Mesangial Matrix Accumulation Research Articles

Scutellarin ameliorates diabetic nephropathy via TGF-β1 signaling pathway

Breviscapine, a natural flavonoid mixture derived from the traditional Chinese herb Erigeron breviscapus (Vant.) Hand-Mazz, has demonstrated a promising potential in improving diabetic nephropathy (DN). However, the specific active constituent(s) responsible for its therapeutic effects and the underlying pharmacological mechanisms remain unclear. In this study, we aimed to investigate the impact of scutellarin, a constituent of breviscapine, on streptozotocin-induced diabetic nephropathy and elucidate its pharmacological mechanism(s). Our findings demonstrate that scutellarin effectively ameliorates various features of DN in vivo, including proteinuria, glomerular expansion, mesangial matrix accumulation, renal fibrosis, and podocyte injury. Mechanistically, scutellarin appears to exert its beneficial effects through modulation of the transforming growth factor-β1 (TGF-β1) signaling pathway, as well as its interaction with the extracellular signal-regulated kinase (Erk) and Wnt/β-catenin pathways.Graphical

Phillyrin alleviates high glucose-induced oxidative stress and inflammation in HBZY-1 cells through inhibition of the PI3K/Akt signaling pathway.

Phillyrin, the major lignin compound of Forsythia suspense (Thunb.) Vahl, has been shown the effects of anti-inflammatory and antioxidant. Our study was aimed to explore the protective effect of phillyrin on glomerular mesangial cells (HBZY-1) and the potential mechanism. Cell viability, cytokine production, levels of reactive oxygen radicals (ROS), glutathione (GSH), malondialdehyde (MDA), and superoxide dismutase (SOD), as well as autophagy and apoptosis levels were determined to verify the mechanism of phillyrin on HBZY-1 cells. Our result indicated that phillyrin significantly inhibited HG-induced HBZY-1 proliferation by inhibiting Bcl-2 expression and upregulating Bad, cleaved caspase-3, and -9 expression. Also, phillyrin suppressed HG-induced mesangial extracellular matrix accumulation by inhibiting the expression of fibronectin and transforming growth factor-β1. Further, phillyrin inhibited oxidative stress and inflammation by decreasing ROS, MDA, TNF-α, IL-1β, and IL-6 contents and increasing SOD and GSH expression. Phillyrin also promoted autophagy by increasing LC3-II/LC3-I ratio and down-regulating p62 expression. Furthermore, WB assay showed that phillyrin inhibited oxidative stress caused by HG via activating Nrf2 signaling pathway, while attenuated proliferation and inflammation in HBZY-1 cells through inactivating PI3K/Akt/mTOR and NF-κB pathways. All results showed that phillyrin might be a promising therapeutic agent for the treatment of DN.

Development of angiogenic periglomerular microvessels after acute glomerular lesions in IgA nephropathy.

To clarify the clinicopathological characteristics and role of periglomerular angiogenesis in IgA nephropathy. The renal biopsy specimens of 114 patients with IgA nephropathy were examined. Among them, 46 (40%) showed periglomerular angiogenesis around the glomeruli. CD34 and α-smooth muscle actin (α-SMA) staining in serial sections revealed that these vessels contained CD34+ α-SMA+ microarterioles along with CD34+ α-SMA- capillaries. We termed these "periglomerular microvessels (PGMVs)". Patients with PGMVs (PGMV group) had clinically and histologically more severe disease than those without PGMVs (non-PGMV group) at the time of biopsy. Even after adjusting for age, there were significant differences in the degree of proteinuria and estimated glomerular filtration rate reduction between the PGMV and non-PGMV groups. The PGMV group showed a higher incidence of segmental and global glomerulosclerosis and crescentic lesions than the non-PGMV group (P < 0.01). Here, PGMVs were undetectable in the acute and active inflammation phase, but were observed in the acute to chronic or chronic glomerular remodelling phase. PGMVs mainly developed around glomerular adherent lesions to the Bowman's capsule with small or minimal glomerular sclerotic lesions. Conversely, they were rarely observed in segmental sclerosis areas. The PGMV group is clinically and pathologically more severe than the non-PGMV group; however, they were undetectable in segmental sclerosis with mesangial matrix accumulation. PGMVs might occur after acute/active glomerular lesions, suggesting that PGMVs may inhibit segmental glomerulosclerosis progression and could be a marker for good repair response after acute/active glomerular injury in severe IgA nephropathy cases.

