#3666 THE PROTECTIVE ROLE OF BUTYRATE IN KIDNEY INJURY
Authors: Favero Chiara , Giordano Laura , Masereeuw Rosalinde , Ortiz Alberto , Sanchez-Nino Maria Dolores 
Published: June 2023

Abstract

Background and Aims
Butyrate is a well-known short-chain fatty acid (SCFAs) produced by the microbial fermentation of indigestible fibers. It contributes to colon homeostasis and controls inflammatory responses and homeostasis in other tissues. It is thought to play an important role in the pathogenesis of kidney disease and contributes to the connections between diet, gut microbiota, and kidney disease. However, a detailed characterization of its function in kidney injury is required. TWEAK is involved in tissue injury, driving inflammation, proliferation, fibrosis and inducing death of renal tubular epithelial cells under certain microenvironmental conditions, such a proinflammatory milieu composed of TNFα and INFγ. It downregulates nephroprotector factors and upregulate the expression of inflammatory markers.

Method
A dose-response curve evaluated cell viability in murine tubular cells (MCT) at different butyrate concentrations using MTT assay. For this, cells were pre-treated with butyrate 0.5, 1 or 5 mM for one hour. After that, they were treated either with TWEAK (tumor necrosis factor-like weak inducer of apoptosis), a member of the TNF superfamily or TTI (TWEAK 100 ng/mL, TNfα 30 ng/mL and INFΓ 30 UI/mL). Through RT-PCR and Western blotting, mRNA and protein levels of various markers were quantified at 3, 6 and 24 h. Results were validated in a kidney-on-a-chip, a microphysiological system that intends to recapitulate key functional characteristics of the kidney, among them, the dynamic conditions in which cells are cultured, mimicking the fluid flows that occurs in the body and with the benefit of representing an alternative to animal testing. It is composed of a hollow fiber membrane, precoated with L-DOPA (2 mg/mL) and collagen IV (1 mg/mL) seeded with human conditionally immortalized proximal tubule epithelial cells (cipTEC-OAT1). After ten days, cells were exposed to butyrate (1 mM) in the basolateral compartment and TWEAK (100 ng/mL) in the apical compartment for 24 h. Cell toxicity, cell viability and cell permeability were measured at 24 h through LDH release, and PrestoBlue and FITC-Inulin assays, respectively. Cells and supernatant were collected to perform RT-PCR and ELISA for relevant markers.

Results
When MCT cells were pre-treated with butyrate and then injured either with TWEAK or TTI (TWEAK, TNF-α, IFN-Γ), the expression of klotho, a nephroprotector and anti-aging gene, and peroxisome proliferator-activated receptor Γ coactivator-1α (PGC-1α), a transcription factor that promotes mitochondrial biogenesis, were preserved, as opposed to decreased by cytokines only. Moreover, cells pre-treated with butyrate had decreased expression of inflammatory markers such as MCP1 as compared to increased expression upon cytokine stimulation. In the kidney-on-a-chip, cell viability was reduced after exposure to TWEAK compared to the cells treated only with butyrate, while cells treated with butyrate and TWEAK had lower toxicity as assessed by LDH release. Furthermore, results for Klotho, PGC-1α and the inflammation markers MCP1 and IL6 were aligned with those in MCT cells.

Conclusion
In summary, in-vitro, butyrate prevents injury caused by TWEAK, preserves protective factors such as the anti-aging factor Klotho and the master regulator of mitochondrial biogenesis PGC-1α and decreases inflammation. Furthermore, these results were validated in a kidney-on-a-chip microphysiological system. Characterization of the mechanism behind the protection of butyrate for kidney injury is ongoing.