*5.1. Pre-Clinical Observations*

In vitro studies in cellular models were used to assess the relationship between SCFAs and inflammation and oxidative stress. Huang et al. evaluated the effect of SCFAs on oxidative stress and inflammation induced by high levels of glucose and lipopolysaccharide (LPS) in mouse glomerular mesangial cells (CMG) (SV-40 MES 13) in the presence of acetate and butyrate or GPR43 agonist. The results indicated that both the treatment with SCFA and the treatment with the GPR43 agonist reduced MCP-1, IL-1β, and ICAM-1 levels. Moreover, both acetate and butyrate and the agonist GPR43 inhibited the generation of ROS and MDA and reversed the decrease in SOD induced by high levels of glucose and LPS. These pieces of evidence support the hypothesis that both SCFAs and the GPR43 signalling pathway may act as potential therapeutic targets in inflammation and oxidative

stress in glomerular mesangial cells [143]. Andrade-Olivera and colleagues also confirmed that SCFAs modulated the inflammatory process. In renal tubular epithelial cells (TECs) stimulated with an inflammatory cocktail (LPS, zymosan, and TNF-α) and treated with butyrate, propionate, and acetate indicated that SCFAs reduce NF-κB activation, nitric oxide production, and ROS production in TECs. Furthermore, the translocation of hypoxiainducible factor (HIF)-1 α transcription factor to the nucleus, a hallmark of hypoxia, was also reduced due to the role of SCFA. Therefore, treatment with SCFAs seems to counteract the inflammatory response and hypoxia in renal tubular epithelial cells. SCFAs could also modulate the inflammatory response, regulating immune cells and reducing the expression of the costimulatory molecules, CD80 and CD40, in bone marrow dendritic cells (DCs), and reducing CD8+ and CD4+ cell proliferation after treating antigen-presenting cells (APCs) from RAGKO mice with LPS, with or without SCFAs, for 24 h. Other studies performed in animal models of renal disease evaluated the effects of SCFA. In particular, acetate showed to have beneficial effects in preserving the structure of the kidney, reducing ROS, cytokines, and chemokines. Then, low mRNA levels of toll-like receptor 4, and its endogenous ligand, lower the activation of the NF-κB pathway, wherein low levels of activated neutrophils and macrophages, a low frequency of infiltrating macrophages, and a low frequency of activated DCs were observed. Acetate also increased the expression of GPR43 by modulating the expression of genes encoding for enzymes involved in epigenetic modifications and inhibited the activity of HDACs [144]. Butyrate appears to modulate the inflammatory response in vitro, also modifying the profibrotic cytokine transforming growth factor beta (TGF- β1) generation on immortalised human renal proximal tubular epithelial cells (HK-2 cells). There is strong evidence that this cytokine is involved in renal fibrosis in all renal diseases, and butyrate reduces the basal generation of TGF-β1 in renal tubular epithelial cells; in addition, butyrate mediates its effect through the inhibition of ERK/MAP kinase. This evidence was useful in confirming the role of butyrate in preventing renal fibrosis through the reduction of TGF-β1 and provided a useful basis for subsequent studies on dietary supplementation with Acacia(sen) SUPERGUM™ (gum arabic) that, increasing systemic levels of butyrate, may therefore have a potential beneficial effect in renal disease through the suppression of TGF-β1 activity [145–148]. SCFAs are, therefore, able to directly modulate some of the pro-inflammatory and oxidative stress parameters, as also demonstrated by other studies [149,150].

The effect of SCFAs in modulating inflammation and oxidative stress response was also reported in in vivo studies in animal models of chronic renal failure, which also correlated with a number of secondary complications.