IL-17C neutralization protects the kidney against acute injury and chronic injury.

Interleukin-17C (IL-17C), a member of the IL-17 cytokine family, plays a pathogenic role in kidney diseases. Our previous studies have shown that pre-administration of IL-17C neutralizing antibody attenuated acute kidney injury (AKI, a common acute inflammation associated renal disease). In this study, we explored whether post-ischemia reperfusion (IR) of IL-17C blockade has therapeutic effects on AKI and whether IL-17C is involved in the pathogenesis of diabetic nephropathy (DN), a major type of chronic inflammation-associated kidney disease. 12-week-old male C57BL/6JGpt mice were treated with IL-17C neutralizing antibody or normal IgG control antibody at 3h after reperfusion. Renal injury, inflammation, and oxidative stress were assessed. Additionally, we examined renal IL-17C expression in patients with DN and db/db mice and evaluated albuminuria, mesangial matrix accumulation and podocyte loss in db/db mice with IL-17C neutralization. Knockdown of NF-κB p65 using siRNA, and blocking Hypoxia-inducible factor-1α (HIF-1α) using YC-1 in mice and HIF-1α Decoy in HK2 cells were investigated to explore the possible signaling pathway involved in IL-17C regulation. We found that delayed IL-17C neutralization had similar reno-protective effects on renal ischemia-reperfusion injury (IRI). Additionally, renal IL-17C expression was increased in patients with DN and db/db mice, while IL-17C blockade significantly attenuated DN, accompanied with blunted albuminuria, mesangial matrix accumulation, and podocyte loss. Moreover, IL-17C neutralization significantly repressed the expression of downstream pro-inflammatory cytokines, inflammatory cell infiltration, and Th17/IL-17A activation both in mice with renal IRI and DN. Mechanistical studies demonstrated that hypoxia or high glucose-induced IL-17C up-regulation was predominantly mediated by NF-κB pathway. IL-17C participates in the pathogenesis of AKI and DN and inhibition of IL-17C shows potential as a therapeutic strategy for AKI and DN. The National Natural Science Foundation of China (81770741, 81700601 and 81870504).

Scutellarin ameliorates diabetic nephropathy via TGF-β1 signaling pathway.

Breviscapine, a natural flavonoid mixture derived from the traditional Chinese herb Erigeron breviscapus (Vant.) Hand-Mazz, has demonstrated a promising potential in improving diabetic nephropathy (DN). However, the specific active constituent(s) responsible for its therapeutic effects and the underlying pharmacological mechanisms remain unclear. In this study, we aimed to investigate the impact of scutellarin, a constituent of breviscapine, on streptozotocin-induced diabetic nephropathy and elucidate its pharmacological mechanism(s). Our findings demonstrate that scutellarin effectively ameliorates various features of DN in vivo, including proteinuria, glomerular expansion, mesangial matrix accumulation, renal fibrosis, and podocyte injury. Mechanistically, scutellarin appears to exert its beneficial effects through modulation of the TGF-β1 signaling pathway, as well as its interaction with the Erk and Wnt/β-catenin pathways.

Protective effect and mechanism of electroacupuncture of "Biao-Ben" acupoints combination for mitochondrial dysfunction in diabetic nephropathy rats

