Twist1 downregulation of PGC-1α decreases fatty acid oxidation in tubular epithelial cells, leading to kidney fibrosis

Rationale: A deficiency of fatty acid oxidation (FAO) is the metabolic hallmark in proximal tubular cells (PTCs) in renal fibrosis owing to utilization of fatty acids by PTCs as the main energy source. Lipid accumulation may promote lipotoxicity-induced pathological injury in renal tissue. However, the molecular mechanism underlying lipotoxicity and renal tubulointerstitial fibrosis (TIF) remains unclear. Twist1 has been identified to play an essential role in fatty acid metabolism. We hypothesized that Twist1 may regulate FAO in PTCs and consequently facilitate lipotoxicity-induced TIF. Methods: We used hypoxia-induced Twist1 overexpression to incite defective mitochondrial FAO in PTCs, and used renal ischemia-reperfusion or unilateral ureteral obstruction to induce renal injury in mice. We used knockout cells, mice of Twist1, and Harmine to determine the role of Twist1 in FAO and TIF. Results: Overexpression of Twist1 downregulates the transcription of PGC-1α and further inhibits the expression of FAO-associated genes, such as PPARα, CPT1 and ACOX1. Consequently, reduced FAO and increased intracellular lipid droplet accumulation in a human PTC line (HK-2), leads to mitochondrial dysfunction, and production of increased profibrogenic factors. Twist1 knockout mice with renal injury had increased expression of PGC-1α, which restored FAO and obstructed progression of TIF. Strikingly, pharmacological inhibition of Twist1 by using Harmine reduced lipid accumulation and restored FAO in vitro and in vivo. Conclusion: Our findings suggest that Twist1-mediated inhibition of FAO in PTCs results in TIF and suggest that Twist1-targeted inhibition could provide a potential strategy for the treatment of renal fibrosis.

GO term analysis of upregulated pathways in UUO was performed. The first layer indicates top 20 GO term and the number of the genes is shown in the outer layer. The second layer indicates the number of the genes in the 5 genome background and Q values for enrichment of the upregulated genes for the specified biological process.
The third layer indicates the ratio of the upregulated genes and downregulated genes. The inner layer indicates the enrichment factor of each GO term. GO, Gene Ontology (top). Description of the GO term (bottom).  Quantitative RT-PCR analysis of Ppara following 48 h of transfection of pcDNA3.1-PGC-1α, pcDNA3.1, pSilencer or si PGC-1α. All plasmid treatment groups are normalized to untreated group. Data, mean ± SEM (n = 3 technical replicates). (C) HK-2 cells were transfected with 40 ng PPARα promoter and 20 ng pcDNA3.1-Twist1, 20 ng pcDNA3.1-PGC-1α or PPARα agonist Fenofibrate 1 μM for 48 h. The relative value of luciferase activity relative to renilla luciferase activity was analyzed. HK2 cells were co-transfected with pGL3-CMV, 7 pcDNA3.1 Twist1 and pRL-TK as the positive control group, and HK2 cells were co-transfected with pGL3-PPAR α and pRL-TK plasmid as the negative control group. The experiment was repeated 3 times to calculate the average value. * P < 0.05 compared with the negative control, # P < 0.05 compared with the positive control.

Figure S6 Construction of UIRI and UUO models with PT-Twist1 -/mice.
Related to Figure 6. (A) UUO models were built with WT, PT-Twist1 +/+ and PT-Twist1 -/mice with kidneys harvested on day 14, (B) UIRI models were built for the same groups with kidneys harvested on day 28. H&E and Masson were used to detect pathological and fibrotic changes in kidney tissue in each group, bar = 20 μm. Renal tubule health status was analyzed in WT, PT-Twist1 +/+ and PT-Twist1 -/mice with UIRI (C) and UUO (D). Renal tubule health status 9 was analyzed in WT, PT-Twist1 +/+ and PT-Twist1 -/mice with UIRI (C) and UUO (D). The positive rate of
Table S1 Gene full name and sequences of primers used in this study. Figure 5 and Figure S4.  Table S3. Primer sequence of PGC1 promoter region.

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