Acute Kidney Injury (AKI) is an important risk factor for CKD. Therefore, Liu et al. used a mouse model of folic acid nephropathy to examine the effect of dietary fibre, from which SCFAs are derived after microbial fermentation, on the development of AKI and, consequently, on the progression of CKD. Wild-type and knockout mice for GPR41, GPR43, or GPR109A receptors in which folic acid nephropathy had been induced were fed fibre-rich diets or treated with SCFAs. The gut microbiota was examined by RNA sequencing, and an increase in *Bifidobacterium* and *Prevotella* was observed, which also increased the concentration of SCFAs in both faeces and serum. After 28 days, the animals showed improved kidney function, fewer tubular lesions, and fewer interstitial fibrosis; chronic inflammation was evaluated by the gene expression analysis of various inflammatory parameters, such as TLR-2, TLR-4, pro-inflammatory cytokines (e.g., TNF-α, IL-6, IL-18, IL-1β, IL-4, IL-10, and IFNγ), and anti-inflammatory cytokine IL-10, the activation of NLRP3 inflammasome, chemokines (e.g., CXCL2, CCL2, and CXCL10), TGF-β1 expression and pro-inflammatory enzymes (e.g., iNOS). The SCFAs treatment led to similar protection through the inhibition of HDAC and GPR41-, GPR43-, and GPR109A-dependent signalling. Thus, both dietary manipulation and SCFAs have been shown to significantly reduce the damage of AKI and, thus, the risk of CKD progression [151]. Diabetic nephropathy is a chronic inflammatory condition that often overlaps with CKD, in the pathogenesis of which oxidative stress and NF-κB signalling are mainly observed. Huang et al. evaluated the role of acetate,

propionate, and butyrate both in vitro on GMC cells (SV-40 MES 13) and in different animal models such as mice with type 2 diabetes (T2D) induced by streptozotocin (STZ), diabetic nephropathy (DN), and GMC cells of high-glucose mice, but also in a high-fat diet (HFD). In GMCs, SCFAs inhibited oxidative stress by reducing ROS and MDA and increased SOD, reduced NF-κB activation, enhanced the interaction between β-arrestin-2 and I-κBα, and reduced the release of MCP-1 and IL-1 β. For in vivo studies, however, the kidneys were used for the pathology assessment, and biochemical analyses were performed. The results showed that SCFAs, particularly butyrate, improved hyperglycaemia and insulin resistance, reduced proteinuria, serum creatinine, urea nitrogen and cystatin C, inhibited mesangial matrix accumulation and renal fibrosis, and blocked NF-κB activation in mice by GPR43-mediated signalling. SCFAs ameliorated the renal damage of DT2 and demonstrated antioxidant and anti-inflammatory effects mediated by the overexpression of GPR43 [152]. These results were also confirmed by another study. Diabetes was induced by STZ in wild-type C57BL/6 and GPR43 or GPR109A knockout mice, and then they were fed fibre-rich diets followed by sodium acetate, sodium propionate, and sodium butyrate. After 12 weeks, stool, urine, and plasma samples were collected and examined. The results indicated that diabetic mice fed a high-fibre diet had less albuminuria, glomerular hypertrophy, podocyte lesions, and fibrosis and were less likely to develop diabetic nephropathy and, consequently, CKD. The fibre also promoted the expansion of SCFA-producing bacteria such as *Prevotella* and *Bifidobacterium*, which increased the faecal and systemic SCFA concentrations, and reduced the expression of genes encoding for inflammatory cytokines, chemokines, and fibrosis-promoting proteins in diabetic kidneys. In vitro studies used TEC cells and podocytes isolated from C57BL/6 mice, both treated with either acetate, propionate, or butyrate. The results indicated that SCFAs modulated inflammation by reducing the chemokines CCL2 and CXCL10 and the cytokines IL-6 and TNF-α. In addition, the expression of the fibrosis-related genes TGF-β and fibronectin was also reduced [153]. These effects depended on the modulation of inflammation by SCFAs through the GPR41 and GPR43 receptors, as also shown in other studies. Indeed, butyrate, through the activation of the GPR109A receptor in renal podocytes, influences the gene transcription of pro-inflammatory cytokines and controls inflammatory responses. This GPR109A receptor phenotype in renal podocytes was associated with an increase in podocyte-related proteins and a normalised pattern of acetylation and methylation at the promoter sites of genes that are essential for podocyte function. Thus, the protective effect of butyrate-dependent GPR109A signalling ameliorated proteinuria by preserving the podocytes on the glomerular basement membrane and attenuating glomerulosclerosis and tissue inflammation [154,155]. There is a large body of evidence that CKD is associated with impaired function and decreased integrity of the intestinal epithelial barrier. Indeed, chronic low-grade inflammation and marked alteration of the intestinal microbiota can be observed in the intestine, which, by further producing toxic metabolites, promotes increased inflammation and its progression to the systemic level. Hung et Suzuki conducted a study in which they evaluated whether fermentable dietary fibre (DF), such as unmodified guar gum (GG) and partially hydrolysed GG (PHGG), could cause an increase in SCFA concentrations and, consequently, restore intestinal barrier permeability and function, thereby also improving inflammation in cases of CKD. Thirty-three seven-week-old male mice were fed a diet supplemented with adenine for 14 days to induce CKD and were subsequently examined. Twenty-seven of these mice were then divided into three groups (CKD, CKD+GG, and CKD+PHGG), while six mice received a control diet. Pro-inflammatory parameters, such as TNF-α and IL-1β, tight junction proteins, such as zonula occludens (ZO)-1, ZO-2 and occludin, serum urea and creatinine, intestinal barrier permeability, SCFA levels, and bacterial populations were examined. The results indicated that in the mice fed with GG and PGHH, not only was inflammation reduced, but high caecal levels of SCFAs, intestinal barrier function, and bacterial population composition, in particular *Lactobacillus*, were also improved. Thus, SCFAs, produced through the intestinal fermentation of PHGG and GG and transported into the circulatory system, have been shown to suppress inflammation and renal fibrosis

