Atractylodes lancea and Magnolia officinalis combination protects against high fructose-impaired insulin signaling in glomerular podocytes through upregulating Sirt1 to inhibit p53-driven miR-221.

ETHNOPHARMACOLOGICAL RELEVANCE: In traditional Chinese medicine, a long term of improper diet causes the Dampness and disturbs Zang-Fu's functions including Kidney deficiency. Atractylodes lancea (Atr) and Magnolia officinalis (Mag) as a famous herb pair are commonly used to transform Dampness, with kidney protection. AIM OF THE STUDY: To explore how Atr and Mag protected against insulin signaling impairment in glomerular podocytes induced by high dietary fructose feeding, a major contributor for insulin resistance in glomerular podocyte dysfunction. MATERIALS AND METHODS: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyze constituents of Atr and Mag. Rat model was induced by 10% fructose drinking water in vivo, and heat-sensitive human podocyte cells (HPCs) were exposed to 5 mM fructose in vitro. Animal or cultured podocyte models were treated with different doses of Atr, Mag or Atr and Mag combination. Western blot, qRT-PCR and immunofluorescence assays as well as other experiments were performed to detect adiponectin receptor protein 1 (AdipoR1), protein kinase B (AKT), Sirt1, p53 and miR-221 levels in rat glomeruli or HPCs, respectively. RESULTS: Fifty-five components were identified in Atr and Mag combination. Network pharmacology analysis indicated that Atr and Mag combination might affect insulin signaling pathway. This combination significantly improved systemic insulin resistance and prevented glomerulus morphological damage in high fructose-fed rats. Of note, high fructose decreased IRS1, AKT and AdipoR1 in rat glomeruli and cultured podocytes. Further data from cultured podocytes with Sirt1 inhibitor/agonist, p53 agonist/inhibitor, or miR-221 mimic/inhibitor showed that high fructose downregulated Sirt1 to stimulate p53-driven miR-221, resulting in insulin signaling impairment. Atr and Mag combination effectively increased Sirt1, and decreased p53 and miR-221 in in vivo and in vitro models. CONCLUSIONS: Atr and Mag combination improved insulin signaling in high fructose-stimulated glomerular podocytes possibly through upregulating Sirt1 to inhibit p53-driven miR-221. Thus, the regulation of Sirt1/p53/miR-221 by this combination may be a potential therapeutic approach in podocyte insulin signaling impairment.

SSBP1 drives high fructose-induced glomerular podocyte ferroptosis via activating DNA-PK/p53 pathway.

High fructose consumption is a significant risking factor for glomerular podocyte injury. However, the causes of high fructose-induced glomerular podocyte injury are still unclear. In this study, we reported a novel mechanism by which high fructose induced ferroptosis, a newly form of programmed cell death, in glomerular podocyte injury. We performed quantitative proteomic analysis in glomeruli of high fructose-fed rats to identify key regulating proteins involved in glomerular injury, and found that mitochondrial single-strand DNA-binding protein 1 (SSBP1) was markedly upregulated. Depletion of SSBP1 could alleviate high fructose-induced ferroptotic cell death in podocytes. Subsequently, we found that SSBP1 positively regulated a transcription factor p53 by interacting with DNA-dependent protein kinase (DNA-PK) and p53 to drive ferroptosis in high fructose-induced podocyte injury. Mechanically, SSBP1 activated DNA-PK to induce p53 phosphorylation at serine 15 (S15) to promote the nuclear accumulation of p53, and thereby inhibited expression of ferroptosis regulator solute carrier family 7 member 11 (SLC7A11) in high fructose-exposed podocytes. Natural antioxidant pterostilbene was showed to downregulate SSBP1 and then inhibit DNA-PK/p53 pathway in its alleviation of high fructose-induced glomerular podocyte ferroptosis and injury. This study identified SSBP1 as a novel intervention target against high fructose-induced podocyte ferroptosis and suggested that the suppression of SSBP1 by pterostilbene may be a potential therapy for the treatment of podocyte ferroptosis in glomerular injury.

Atractylodis rhizoma water extract attenuates fructose-induced glomerular injury in rats through anti-oxidation to inhibit TRPC6/p-CaMK4 signaling.

