks, respectively; and db/db mice treated with NCS (20 mg/kg daily via i.p.) from weeks 18 to 22. Increased HuR expression was observed in diabetic kidneys from db/db mice, which was mitigated by NCS treatment. Untreated db/db mice exhibited obesity, progressive hyperglycemia, albuminuria, kidney hypertrophy and glomerular mesangial matrix expansion, increased renal production of fibronectin and a-smooth muscle actin, and decreased glomerular WT-1+-podocytes and nephrin expression. NCS treatment did not affect mouse body weight, but reduced blood glucose and HbA1c levels and halted the DN progression observed in untreated db/db mice. Renal production of inflammatory and oxidative stress markers (NF-κBp65, TNF-a, MCP-1) and urine MDA levels increased during disease progression in db/db mice but were halted by NCS treatment. Additionally, the Wnt1-signaling-pathway downstream factor, Wisp1, was identified as a key downstream mediator of HuR-dependent action and found to be markedly increased in db/db mouse kidneys, which was normalized by NCS treatment. These findings suggest that inhibition of HuR with NCS is therapeutic for DN by improving hyperglycemia, renal inflammation, and oxidative stress. The reduction in renal Wisp1 expression also contributes to its renoprotective effects. This study supports the potential of repurposing HuR inhibitors as a novel therapy for DN.

Astaxanthin attenuates diabetic kidney injury through upregulation of autophagy in podocytes and pathological crosstalk with mesangial cells

Objectives: Astaxanthin (ATX) is a strong antioxidant drug. This study aimed to investigate the effects of ATX on podocytes in diabetic nephropathy and the underlying renal protective mechanism of ATX, which leads to pathological crosstalk with mesangial cells.Methods: In this study, diabetic rats treated with ATX exhibited reduced 24-h urinary protein excretion and decreased blood glucose and lipid levels compared to vehicle-treated rats. Glomerular mesangial matrix expansion and renal tubular epithelial cell injury were also attenuated in ATX-treated diabetic rats compared to control rats.Results: ATX treatment markedly reduced the α-SMA and collagen IV levels in the kidneys of diabetic rats. Additionally, ATX downregulated autophagy levels. In vitro, compared with normal glucose, high glucose inhibited LC3-II expression and increased p62 expression, whereas ATX treatment reversed these changes. ATX treatment also inhibited α-SMA and collagen IV expression in cultured podocytes. Secreted factors (vascular endothelial growth factor B and transforming growth factor-β) generated by high glucose-induced podocytes downregulated autophagy in human mesangial cells (HMCs); however, this downregulation was upregulated when podocytes were treated with ATX.Conclusions: The current study revealed that ATX attenuates diabetes-induced kidney injury likely through the upregulation of autophagic activity in podocytes and its antifibrotic effects. Crosstalk between podocytes and HMCs can cause renal injury in diabetes, but ATX treatment reversed this phenomenon.

GTS-21, a selective alpha7 nicotinic acetylcholine receptor agonist, ameliorates diabetic nephropathy in Leprdb/db mice

Diabetic nephropathy (DN) is a serious complicating factor in human type 2 diabetes mellitus (T2DM), and it commonly results in end-stage renal disease (ESRD) that requires kidney dialysis. Here, we report that the α7 nicotinic acetylcholine receptor (α7nAChR) agonist GTS-21 exerts a novel anti-inflammatory action to ameliorate DN, as studied using an inbred strain of Leprdb/db mice in which hyperglycemia and obesity co-exist owing to defective leptin receptor (Lepr) signaling. For this analysis, GTS-21 was administered to 10–12 week-old male and female mice as a 4 mg/kg intraperitoneal injection, twice-a-day, for 8 weeks. Kidney function and injury owing to DN were monitored by determination of plasma levels of BUN, creatinine, KIM-1 and NGAL. Histologic analysis of glomerular hypertrophy and mesangial matrix expansion were also used to assess DN in these mice. Concurrently, renal inflammation was assessed by measuring IL-6 and HMGB1, while also quantifying renal cell apoptosis, and apoptotic signaling pathways. We found that Leprdb/db mice exhibited increased markers of BUN, creatinine, NGAL, KIM-1, IL-6, cytochrome C, and HMGB-1. These abnormalities were also accompanied by histologic kidney injury (mesangial matrix expansion and apoptosis). Remarkably, all such pathologies were significantly reduced by GTS-21. Collectively, our results provide new evidence that the α7nAChR agonist GTS-21 has the ability to attenuate diabetes-induced kidney injury. Additional studies are warranted to further investigate the involvement of the vagal cholinergic anti-inflammatory reflex pathway (CAP) in ameliorating diabetic nephropathy.