directly [156]. In another study, the effects of the prebiotic fibre, xylooligosaccharide (XOS), on renal function and gut microbiota in mice with adenine-induced CKD were evaluated. The mice were fed adenine for 3 weeks to induce CKD and then fed XOS for a further 3 weeks. The results indicated that XOS reduced the renal damage in CKD mice, improved intestinal bacterial populations, increased the caecal production of SCFAs and reduced the levels of the uremic toxin IS [157].

The study sections and results are summarised in Table 2.

**Table 2.** Pre-clinical studies report a related improvement in SCFA levels both in in vitro and animal models.




Abbreviations: ROS, Reactive Oxygen Species; SOD, Super Oxide Dismutase; MDA, Malondialdehyde; IL, Interleukin; MCP-1, Monocyte Chemoattractant Protein-1; ICAM-1, Intracellular Adhesion Molecule-1; NF-κB, Nuclear Factor Kappa-light-chain-enhancer of Activated B cells; NO, Nitric Oxide; TLR, Toll-Like Receptor; TNF-α, Tumor Necrosis Factor alpha; *TGF-β*, Transforming Growth Factor beta; INF-γ, Interferon gamma; MMP, Matrix Metallopeptidase; KIM-1, Kidney Injury Molecule; HDAC, Histone Deacetylase; GAPDH, Glyceraldehyde 3-Phosphate Dehydrogenase; GPR, G-Protein-coupled Receptor; FBG, Fasting Blood Glucose; ACR, Random Urine Albumin-Creatinine ratios; FINS, Fasting Insulin Levels; BUN, Urea Nitrogen Levels; SCr, Serum Creatinine; TC, Total Cholesterol; TG, Triglycerides; LDL, Low-Density Lipoprotein; LDL-C, Low-Density Lipoprotein Cholesterol; CCL2, C-C Motif Chemokine Ligand 2; CXCL10, C-X-C motif chemokine ligand 10; SCFAs, Short-chain Fatty Acids; Fsp1, Fibroblast-Specific Protein 1; Col4α1, Kidney Collagen type IV alpha 1; ActaII, Actin alpha 2, Smooth Muscle; Col1A1, Kidney Collagen type I; IgA, Immunoglobulin A; ZO, JAMA; Defa5, Ileal Defensins alpha; Pla2g2a, Phospholipase A2; Reg3γ, Regenerating islet-Derived Protein 3 gamma; Cgtf, Connective tissue growth factor; Arg, Arginase; XOS; Timp1, Metallopeptidase Inhibitor 1; IS, Indoxyl Sulfate; pCS, pCresil Sulfate.