To study the protective effect of electroacupuncture (EA) at "Biao"-acupoints, "Fenglong"(ST40) and "Zhongwan"(CV12), used for treating symptoms of the disease, and "Ben"-acupoints, "Zusanli"(ST36) and "Guanyuan"(CV4), for treating the root cause of the disease on oxidative stress injury of renal mitochondria through SIRT1/PGC-1α signal pathway in rats with diabetic nephropathy (DN). A total of 33 male Wistar rats were randomized into normal (n=10), model (n=12) and EA (n=11) groups.The DN model was established by feeding the rats with high-sugar and high-fat diet for 6 weeks combined with streptozotocin (STZ, 35 mg/kg, i.p.). EA (4 Hz/60 Hz, 1 mA)was applied to ST36-ST40 and CV4-CV12 for 15 min, once every other day for 8 weeks. The rats' body weight was recorded, urine in 24 hours (24-h UP) was collected to measure the urine protein level, and the fasting blood glucose (FBG) level detected by using a glucometer. The levels of serum glycosylated hemoglobin (HbA1c), creatinine (Scr) and urea nitrogen (BUN) were assayed using immunoturbidi-metry, picric acid method and urease method, respectively, and those of serum triglyceride (TG), total cholesterol (TC), low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol (HDL-C) detected using an automatic biochemical analyzer. The kidney tissue was collected for assaying the activity of superoxide dismutase (SOD) with xanthine oxidase method, glutathione (GSH) activity with dithio-dinitrobenzoic acid method, catalase （CAT） activity with ammonium molybdate spectrometric method, and malondialdehyde (MDA) content with thiobarbituric acid method. Histopathological changes of the kidney tissue were observed by microscope after hematoxylin-eosin staining (HE), periodate Schiff staining (PAS) and Masson staining, separately, and its subcellular structure was observed under transmission electron microscopy. The expression levels of renal SIRT1 and PGC-1α mRNAs and proteins were detected by quantitative real-time PCR and Western blot, and the immunoactivity of renal α-smooth muscle actin (α-SMA), and immunofluorescence density of renal collagen Ⅰ (Col Ⅰ), collagen Ⅳ(Col Ⅳ) and fibronec-tin (FN) detected by immunohistochemistry and immunofluorescence assay, respectively. Compared with the normal group, the levels of FBG, HbA1c, BUN, Scr, 24-h UP, TG, TC, LDL-C, MDA, α-SMA, Col Ⅰ, Col Ⅳ and FN proteins were significantly increased (P<0.01), and the levels of body weight, HDL-C, SOD, GSH, CAT, SIRT1 and PGC-1α mRNAs and proteins were decreased in the model group (P<0.01, P<0.05). In comparison with the model group, the levels of FBG, HbA1c, BUN, Scr, 24-h UP, TG, TC, LDL-C and MDA, and the expressions of α-SMA, Col Ⅰ, Col Ⅳ and FN proteins were markedly down-regulated (P<0.05, P<0.01), and those of body weight, HDL-C, SOD, GSH, CAT, and the expressions of SIRT1 and PGC-1α mRNAs and proteins significantly up-regulated (P<0.01, P<0.05) in the EA group. HE staining showed mesangial dilatation, glomerular hypertrophy and mesangial matrix accumulation; PAS staining showed an increase of the glomerular extracellular matrix deposition; and Masson staining displayed an enhancement of glomerular fibrosis and interstitial space expansion; and electron microscope revealed foot process fusion, basement membrane thickening and organelle injury in the rat's kidney of the model group, which were relatively milder in the EA group. EA of ST36-ST40 and CV4-CV12, "Biao-Ben" acupoints combination, can alleviate oxidative stress and mitochondrial dysfunction in DN rats, which may be associated with its functions in up-regulating SIRT1/PGC-1α signaling, and decreasing renal fibrosis.

Krill Oil Turns Off TGF-β1 Profibrotic Signaling in the Prevention of Diabetic Nephropathy.

Diabetic nephropathy (DN), a severe microvascular complication of diabetes mellitus (DM), results in high mortality due to the lack of effective interventions. The current study investigated the preventive effect of krill oil (KO) on DN using a type 2 DM mouse model induced by streptozotocin and high-fat diet for 24 weeks. The diabetic mice developed albuminuria, mesangial matrix accumulation, glomerular hypertrophy, and fibrosis formation, with an increase in renal proinflammatory, oxidative and profibrotic gene expression. KO significantly prevented these effects but did not improve hyperglycemia and glucose intolerance. In high-glucose-treated mesangial cells (MCs), KO preferably modulated TGF-β1 signaling as revealed by RNA-sequencing. In TGF-β1-treated MCs, KO abolished SMAD2/3 phosphorylation and nuclear translocation and activated Smad7 gene expression. The action of KO on the SMADs was confirmed in the diabetic kidneys. Therefore, KO may prevent DN predominantly by suppressing the TGF-β1 signaling pathway.