BACKGROUND: Atractylodis rhizoma, an aromatic herb for resolving dampness, is used to treat Kidney-related edema in traditional Chinese medicine for thousands years. This herb possesses antioxidant effect. However, it is not yet clear how Atractylodis rhizoma prevents glomerular injury through its anti-oxidation. PURPOSE: Based the analysis of Atractylodis rhizoma water extract (ARE) components and network pharmacology, this study was to explore whether ARE prevented glomerular injury via its anti-oxidation to inhibit oxidative stress-driven transient receptor potential channel 6 (TRPC6) and its downstream molecule calcium/calmodulin-dependent protein kinase IV (CaMK4) signaling. METHODS: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to analyze ARE components. Network pharmacology analysis was preliminarily performed. Male Sprague-Dawley rats were given 10% fructose drinking water (100 mL/d) for 16 weeks. ARE at 720 and 1090 mg/kg was orally administered to rats for the last 8 weeks. Hydrogen peroxide (H2O2) and malondialdehyde (MDA) level, and superoxide dismutase (SOD) activity in rat kidney cortex were detected, respectively. In rat glomeruli, redox-related factors forkhead box O3 (FoxO3), SOD2 and catalase (CAT), podocyte slit diaphragm proteins podocin and nephrin, cytoskeleton proteins CD2-associated protein (CD2AP) and α-Actinin-4, as well as TRPC6, p-CaMK4 and synaptopodin protein levels were analyzed by Western Blotting. SOD2 and CAT mRNA levels were detected by qRT-PCR. RESULTS: 36 components were identified in ARE. Among them, network pharmacology analysis indicated that ARE might inhibit kidney oxidative stress. Accordingly, ARE up-regulated nuclear FoxO3 expression, and then increased SOD2 and CAT at mRNA and protein levels in glomeruli of fructose-fed rats. It reduced H2O2 and MDA levels, and increased SOD activity in renal cortex of fructose-fed rats. Subsequently, ARE down-regulated TRPC6 and p-CaMK4, and up-regulated synaptopodin in glomeruli of fructose-fed rats. Furthermore, ARE increased podocin and nephrin, as well as CD2AP and α-Actinin-4, being consistent with its reduction of urine albumin-to-creatinine ratio and improvement of glomerular structure injury in this animal model. CONCLUSIONS: These results suggest that ARE may prevent glomerular injury in fructose-fed rats possibly by reducing oxidative stress to inhibit TRPC6/p-CaMK4 signaling and up-regulate synaptopodin expression. Therefore, ARE may be a promising drug for treating high fructose-induced glomerular injury in clinic.

IL-6/STAT3 signaling activation exacerbates high fructose-induced podocyte hypertrophy by ketohexokinase-A-mediated tristetraprolin down-regulation.

Glomerular hypertrophy is a crucial factor of severe podocyte damage and proteinuria. Our previous study showed that high fructose induced podocyte injury. The current study aimed to explore a novel molecular mechanism underlying podocyte hypertrophy induced by high fructose. Here we demonstrated for the first time that high fructose significantly initiated the hypertrophy in rat glomeruli and differentiated human podocytes (HPCs). Consistently, it induced inflammatory response with the down-regulation of anti-inflammatory factor zinc-finger protein tristetraprolin (TTP) and the activation of interleukin-6 (IL-6)/signal transducer and activator of transcription 3 (STAT3) signaling in these animal and cell models. Subsequently, high-expression of microRNA-92a-3p (miR-92a-3p) and its target protein cyclin-dependent kinase inhibitor p57 (P57) down-regulation, representing abnormal proliferation and apoptosis, were observed in vivo and in vitro. Moreover, high fructose increased ketohexokinase-A (KHK-A) expression in rat glomeruli and differentiated HPCs. Exogenous IL-6 stimulation up-regulated IL-6/STAT3 signaling and miR-92a-3p, reduced P57 expression and promoted podocyte proliferation, apoptosis and hypertrophy in vitro. The data from anti-inflammatory agent maslinic acid treatment or TTP siRNA transfection showed that high fructose may decrease TTP to activate IL-6/STAT3 signaling in podocyte overproliferation and apoptosis, causing podocyte hypertrophy. Whereas, KHK-A siRNA transfection remarkably restored high fructose-induced TTP down-regulation, IL-6/STAT3 signaling activation, podocyte overproliferation, apoptosis and hypertrophy in differentiated HPCs. Taken together, these results suggested that high fructose possibly increased KHK-A expression to down-regulate TTP, subsequently activated IL-6/STAT3 signaling to interfere with podocyte proliferation and apoptosis by up-regulating miR-92a-3p to suppress P57 expression, causing podocyte hypertrophy. Therefore, the inactivation of IL-6/STAT3 to relieve podocyte hypertrophy mediated by inhibiting KHK-A to increase TTP may be a novel strategy for high fructose diet-associated podocyte injury and proteinuria.

Polydatin enhances glomerular podocyte autophagy homeostasis by improving Nrf2-dependent antioxidant capacity in fructose-fed rats.