Endothelial ADAM17 Expression in the Progression of Kidney Injury in an Obese Mouse Model of Pre-Diabetes

Disintegrin and metalloproteinase domain 17 (ADAM17) activates inflammatory and fibrotic processes through the shedding of various molecules such as Tumor Necrosis Factor-α (TNF-α) or Transforming Growht Factor-α (TGF-α). There is a well-recognised link between TNF-α, obesity, inflammation, and diabetes. In physiological situations, ADAM17 is expressed mainly in the distal tubular cell while, in renal damage, its expression increases throughout the kidney including the endothelium. The aim of this study was to characterize, for the first time, an experimental mouse model fed a high-fat diet (HFD) with a specific deletion of Adam17 in endothelial cells and to analyse the effects on different renal structures. Endothelial Adam17 knockout male mice and their controls were fed a high-fat diet, to induce obesity, or standard rodent chow, for 22 weeks. Glucose tolerance, urinary albumin-to-creatinine ratio, renal histology, macrophage infiltration, and galectin-3 levels were evaluated. Results showed that obese mice presented higher blood glucose levels, dysregulated glucose homeostasis, and higher body weight compared to control mice. In addition, obese wild-type mice presented an increased albumin-to-creatinine ratio; greater glomerular size and mesangial matrix expansion; and tubular fibrosis with increased galectin-3 expression. Adam17 deletion decreased the albumin-to-creatinine ratio, glomerular mesangial index, and tubular galectin-3 expression. Moreover, macrophage infiltration in the glomeruli of obese Adam17 knockout mice was reduced as compared to obese wild-type mice. In conclusion, the expression of ADAM17 in endothelial cells impacted renal inflammation, modulating the renal function and histology in an obese pre-diabetic mouse model.

Glomerulotubular pathology in dogs with subclinical ehrlichiosis.

Subclinical stage of ehrlichiosis is characterized by absence of clinical or laboratory alterations; however, it could lead to silent glomerular/tubular changes and contribute significantly to renal failure in humans and animals. The aim of this study was to evaluate glomerular and tubular alterations in dogs with subclinical ehrlichiosis. We evaluated renal biopsies of 14 bitches with subclinical ehrlichiosis and 11 control dogs. Samples were obtained from the left kidney, and the tissue obtained was divided for light microscopy, immunofluorescence, and transmission electron microscopy. Abnormalities were identified by light microscopy in 92.9% of dogs with ehrlichiosis, but not in any of the dogs of the control group. Mesangial cell proliferation and synechiae (46.1%) were the most common findings, but focal segmental glomerulosclerosis and ischemic glomeruli (38.4%), focal glomerular mesangial matrix expansion (30.7%), mild to moderate interstitial fibrosis and tubular atrophy (23%), and glomerular basement membrane spikes (23%) were also frequent in dogs with ehrlichiosis. All animals with ehrlichiosis exhibited positive immunofluorescence staining for immunoglobulins. Transmission electron microscopy from dogs with ehrlichiosis revealed slight changes such as sparse surface projections and basement membrane double contour. The subclinical phase of ehrlichiosis poses a higher risk of development of kidney damage due to the deposition of immune complexes.

Hyperoside Suppresses Renal Inflammation by Regulating Macrophage Polarization in Mice With Type 2 Diabetes Mellitus.