Yishen Capsule Alleviated Symptoms of Diabetic Nephropathy via NOD-like Receptor Signaling Pathway

PurposeTo explore the mechanism of Yishen capsule against diabetic nephropathy (DN) based on the analysis of transcriptomics.Material and MethodsSD rats (Male, SPF grade) were randomly divided into four groups, the normal group, the DN group, the Yishen capsule group and the resveratrol group. Urine and renal tissue samples were collected after feeding with physiological saline and above drugs for 8 weeks. 24-hour urine microalbumin protein was detected by ELISA. HE staining and PAS staining were performed on renal tissues. Differential gene expression in renal tissues was analyzed by transcriptome sequencing. The differentially expressed genes were analyzed by GO enrichment and KEGG enrichment, and verified by RT-PCR and immunohistochemistry staining.ResultsThe level of 24-hour urinary microalbumin in DN group was increased, while Yishen capsule treatment reversed the increasement of urinary microalbumin. Mesangial cell proliferation, matrix accumulation, edema and vacuolar degeneration of renal tubular epithelial cells and glycogen accumulation were observed in DN group. However, pathological phenotypes mentioned above were alleviated after Yisen capsule administration. This result indicates that Yishen capsule reversed pathological phenotypes of DN in rats. The expression of 261 genes were changed in Yishen capsule group compared with DN group. GO enrichment analysis and KEGG pathway analysis showed that these genes were implicated in pathways, including mineral absorption, adipocytokine signaling pathway, fatty acid biosynthesis, thyroid hormone synthesis, renin–angiotensin system, and NOD-like receptor signaling pathway. Based on previous reported study, the expression of key factors in NOD-like receptor signaling pathway was verified. RT-PCR and immunohistochemistry staining showed that the expression of NLRP3, Caspase-1 and IL-1β in renal tissues of DN group were increased (P < 0.05), which were decreased in Yishen capsule group (P < 0.05).ConclusionYishen capsule reduced microalbuminuria and alleviated pathological changes in DN rats, which may be achieved by regulating NOD-like receptor signaling pathway.

MO405: Leucine-Rich Alpha-2-Glycoprotein as a Potential Marker of Mesangial Cell Proliferation in Immunoglobulin a Nephropathy

Abstract BACKGROUND AND AIMS Leucine-rich alpha-2-glycoprotein (LRG1) is a novel proangiogenic factor involved in the abnormal angiogenesis and renal fibrosis in diabetic nephropathy by potentiating endothelial transforming growth factor-beta/activin receptor-like kinase 1 (TGF-β/ALK1) signalling [1]. TGF-β mediates mesangial matrix accumulation culminating in the development of glomerulosclerosis [2], which also occurs in immunoglobulin A nephropathy (IgAN) patients. Our study aims to evaluate LRG1 as a marker of mesangial cell proliferation in IgAN patients. METHODS: Adults with a morphologically confirmed IgAN by the presence of IgA deposits by immunofluorescence microscopy were included in the study. Diabetes mellitus, oncology, acute inflammation processes were exclusion criteria. Serum LRG1 was detected using ELISA. RESULTS A total of 73 patients with IgAN were included [mean age 41 years, interquartile range (IQR) 35–47; range, 21–65; 65.8% men]. The median estimated glomerular filtration rate (eGFR) was 44 (IQR 24–83, range 4–133) mL/min/1.73m2. Kidney biopsies were analyzed according to the Oxford classification: M0 in 10 (13,7%), M1 in 56 (76.7%) patients. The median serum LRG1 was 49 µg/mL (IQR 40.7–57.7, range 29–133.3). Bivariate analyses showed that plasma LRG1 was negatively associated with eGFR (rho = −0.342, P = 0.003). A linear model showed that GFR decline for 1 mL/min was associated with an increase of LRG1 for 0.194 µg/mL. In IgAN patients with an M1-score LRG1 was higher (P = 0027, Mann–Whitney U test), the area under the ROC curve value for serum LRG1 was 0.739. CONCLUSION Serum LRG1 increases with GFR decline in patients with IgAN and at the same time correlates with mesangial hypercellularity according to Oxford classification. Therefore, LRG1 might be a potential marker of advanced disease in IgAN.