High fructose is considered a causative factor for oxidative stress and autophagy imbalance that cause kidney pathogenesis. Antioxidant polydatin isolated from Polygonum cuspidatum has been reported to protect against kidney injury. In this study, polydatin was found to ameliorate fructose-induced podocyte injury. It activated mammalian target of rapamycin complex 1 (mTORC1) and suppressed autophagy in glomeruli of fructose-fed rats and in fructose-exposed conditionally immortalized human podocytes (HPCs). Polydatin also enhanced nuclear factor-E2-related factor 2 (Nrf2)-dependent antioxidant capacity to suppress fructose-induced autophagy activation in vivo and in vitro, with the attenuation of fructose-induced up-regulation of cellular light chain 3 (LC3) II/I protein levels. This effect was abolished by Raptor siRNA in fructose-exposed HPCs. These results demonstrated that polydatin ameliorated fructose-induced autophagy imbalance in an mTORC1-dependent manner via improving Nrf2-dependent antioxidant capacity during podocyte injury. In conclusion, polydatin with anti-oxidation activity suppressed autophagy to protect against fructose-induced podocyte injury.

Pterostilbene alleviates fructose-induced renal fibrosis by suppressing TGF-ß1/TGF-ß type I receptor/Smads signaling in proximal tubular epithelial cells.

High dietary fructose is a key causative factor in the development of renal fibrosis. Pterostilbene has anti-fibrotic effect. Understanding the action mechanism of pterostilbene in fructose-induced renal fibrosis remains as a challenge. Here, fructose feeding was found to promote the progress of epithelial-to-mesenchymal transition (EMT) of proximal tubule epithelial cells (PTECs) and collagen deposition in renal cortex of rats with tubulointerstitial fibrosis. Simultaneously, it impaired insulin receptor (IR)/insulin receptor substrate-1 (IRS-1)/protein kinase B (Akt) pathway, and increased transforming growth factor-beta 1 (TGF-ß1) and TGF-ß type I receptor to enhance phosphorylation of drosophila mothers against decapentaplegic homolog 2 (Smad2) and Smad3, and Smad4 expression in rat kidney cortex. These changes were also observed in cultured PTECs HK-2 cells exposed to 5 mM fructose. The data from fructose-exposed HK-2 cells co-incubated with TGF-ß type I receptor inhibitor further demonstrated that the activation of TGF-ß1/TGF-ß type I receptor/Smads signaling promoted renal tubular EMT and collagen accumulation. Pterostilbene was found to ameliorate fructose-induced renal fibrosis in rats. Importantly, pterostilbene improved IR/IRS-1/Akt pathway impairment and suppressed TGF-ß1/TGF-ß type I receptor/Smads signaling activation in vivo and in vitro, being consistent with its reduction of EMT and collagen deposition. Upregulation of IR/Akt signaling by pterostilbene was also confirmed in Akt inhibitor (MK-2206 2HCl) or IR inhibitor (GSK1904529A)-treated HK-2 cells. Taken together, pterostilbene may be a promising therapeutic agent for the treatment of fructose-induced kidney fibrosis with insulin signaling impairment.

Magnesium isoglycyrrhizinate ameliorates fructose-induced podocyte apoptosis through downregulation of miR-193a to increase WT1.

High fructose intake is a risk of glomerular podocyte dysfunction. Podocyte apoptosis has emerged as a major cause of podocyte loss, exacerbating proteinuria. Magnesium isoglycyrrhizinate (MgIG) is usually used as a hepatoprotective agent in clinic. Liver and kidney injury often occurs in human diseases. Recent report shows that MgIG improves kidney function. In this study, we found that MgIG significantly alleviated kidney dysfunction, proteinuria and podocyte injury in fructose-fed rats. It also restored fructose-induced podocyte apoptosis in rat glomeruli and cultured differentiated podocytes. Of note, high-expression of miR-193a, downregulation of Wilms' tumor protein (WT1) and RelA, as well as upregulation of C-Maf inducing protein (C-mip) were observed in these animal and cell models. The data from the transfection of miR-193a mimic, miR-193a inhibitor, WT1 siRNA or LV5-WT1 in cultured differentiated podocytes showed that fructose increased miR-193a to down-regulate WT1, and subsequently activated C-mip to suppress RelA, causing podocyte apoptosis. These disturbances were significantly attenuated by MgIG. Taken together, these results provide the first evidence that MgIG restrains fructose-induced podocyte apoptosis at least partly through inhibiting miR-193a to upregulate WT1, supporting the application of MgIG with a novel mechanism-of-action against podocyte apoptosis associated with fructose-induced kidney dysfunction.