Accumulating evidence reveals that both inflammation and lymphocyte dysfunction play a vital role in the development of diabetic nephropathy (DN). Hyperoside (HPS) or quercetin-3-O-galactoside is an active flavonoid glycoside mainly found in the Chinese herbal medicine Tu-Si-Zi. Although HPS has a variety of pharmacological effects, including anti-oxidative and anti-apoptotic activities as well as podocyte-protective effects, its underlying anti-inflammatory mechanisms remain unclear. Herein, we investigated the therapeutic effects of HPS on murine DN and the potential mechanisms responsible for its efficacy. We used C57BLKS/6J Lep db/db mice and a high glucose (HG)-induced bone marrow-derived macrophage (BMDM) polarization system to investigate the potentially protective effects of HPS on DN. Our results showed that HPS markedly reduced diabetes-induced albuminuria and glomerular mesangial matrix expansion, accompanied with a significant improvement of fasting blood glucose level, hyperlipidaemia and body weight. Mechanistically, pretreatment with HPS effectively regulated macrophage polarization by shifting proinflammatory M1 macrophages (F4/80+CD11b+CD86+) to anti-inflammatory M2 ones (F4/80+CD11b+CD206+) in vivo and in bone marrow-derived macrophages (BMDMs) in vitro, resulting in the inhibition of renal proinflammatory macrophage infiltration and the reduction in expression of monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor (TNF-α) and inducible nitric oxide synthase (iNOS) while increasing expression of anti-inflammatory cytokine Arg-1 and CD163/CD206 surface molecules. Unexpectedly, pretreatment with HPS suppressed CD4+ T cell proliferation in a coculture model of IL-4-induced M2 macrophages and splenic CD4+ T cells while promoting their differentiation into CD4+IL-4+ Th2 and CD4+Foxp3+ Treg cells. Taken together, we demonstrate that HPS ameliorates murine DN via promoting macrophage polarization from an M1 to M2 phenotype and CD4+ T cell differentiation into Th2 and Treg populations. Our findings may be implicated for the treatment of DN in clinic.

Ginsenoside Rb1 alleviates diabetic kidney podocyte injury by inhibiting aldose reductase activity.

Panax notoginseng, a traditional Chinese medicine, exerts beneficial effect on diabetic kidney disease (DKD), but its mechanism is not well clarified. In this study we investigated the effects of ginsenoside Rb1 (Rb1), the main active ingredients of Panax notoginseng, in alleviating podocyte injury in diabetic nephropathy and the underlying mechanisms. In cultured mouse podocyte cells, Rb1 (10 μM) significantly inhibited high glucose-induced cell apoptosis and mitochondrial injury. Furthermore, Rb1 treatment reversed high glucose-induced increases in Cyto c, Caspase 9 and mitochondrial regulatory protein NOX4, but did not affect the upregulated expression of aldose reductase (AR). Molecular docking analysis revealed that Rb1 could combine with AR and inhibited its activity. We compared the effects of Rb1 with eparestat, a known aldose reductase inhibitor, in high glucose-treated podocytes, and found that both alleviated high glucose-induced cell apoptosis and mitochondrial damage, and Rb1 was more effective in inhibiting apoptosis. In AR-overexpressing podocytes, Rb1 (10 μM) inhibited AR-mediated ROS overproduction and protected against high glucose-induced mitochondrial injury. In streptozotocin-induced DKD mice, administration of Rb1 (40 mg·kg-1·d-1, ig, for 7 weeks) significantly mitigated diabetic-induced glomerular injuries, such as glomerular hypertrophy and mesangial matrix expansion, and reduced the expression of apoptotic proteins. Collectively, Rb1 combines with AR to alleviate high glucose-induced podocyte apoptosis and mitochondrial damage, and effectively mitigates the progression of diabetic kidney disease.