Loss of urotensin II receptor diminishes hyperglycemia and kidney injury in streptozotocin-treated mice.

Beyond the CNS, urotensin II (UII) and its receptor (UT) are functionally expressed in peripheral tissues of the endocrine, cardiovascular, and renal systems. The expression levels of UII and UT in the kidney and circulating UII levels are increased in diabetes. UII also promotes mesangial proliferation and matrix accumulation in vitro. Here, we evaluate the effect of UT deletion on the development of hyperglycemia and diabetic kidney disease (DKD) in streptozotocin (STZ)-treated mice. Ten-week-old WT and UT knockout (KO) mice were injected with STZ for 5 days to induce diabetes. Blood glucose levels were measured weekly, and necropsy was performed 12 weeks after STZ injection. UT ablation slowed hyperglycemia and glucosuria in STZ-treated mice. UT KO also ameliorated STZ-induced increase in HbA1c, but not STZ-induced decrease in plasma insulin levels. However, STZ-induced increases in plasma glucagon concentration and immunohistochemical staining for glucagon in pancreatic islets were lessened in UT KO mice. UT ablation also protected against STZ-induced kidney derangements, including albuminuria, mesangial expansion, glomerular lesions, and glomerular endoplasmic reticulum stress. UT is expressed in a cultured pancreatic alpha cell line, and its activation by UII triggered membrane depolarization, T- and L-type voltage-gated Ca2+channel-dependent Ca2+influx, and glucagon secretion. These findings suggest that apart from direct action on the kidneys to cause injury, UT activation by UII may result in DKD by promoting hyperglycemia via induction of glucagon secretion by pancreatic alpha cells.

Regression of diabetic nephropathy by treatment with empagliflozin in BTBR ob/ob mice.

The sodium-glucose cotransporter-2 (SGLT2) inhibitor empagliflozin lowers blood glucose via reduced tubular reabsorption of filtered glucose and is an important new therapy for diabetic nephropathy (DN). This study tested whether treatment with empagliflozin would ameliorate proteinuria and the pathologic alterations of DN including podocyte number and integrity in the leptin-deficient BTBR ob/ob mouse model of DN. Study cohorts included wild-type (WT) BTBR mice, untreated diabetic BTBR ob/ob mice and mice treated with empagliflozin for 6 weeks after development of established DN at 18 weeks of age. Hyperglycemia, proteinuria, serum creatinine, accumulation of mesangial matrix and the extent of mesangiolysis were reversed with empagliflozin treatment. Treatment with empagliflozin resulted in an increased podocyte number and podocyte density, improvement in the degree of podocyte foot process effacement and parietal epithelial cell activation. SGLT2 inhibition reduced renal oxidative stress, measured by urinary excretion of markers of RNA/DNA damage and in situ demonstration of decreased carbonyl oxidation. There was no discernable difference in accumulations of advanced glycation end-products by immunohistochemistry. The structural improvements seen in BTBR ob/ob mice treated with empagliflozin provide insights into potential long-term benefits for humans with DN, for whom there is no comparable biopsy information to identify structural changes effected by SGLT2 inhibition. The findings suggest SGLT2 inhibition may ameliorate DN through glucose lowering-dependent and -independent mechanisms that lead to podocyte restoration and delay or reversal of disease progress.

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.

