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Deficiency of CFB attenuates renal tubulointerstitial damage via inhibiting ceramide synthesis in diabetic kidney disease

ABSTRACT: Accumulating evidence suggests the pathogenic role of immunity and metabolism in diabetic kidney disease (DKD). Herein, we aimed to investigate the effect of complement factor B (CFB) on lipid metabolism in the development of DKD. We found that in patients with diabetic nephropathy (DN), the staining of Bb, CFB, C3a, C5a, and C5b-9 were significantly elevated in renal tubulointerstitium. Cfb knockout diabetic mice had significantly milder tubulointerstitial injury and less ceramide biosynthesis. The in vitro study demonstrated that cytokine secretion, endoplasmic reticulum stress, oxidative stress, and cell apoptosis were ameliorated in siCFB HK-2 cells under high glucose conditions. Exogenous ceramide supplementation attenuated the protective effect of CFB knockdown in HK-2 cells, while inhibiting ceramide synthases (CERS) with fumonisin B1 in CFB-overexpressing cells rescued the cell injury. CFB knockdown could downregulate the expression of NF-κB p65, which initiates the transcription of CERS3. Furthermore, C3 knockdown abolished CFB-mediated cytokine secretion, NF-κB signaling activation, and subsequently ceramide biosynthesis. Thus, CFB deficiency inhibited activation of the complement alternative pathway and attenuated kidney damage in DKD, especially tubulointerstitial injury, via inhibiting the NF-κB signaling pathway, further blocking the transcription of CERS, which regulates the biosynthesis of ceramide. CFB may be a promising therapeutic target of DKD.

ORGANISM(S): Mus musculus

PROVIDER: GSE217153 | GEO | 2023/02/01

REPOSITORIES: GEO

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Project description:Aim： Use microarray analysis to understand the molecular mechanism underlying the effect of aristolochic acid (AA), a major active component of plants from the Aristolochiaceae family, in normal human kidney (HK-2) cells. Methods： HK-2 cells were treated with AA for 24 hours and cell viability was measured by a 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide assay. Complementary DNA microarrays were used to investigate the gene expression pattern of HK-2 cells exposed to AA and the results of this study were in triplicate. Quantitative real-time RT-PCR assay was used to verify the microarray data for selected nuclear factor kappa B (NF-κB)-regulated genes. Furthermore, subcellular localization of NF-κB p65 was visualized by immunofluorescence confocal microscopy in HK-2 cells. NF-κB activity was examined by luciferase reporter assay in HK-2/NF-κB transgenic cells. Results： AA exhibited a dose-dependent cytotoxic effect in HK-2 cells and induced alterations in gene expression profiles related to DNA damage response, stress response, etc. In addition, 9 biological pathways associated with immunomodulatory functions were down-regulated in AA-treated HK-2 cells. Network analysis revealed that NF-κB played a central role in the network topology. Among NF-kB-regulated genes, 8 differentially expressed genes were verified by real-time RT-PCR. The inhibition of NF-κB activity by AA was further confirmed by immunofluorescence confocal microscopy and by NF-κB luciferase reporter assay. Conclusion： Our data revealed that AA could suppress NF-κB activity in normal human cells, perhaps partially accounting for the reported anti-inflammatory effects of some plants from the genus Aristolochia. HK-2 cells were grown in keratinocyte serum-free basal medium (Gibco) supplemented with 5 ng/ml of recombinant epidermal growth factor and 50 μg/ml of bovine pituitary extract without antibiotics in 5 % CO2 at 370C. HK-2 cells were seeded in 25-T flasks and incubated for 24 h before aristolochic acid treatment. Aristolochic acid (10 90 μM) were added to HK-2 cells for 24 h. The control cells received equal amounts of water only.

Project description:Background: Diabetic kidney disease (DKD) is the leading cause of end-stage renal disease worldwide with limited treatment options. The intricate pathogenesis of dysregulated lipid metabolism leading to the development of DKD remains obscure. Lipophagy, which refers to the autophagic degradation of intracellular lipid droplets, has been found to be impaired in DKD, resulting in renal tubule dysfunction and ectopic lipid deposition (ELD). The lipotoxity in renal tubules is closely related to the pathogenesis of DKD. Therefore, it is crucial to develop effective and safe agents that can restore autophagic flux and lipophagy in renal tubules for potential preventive interventions. Purpose: This study seeks to investigate the preventive effect of sodium butyrate (NaB) on ELD in the progression of DKD and elucidate the underlying mechanisms involved.Methods: The beneficial effects of NaB were investigated in glucolipotoxicity (GLT)-stimulated HK-2 cells and the streptozotocin (STZ) injection combined with a high-fat diet induced DKD model mice. Additionally, we managed to silence transcription factor EB (TFEB) in HK-2 cells and establish the renal specific silence model by retrograde ureteral infusion of AAV9-shTFEB in mice. We investigated the expression profile of RNAs in HK-2 cells through. The mechanism of NaB is demonstrated by RNA sequencing (RNA-seq), pulldown MS assay, SPR experiments etc.Results: Our findings revealed that serum butyrate level is reduced in DKD patients and is statistically correlated with urinary protein excretion and eGFR. Sodium butyrate (NaB) dose-dependently improved lipid deposition in HK-2 cells and renal tubular epithelial cells of DKD model mice. This effect may be attributed to the promotion of TFEB dephosphorylation, as NaB can directly interact with PPP2R1A to activate PP2A phosphorylation activity. Therefore, NaB may play a role in improving renal lipid deposition in DKD through the PP2A-TFEB pathway. Conclusion: NaB acts as a PP2A-TFEB axis activator that restores lipophagy to ameliorate ELD and renal dysfunction in DKD.

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Deficiency of CFB attenuates renal tubulointerstitial damage via inhibiting ceramide synthesis in diabetic kidney disease

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