FC 088DUAL BLOCKADE OF ENDOTHELIN A RECEPTOR (ETA) AND SODIUM-GLUCOSE COTRANSPORTER 2 (SGLT2) TO PREVENT DIABETIC KIDNEY DISEASE PROGRESSION ON A TYPE 2 MURINE MODEL

Abstract Background and Aims Sodium-glucose cotransporter 2 inhibitors (SGLT2i) and endothelin A receptor (ETA) antagonist have shown nephroprotective effects in diabetic kidney disease (DKD) through blood pressure and urinary albumin loss reduction. The protective impact and the pathways through which they exert this protection have not yet been elucidated. This study aimed to investigate the effects of the add-on therapy of SGLT2i and ETA antagonists on a type 2 diabetes murine model. Method 12 weeks-old db/db mice were treated for 8 weeks with different combinations of empagliflozin 10 mg/Kg/day (SGLT2i), atrasentan 7 mg/Kg/day (ETA antagonist) or ramipril 8 mg/Kg/day. A group of non-diabetic mice (db/m) was included as negative control. In vivo variables were recorded during treatment, including transdermal measured glomerular filtration rate (GFR) and urinary albumin-to-creatinine ratio (UACR). After treatment kidneys were preserved for histopathological studies. Results After 8 weeks of treatment empagliflozin decreased fasting blood glucose alone or in combination with atrasentan or ramipril (234.2 mg/dL mean reduction in three treated groups when compared to db/db). Ramipril decreased blood pressure (BP) in monotherapy or in add-on therapy. Empagliflozin or atrasentan alone did not have any effect on blood pressure, but combination of atrasentan and ramipril had a synergistic effect and reduced both systolic (9.0 mmHg, CI 95%: -16.3 to -1.1; P=0.028) and diastolic BP (11.9 mmHg, CI 95%: -17.7 to -3.1; P=0.005) when compared to ramipril alone. The combination of atrasentan and ramipril significantly reduced UACR (1002 ug/mg, CI 95%: -2312.0 to -32.4; P=0.043). Empagliflozin treatment alone or in combination also reduced UACR (686.0 ug/mg mean reduction in three treated groups), although this reduction was not statistically significant. In the kidney, empagliflozin in monotherapy or combination reduced glomerular mesangial matrix expansion (4.85% mean mesangial reduction in three treated groups). Treatments with atrasentan and ramipril also reduced measangial matrix expansion. Conclusion Both empagliflozin and atrasentan demonstrated possible beneficial effects in DKD by reducing BP, UACR, and mesangial matrix expansion. The add-on therapy did not show greater protective effects in the analysed variables. Further studies are needed to characterize these protective effects and pathways involved.

ACTIVE IMMUNOTHERAPY AGAINST RECEPTOR FOR ADVANCED GLYCATION END PRODUCTS ATTENUATES THE PROGRESSION OF DIABETIC KIDNEY DISEASE

Objective: To combat global increases in the prevalence of diabetic kidney disease (DKD), we aimed to develop a new therapeutic vaccine targeting receptor for advanced glycation end products (RAGE). Design and method: RAGE vaccine was a water-in-oil emulsion composed of Freund's adjuvant and RAGE partial peptide that was conjugated to the carrier protein KLH. Male DBA2/J mice were subcutaneously immunized with three doses of KLH (vehicle) or RAGE vaccine and subsequently induced diabetes by multiple low-dose streptozotocin. To investigate the renal protective effect of RAGE vaccine, we measured urine albumin to creatinine ratio (UACR) and analyzed renal histology using light and electron microscopy. Results: There was no difference in blood glucose between vehicle-treated mice and vaccinated mice. On the other hand, immunization with the RAGE vaccine significantly attenuated the increase in UACR (vehicle group 850.6 ± 208.6 vs vaccine group 333.6 ± 104.7 ug/mg, n = 10/group, p < 0.05). In the histological analyses, total glomerular volume of vaccinated mice was siginificantly less than that of vehicle-treated mice (vehicle group 5.28 ± 0.52 vs vaccine group 3.18 ± 0.17 um3, p < 0.01) and glomerular mesangial matrix expansion was attenuated in vaccine-treated mice (vehicle group 23.6 ± 1.6 vs vaccine group 18.8 ± 0.8%, p < 0.01). Ultrastructural morphometric study (n = 3/group) also indicated that glomerular basement membrane thickness was significantly suppressed in vaccinated mice compared with vehicle-treated mice (vehicle group 215.3 ± 1.9 vs vaccine group 181.3 ± 1.8 nm, p < 0.01). Conclusions: These results suggest that active immunotherapy targeting RAGE is effective for the attenuation of DKD in streptozotocin-induced diabetic mice.