IL-17C Neutralization Protects the Kidney Against Acute Injury and Chronic Injury

Background: Interleukin-17C (IL-17C), a member of the IL-17 cytokine family, plays a pathogenic role in kidney diseases. Our previous studies have shown that pre-administration of IL-17C neutralizing antibody protected against acute renal ischemia/reperfusion injury (IRI) in mice. However, whether post-IR of IL-17C blockade has therapeutic effects on AKI and whether IL-17C is involved in the pathogenesis of diabetic nephropathy (DN), a major type of chronic kidney disease, remain unknown. Methods: 12-week-old male C57BL/6JGpt mice were treated with IL-17C neutralizing antibody or normal IgG1 control antibody at 3 hours after reperfusion. Renal injury, inflammation and oxidative stress were assessed. Additionally, we examined renal IL-17C expression in patients with DN and db/db mice and evaluated albuminuria, mesangial matrix accumulation and podocyte loss in db/db mice with IL-17C neutralization. To further explore the possible signaling pathway involved in IL-17C regulation, NF-κB and HIF-1α pathways were investigated in vitro and in vivo. Findings: Renal dysfunction, oxidative stress and renal tubular injury were remarkably blunted by post-IR administration of an IL-17C neutralizing antibody in mice with renal IRI. Additionally, we found that renal IL-17C expression was significantly increased in patients with DN and db/db mice while IL-17C blockade significantly attenuated diabetic nephropathy, accompanied with blunted albuminuria, mesangial matrix accumulation and podocyte loss. Moreover, IL-17C neutralization significantly repressed the expression of downstream proinflammatory cytokines, inflammatory cells infiltration and Th17/IL-17A activation both in mice with renal IRI and DN. Mechanistical studies demonstrated that hypoxia and high glucose both resulted in IL-17C upregulation in renal tubular cells through NF-κB pathway other than HIF-1α pathway. Interpretation: IL-17C participates in the pathogenesis of AKI and DN and inhibition of IL-17C shows potential as a therapeutic strategy for AKI and DN. Funding Information: This work was supported by the National Natural Science Foundation of China (81770741, 81700601 and 81870504). Declaration of Interests: The authors have declared that no conflict of interest exists. Ethics Approval Statement: Protocols involving the use of human medical information and kidney biopsy samples were approved by the Ethics Committee of Shanghai Jiao Tong University Affiliated Sixth People’s Hospital. The study was conducted in accordance with the World Medical Association Declaration of Helsinki, and all subjects signed the informed consent. All animal experiments and procedures were approved by the Institutional Animal Care and Use Committee of Shanghai Jiao Tong University Affiliated Sixth People’s Hospital.

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.

The effects of carbon monoxide releasing molecules on paraquat-induced pulmonary interstitial inflammation and fibrosis

Paraquat, an herbicide used extensively worldwide, can cause severe toxicity in humans and animals, leading to irreversible, lethal lung fibrosis. The potential of CO-releasing molecules (CORMs), substances that release CO (Carbon monoxide) within animal tissues, for treating paraquat-induced ROS generation and inflammation is investigated here. Our results show that the fast CO releaser CORM-3 (4-20 μM) acts as a potential scavenger of free radicals and decreases fibrosis progression by inhibiting paraquat-induced overexpression of connective tissue growth factor and angiotensin II in MRC-5 cells. The slow CO releaser CORM-A1 (5 mg/kg) clearly decreased expression of the lung profibrogenic cytokines COX-2, TNF-α, and α-SMA and serum hydroxyproline, resulting in a lower mortality rate in paraquat-treated mice. Mice treated with higher-dose CORM-A1 (10 mg/kg) had relatively intact lung lobes and fewer fibrotic patches by gross observation, with less collagen deposition, mesangial matrix accumulation, and pulmonary fibrosis resulting from the mitigation of TGF-β overexpression.In conclusion, our data demonstrate for the first time that CORM-A1 alleviated the development of the fibrotic process and improved survival rate in mice exposed to PQ, would be an attractive therapeutic approach to attenuate the progression of pulmonary fibrosis following PQ exposure.

Salvianolate ameliorates oxidative stress and podocyte injury through modulation of NOX4 activity in db/db mice.