Piperazine ferulate attenuates high glucose‑induced mesangial cell injury via the regulation of p66Shc.

Diabetic nephropathy (DN) is a severe microvascular complication of diabetes. Hyperglycemia-induced glomerular mesangial cells injury is associated with microvascular damage, which is an important step in the development of DN. Piperazine ferulate (PF) has been reported to exert protective effects against the progression of DN. However, whether PF prevents high glucose (HG)-induced mesangial cell injury remains unknown. The aim of the present study was to investigate the effects of PF on HG-induced mesangial cell injury and to elucidate the underlying mechanisms. Protein and mRNA expression levels were determined via western blot analysis and reverse transcription-quantitative PCR, respectively. IL-6 and TNF-α levels were measured using ELISA. Reactive oxygen species levels and NF-κB p65 nuclear translation were determined via immunofluorescence analysis. Apoptosis was assessed by measuring lactate dehydrogenase (LDH) release, as well as using MTT and flow cytometric assays. The mitochondrial membrane potential of mesangial cells was determined using the JC-1 kit. The results revealed that LDH release were increased; however, cell viability and mitochondrial membrane potential were decreased in the HG group compared with the control group. These changes were inhibited after the mesangial cells were treated with PF. Moreover, PF significantly inhibited the HG-induced production of inflammatory cytokines and the activation of NF-κB in mesangial cells. PF also attenuated the HG-induced upregulation of the expression levels of fibronectin and collagen 4A1. Furthermore, the overexpression of p66Src homology/collagen (Shc) abolished the protective effect of PF on HG-induced mesangial cell injury. In vivo experiments revealed that PF inhibited the activation of inflammatory signaling pathways, glomerular cell apoptosis and mesangial matrix expansion in diabetic mice. Collectively, the present findings demonstrated that PF attenuated HG-induced mesangial cells injury by inhibiting p66Shc.

Highly bioavailable berberine formulation ameliorates diabetic nephropathy through the inhibition of glomerular mesangial matrix expansion and the activation of autophagy

Glomerular mesangial matrix expansion and cell autophagy are the most important factors in the development of kidney damage under diabetic conditions. The activation of AMPK might be an important treatment target for diabetic nephropathy. Berberine has multiple effects on all types of diabetic complications as an activator of AMPK. However, the poor bioavailability of berberine limits its clinical applications. Huang-Gui Solid Dispersion (HGSD), a new formulation of berberine developed in our lab, has 4-fold greater bioavailability than berberine. However, its therapeutic application and mechanism still need to be explored. In the present study, the effect of HGSD on kidney function in type 2 diabetic rats and db/db mice was investigated. The results demonstrated that HGSD improved kidney function in these two animal models, decreased the glomerular volume and increased autophagy. Meanwhile, AMPK phosphorylation levels and autophagy-related protein expression were significantly increased, and extracellular matrix protein deposition-related protein expression was decreased after treatment. The present study indicated that HGSD protected against diabetic kidney dysfunction by inhibiting glomerular mesangial matrix expansion and activating autophagy. The mechanism of HGSD in the treatment of diabetic nephropathy might be connected to the activation of AMPK phosphorylation.

Role of neutrophil extracellular traps in chronic kidney injury induced by bisphenol-A.

Bisphenol-A (BPA) is a common environmental pollutant, and exposure to it is associated with proteinuria and may predict the progression of chronic kidney disease，however, the mechanism is not clear. Neutrophil extracellular traps (NETs) is a DNA skeleton coated with various proteases, and it is associated with various autoimmune nephritis. In this study, we examine whether NETs is involved in BPA-induced chronic kidney injury. In vivo, BPA exposure resulted in impaired renal function and altered renal morphology, including glomerular mesangial matrix expansion and increased renal interstitial fibroblast markers. Meanwhile, more dsDNA can be detected in the serum, and the NETs-associated proteins, MPO and citH3 were deposited in the renal system. In vitro, BPA and NETs treatment caused podocyte injury, a loss of marker proteins, and disorder in the actin skeleton. After NETs inhibition via DNase administration, BPA-induced injuries were significantly relieved. In conclusion, the increase of NETosis in circulation and the renal system during BPA exposure suggests that NETs may be involved in BPA-induced chronic kidney injury.