Podocyte injury is associated with albuminuria and the progression of diabetic nephropathy (DN). NADPH oxidase 4 (NOX4) is the main source of reactive oxygen species (ROS) in the kidney and NOX4 is up‐regulated in podocytes in response to high glucose. In the present study, the effects of Salvianolate on DN and its underlying mechanisms were investigated in diabetic db/db mice and human podocytes. We confirmed that the Salvianolate administration exhibited similar beneficial effects as the NOX1/NOX4 inhibitor GKT137831 treated diabetic mice, as reflected by attenuated albuminuria, reduced podocyte loss and mesangial matrix accumulation. We further observed that Salvianolate attenuated the increase of Nox4 protein, NOX4‐based NADPH oxidase activity and restored podocyte loss in the diabetic kidney. In human podocytes, NOX4 was predominantly localized to mitochondria and Sal B treatment blocked HG‐induced mitochondrial NOX4 derived superoxide generation and thereby ameliorating podocyte apoptosis, which can be abrogated by AMPK knockdown. Therefore, our results suggest that Sal B possesses the reno‐protective capabilities in part through AMPK‐mediated control of NOX4 expression. Taken together, our results identify that Salvianolate could prevent glucose‐induced oxidative podocyte injury through modulation of NOX4 activity in DN and have a novel therapeutic potential for DN.

Short-Chain Fatty Acids Ameliorate Diabetic Nephropathy via GPR43-Mediated Inhibition of Oxidative Stress and NF-κB Signaling.

Diabetic nephropathy (DN) is a chronic low-grade inflammatory disease. Oxidative stress and nuclear factor kappa B (NF-κB) signaling play an important role in the pathogenesis of DN. Short-chain fatty acids (SCFAs) produced from carbohydrate fermentation in the gastrointestinal tract exert positive regulatory effects on inflammation and kidney injuries. However, it is unclear whether SCFAs can prevent and ameliorate DN. In the present study, we evaluated the role and mechanism of the three main SCFAs (acetate, propionate, and butyrate) in high-fat diet (HFD) and streptozotocin- (STZ-) induced type2 diabetes (T2D) and DN mouse models and in high glucose-induced mouse glomerular mesangial cells (GMCs), to explore novel therapeutic strategies and molecular targets for DN. We found that exogenous SCFAs, especially butyrate, improved hyperglycemia and insulin resistance; prevented the formation of proteinuria and an increase in serum creatinine, urea nitrogen, and cystatin C; inhibited mesangial matrix accumulation and renal fibrosis; and blocked NF-κB activation in mice. SCFAs also inhibited high glucose-induced oxidative stress and NF-κB activation and enhanced the interaction between β-arrestin-2 and I-κBα in GMCs. Specifically, the beneficial effects of SCFAs were significantly facilitated by the overexpression GPR43 or imitated by a GPR43 agonist but were inhibited by siRNA-GPR43 in GMCs. These results support the conclusion that SCFAs, especially butyrate, partially improve T2D-induced kidney injury via GPR43-mediated inhibition of oxidative stress and NF-κB signaling, suggesting SCFAs may be potential therapeutic agents in the prevention and treatment of DN.

Protective effects of Citrus limonia oil against cisplatin-induced nephrotoxicity.

Cisplatin is broadly used in the treatment of malignancies. However, the high incidence of nephrotoxicity following cisplatin use deters its clinical utility. Former studies have shown that the essential oils, obtained from Citrus limonia demonstrated significant anti-inflammatory and antioxidant effects. The aim of the current work was to evaluate the protective effects of Citrus limonia oil against cisplatin-induced nephrotoxicity. Thirty-two adult male mice were divided into four groups, eight mice each. The control group received distilled water, and the second group received a single intraperitoneal injection of cisplatin (20mg/kg), while the third and fourth groups received cisplatin plus Citrus limonia oil at 100 or 200mg/kg for 10days, respectively. GC-MS analysis showed that the major components in Citrus limonia oil were D-limonene, 5-methyl-pentadecane, (n)-menthol, 3,7-dimethyl-(E)-2,6-octadienal, 3,7-dimethyl-2,6-octadienal, and nonadecane. Biochemical analysis showed that cisplatin intoxication was associated with significantly increased (p < 0.05) serum levels of urea and creatine and pro-inflammatory cytokines, as well as augmented renal tissue oxidative stress. Light microscopic examination showed loss of renal architecture, atrophied glomeruli, interstitial hemorrhage, dilated cortical tubules with cast formation, and excessive collagen production. Electron microscopic examination revealed compressed and karyorrhectic endothelial nuclei with chromatin condensation in the glomeruli, accumulation of mesangial matrix, and obliteration of glomerular blood capillaries. Co-administration of Citrus limonia oil attenuated these effects in renal histopathological, morphometric, and ultrastructural examinations, frequently in a dose-dependent manner. In conclusion, Citrus limonia oil can ameliorate the toxic effect of cisplatin on mice kidneys, probably through its antioxidant and anti-inflammatory effects.