Sporidiobolus pararoseus wall-broken powder ameliorates oxidative stress in diabetic nephropathy in type-2 diabetic mice by activating the Nrf2/ARE pathway.

In type 2 diabetes mellitus (T2DM), hyperglycemia promotes oxidative stress and eventually leads to diabetic nephropathy (DN). Sporidiobolus pararoseus is reported to exhibit enhanced anti-oxidation properties. However, its role in DN remains obscure. This study aimed to determine the antioxidative effects of a Sporidiobolus pararoseus wall-broken powder (SPP) supplement on DN and investigate the possible underlying mechanisms. A model of T2DM was successfully established, and C57BL/6J male mice were fed a high-fat diet for 4 weeks and then injected with streptozotocin (100 mg per kg per day) for three consecutive days. After eight weeks of intervention, SPP strongly lowered fasting glucose levels, serum creatinine, serum urea nitrogen, urinary albumin and reduced glomerular hypertrophy and mesangial matrix expansion. In addition, SPP increased the activities of SOD, T-AOC, CAT, and GST and decreased the amount of MDA. Furthermore, it was revealed that SPP significantly abrogated oxidative stress not only by activating the Nrf2 gene but also by activating two Nrf2-targeted antioxidative genes (NQO-1 and HO-1) compared with metformin hydrochloride, which is widely accepted as a diabetes drug. Our study showed that SPP has antioxidant properties and delays the progression of DN; the underlying mechanism may be associated with activation of the Nrf2/ARE pathway.

Active vitamin D regulates macrophage M1/M2 phenotypes via the STAT-1-TREM-1 pathway in diabetic nephropathy.

Imbalance of M1/M2 macrophages phenotype activation is a key point in diabetic nephropathy (DN). This study aimed to investigate whether active vitamin D (VD) suppresses macrophage transition to the M1 phenotype via inhibiting the high glucose-induced STAT-1 phosphorylation to reduce TREM-1 expression. In vivo, pathological changes in kidney tissue were detected and the expression of CD68 TREM-1, STAT-1, M1 makers, and M2 makers were acquired in renal tissue of patients with DN and 18w DN rats. In vitro, RAW 264.7 cells were incubated in the presence of high glucose with or without VD. Silencing and overexpression of TREM-1 and silencing and activate of STAT-1 were explored to elucidate the underlying mechanism. The expression of TREM-1 and STAT-1 and the changes of macrophage phenotype were examined separately by western blot and immunofluorescence staining. (a) Expression of TREM-1, p-STAT-1, and M1 markers (iNOS and TNF-α) were increased and positively correlated in kidneys from patients with DN. (b) In DN rats, the enlargement of glomerular surface area, expansion of glomerular mesangial matrix, the expression of CD68, TREM-1, p-STAT-1, and M1 marker (iNOS) were significantly increased in comparison with the normal control group, whereas above changes were markedly decreased in the diabetic group treated with the VD group. (c) In vitro, VD significantly decreased high glucose-induced CD68, TREM-1, p-STAT-1, and M1 marker (iNOS) expression. However, above-mentioned effects of VD are abolished when TREM-1 is overexpressed or STAT-1 is activated. Reductions in STAT-1 expression decreased the TREM-1 expression. VD can inhibit macrophage transition to the M1 phenotype through the STAT-1/TREM-1 pathway.

Luseogliflozin Inhibits HIF-1α Expression in Renal Proximal Tubular Epithelial Cells

Recent clinical trials have demonstrated the renoprotective effects of SGLT2 inhibitors in diabetic nephropathy (DN). However, the mechanisms of SGLT2 inhibitors on prevention of DN have not been fully elucidated. Hypoxia-induced tubulointerstitial fibrosis is considered to be a common pathway for various progressive kidney diseases including DN. Hypoxia inducible factor (HIF)-1α plays an important role in these pathological processes. In this study, we assessed the effects of luseogliflozin, an SGLT2 inhibitor, on HIF-1α expression in both cultured human renal proximal tubular epithelial cells (HRPTECs) and kidney of diabetic mice. Hypoxia (1%O2 for 24h) markedly increased HIF-1α protein in HRPTEC, whereas luseogliflozin inhibited expression of hypoxia-induced HIF-1α protein. In addition, luseogliflozin inhibited mRNA expression for HIF-1α-targeted genes, PAI-1, VEGF and GLUT-1 under hypoxia. The inhibitors of mitochondrial respiratory complex I, rotenone or complex III, antimycin A equally suppressed hypoxia-induced HIF-1α expression. We next treated db/db mice with 15 mg/kg/day luseogliflozin for 8 weeks. Luseogliflozin administration lowered plasma glucose levels and decreased glomerular mesangial matrix expansion as well as glomerular and interstitial fibronectin accumulation in db/db mice. Electron microscopic study revealed that luseogliflozin attenuated the thickening of glomerular basement membrane in db/db mice. Intriguingly, luseogliflozin decreased the length of mitochondria in the renal proximal tubules of the S1 segment. Furthermore, luseogliflozin attenuated diabetes-induced HIF-1α accumulation in the kidney of db/db mice. These results suggest that luseogliflozin inhibits hypoxia-induced HIF-1α accumulation at least partly by suppressing mitochondrial respiration. SGLT2 inhibitors may protect diabetic kidney from hypoxia-induced renal fibrosis by attenuating the expression of HIF-1α and profibrotic molecules. Disclosure R. Bessho: None. Y. Takiyama: None. T. Ota: None.

Intermittent hypoxia causes histological kidney damage and increases growth factor expression in a mouse model of obstructive sleep apnea.

Epidemiological studies demonstrate an association between obstructive sleep apnea (OSA) and accelerated loss of kidney function. It is unclear whether the decline in function is due to OSA per se or to other confounding factors such as obesity. In addition, the structural kidney abnormalities associated with OSA are unclear. The objective of this study was to determine whether intermittent hypoxia (IH), a key pathological feature of OSA, induces renal histopathological damage using a mouse model. Ten 8-week old wild-type male CB57BL/6 mice were randomly assigned to receive either IH or intermittent air (IA) for 60 days. After euthanasia, one kidney per animal was paraformaldehyde-fixed and then sectioned for histopathological and immunohistochemical analysis. Measurements of glomerular hypertrophy and mesangial matrix expansion were made in periodic acid–Schiff stained kidney sections, while glomerular transforming growth factor-β1 (TGF-β1), connective tissue growth factor (CTGF) and vascular endothelial growth factor-A (VEGF-A) proteins were semi-quantified by immunohistochemistry. The antigen-antibody reaction was detected by 3,3′-diaminobenzidine chromogen where the color intensity semi-quantified glomerular protein expression. To enhance the accuracy of protein semi-quantification, the percentage of only highly-positive staining was used for analysis. Levels of TGF-β, CTGF and VEGF-A proteins in the kidney cortex were further quantified by western blotting. Cellular apoptosis was also investigated by measuring cortical antiapoptotic B-cell lymphoma 2 (Bcl-2) and apoptotic Bcl-2-associated X (Bax) proteins by western blotting. Further investigation of cellular apoptosis was carried out by fluorometric terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) staining. Finally, the levels of serum creatinine and 24-hour urinary albumin were measured as a general index of renal function. Our results indicate that mice exposed to IH have an increased glomerular area (by 1.13 fold, p< 0.001) and expansion of mesangial matrix (by 1.8 fold, p< 0.01). Moreover, the glomerular expressions of TGF-β1, CTGF and VEGF-A proteins were 2.7, 2.2 and 3.8-fold higher in mice exposed to IH (p< 0.05 for all). Furthermore, western blotting protein analysis demonstrates that IH-exposed mice express higher levels of TGF-β1, CTGF and VEGF-A proteins by 1.9, 4.0 and 1.6-fold (p< 0.05 for all) respectively. Renal cellular apoptosis was greater in the IH group as shown by an increased cortical Bax/Bcl-2 protein ratio (p< 0.01) and higher fluorometric TUNEL staining (p< 0.001). Finally, 24-hr urinary albumin levels were higher in mice exposed to IH (43.4 μg vs 9.7 μg, p< 0.01), while there were no differences in serum creatinine levels between the two groups. We conclude that IH causes kidney injury that is accompanied by glomerular hypertrophy, mesangial matrix expansion, increased expression of glomerular growth factors and an increased cellular apoptosis.