Classification of glomerular pathological findings using deep learning and nephrologist–AI collective intelligence approach

BackgroundAutomated classification of glomerular pathological findings is potentially beneficial in establishing an efficient and objective diagnosis in renal pathology. While previous studies have verified the artificial intelligence (AI) models for the classification of global sclerosis and glomerular cell proliferation, there are several other glomerular pathological findings required for diagnosis, and the comprehensive models for the classification of these major findings have not yet been reported. Whether the cooperation between these AI models and clinicians improves diagnostic performance also remains unknown. Here, we developed AI models to classify glomerular images for major findings required for pathological diagnosis and investigated whether those models could improve the diagnostic performance of nephrologists. MethodsWe used a dataset of 283 kidney biopsy cases comprising 15,888 glomerular images that were annotated by a total of 25 nephrologists. AI models to classify seven pathological findings: global sclerosis, segmental sclerosis, endocapillary proliferation, mesangial matrix accumulation, mesangial cell proliferation, crescent, and basement membrane structural changes, were constructed using deep learning by fine-tuning of InceptionV3 convolutional neural network. Subsequently, we compared the agreement to truth labels between majority decision among nephrologists with or without the AI model as a voter. ResultsOur model for global sclerosis showed high performance (area under the curve: periodic acid-Schiff, 0.986; periodic acid methenamine silver, 0.983); the models for the other findings also showed performance close to those of nephrologists. By adding the AI model output to majority decision among nephrologists, out of the 14 constructed models, the results of the majority decision showed improvement in sensitivity for 10 models (four of them were statistically significant) and specificity for eight models (five significant). ConclusionOur study showed a proof-of-concept for the classification of multiple glomerular findings in a comprehensive method of deep learning and suggested its potential effectiveness in improving diagnostic accuracy of clinicians.

Restoring mitochondrial superoxide levels with elamipretide (MTP-131) protects db/db mice against progression of diabetic kidney disease

Exposure to chronic hyperglycemia because of diabetes mellitus can lead to development and progression of diabetic kidney disease (DKD). We recently reported that reduced superoxide production is associated with mitochondrial dysfunction in the kidneys of mouse models of type 1 DKD. We also demonstrated that humans with DKD have significantly reduced levels of mitochondrion-derived metabolites in their urine. Here we examined renal superoxide production in a type 2 diabetes animal model, the db/db mouse, and the role of a mitochondrial protectant, MTP-131 (also called elamipretide, SS-31, or Bendavia) in restoring renal superoxide production and ameliorating DKD. We found that 18-week-old db/db mice have reduced renal and cardiac superoxide levels, as measured by dihydroethidium oxidation, and increased levels of albuminuria, mesangial matrix accumulation, and urinary H2O2 Administration of MTP-131 significantly inhibited increases in albuminuria, urinary H2O2, and mesangial matrix accumulation in db/db mice and fully preserved levels of renal superoxide production in these mice. MTP-131 also reduced total renal lysocardiolipin and major lysocardiolipin subspecies and preserved lysocardiolipin acyltransferase 1 expression in db/db mice. These results indicate that, in type 2 diabetes, DKD is associated with reduced renal and cardiac superoxide levels and that MTP-131 protects against DKD and preserves physiological superoxide levels, possibly by regulating cardiolipin remodeling.