Urotensin II-induced store-operated Ca2+ entry contributes to glomerular mesangial cell proliferation and extracellular matrix protein production under high glucose conditions

Glomerular mesangial cell (GMC) proliferation and matrix expansion are pathological hallmarks of a wide range of kidney diseases, including diabetic nephropathy. Although the circulating level of peptide hormone urotensin II (UII) and kidney tissue expression of UII and UII receptors (UTR) are increased in diabetic nephropathy, it remains unclear whether UII regulates GMC growth and extracellular matrix (ECM) accumulation. In this study, we tested the hypothesis that UII-induced Ca2+ signaling controls GMC proliferation and ECM production under normal and high glucose conditions. Mouse GMCs cultured under normal glucose conditions proliferated and synthesized ECM proteins in response to stimulation by mouse UII. UII-induced GMC proliferation and ECM protein synthesis were dependent on TRPC4 channel-mediated store-operated Ca2+ entry (SOCE) and sequential activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and Ca2+/cAMP response element-binding protein (CREB) transcription factor. Under high glucose conditions, GMCs synthesized UII. Moreover, proliferation and ECM production in high glucose-challenged GMCs were attenuated by selective UTR antagonist, TRPC4 channel blocker, and CaMKII and CREB-binding protein/p300 inhibitors. These findings indicate that UII-induced SOCE via TRPC4 channels stimulates CaMKII/CREB-dependent GMC proliferation and ECM protein production. Our data also suggest that UII synthesis contributes to GMC proliferation and ECM accumulation under high glucose conditions.

Dapagliflozin slows the progression of the renal and liver fibrosis associated with type 2 diabetes.

Sodium-glucose cotransporter 2 (SGLT2) inhibitors are a new class of antidiabetic oral agents indicating promising effects on cardiovascular and renal end points. However, the renoprotective effects of SGLT2 inhibitors are not fully understood. Also, metabolic effects of SGLT2 inhibition on other organ systems, such as effects on hepatic steatosis, are not fully understood. This study sought to address these questions by treating 18-wk-old uninephrectomized db/db mice with the selective SGLT2 inhibitor dapagliflozin. Untreated db/db mice developed progressive albuminuria, glomerular mesangial matrix expansion, and fatty liver associated with increased renal expression of TGFβ1, PAI-1, type IV collagen and fibronectin, and liver deposition of fibronectin, type I and III collagen, and laminin. Treatment with dapagliflozin (1 mg·kg-1·day-1) via gel diet from 18 to 22 wk of age not only reduced blood glucose (371.14 ± 55.02 mg/dl in treated db/db vs. 573.53 ± 21.73 mg/dl in untreated db/db, P < 0.05) and Hb A1c levels (9.47 ± 0.79% in treated db/db vs. 12.1 ± 0.73% in untreated db/db, P < 0.05) but also ameliorated the increases in albuminuria and markers of glomerulosclerosis and liver injury seen in untreated db/db mice. Furthermore, both renal expressions of NF-kB p65, MCP-1, Nox4, Nox2, and p47phox and urine TBARS levels and liver productions of myeloperoxidase and reactive oxygen species, the markers of tissue inflammation and oxidative stress, were increased in untreated db/db mice, which were reduced by dapagliflozin administration. These results demonstrate that dapagliflozin not only improves hyperglycemia but also slows the progression of diabetes-associated glomerulosclerosis and liver fibrosis by improving hyperglycemia-induced tissue inflammation and oxidative stress.