Modulation of miR-29a and miR-29b Expression and Their Target Genes Related to Inflammation and Renal Fibrosis by an Oral Nutritional Supplement with Probiotics in Malnourished Hemodialysis Patients

Abstract

Malnutrition is prevalent in patients with chronic kidney disease (CKD), especially those on hemodialysis. Recently, our group described that a new oral nutritional supplement (ONS), specifically designed for malnourished (or at risk) hemodialysis patients with a "similar to the Mediterranean diet" pattern, improved caloric-protein intake, nutritional status and biomarkers of inflammation and oxidation. Our aim in this study was to evaluate whether the new ONS, associated with probiotics or not, may produce changes in miRNA's expression and its target genes in malnourished hemodialysis patients, compared to individualized diet recommendations. We performed a randomized, multicenter, parallel-group trial in malnourished hemodialysis patients with three groups (1: control (C) individualized diet (n = 11); 2: oral nutritional supplement (ONS) + placebo (ONS-PL) (n = 10); and 3: ONS + probiotics (ONS-PR) (n = 10)); the trial was open regarding the intake of ONS or individualized diet recommendations but double-blinded for the intake of probiotics. MiRNAs and gene expression levels were analyzed by RT-qPCR at baseline and after 3 and 6 months. We observed that the expression of miR-29a and miR-29b increased significantly in patients with ONS-PR at 3 months in comparison with baseline, stabilizing at the sixth month. Moreover, we observed differences between studied groups, where miR-29b expression levels were elevated in patients receiving ONS-PR compared to the control group in the third month. Regarding the gene expression levels, we observed a decrease in the ONS-PR group compared to the control group in the third month for RUNX2 and TNFα. TGFB1 expression was decreased in the ONS-PR group compared to baseline in the third month. PTEN gene expression was significantly elevated in the ONS-PR group at 3 months in comparison with baseline. LEPTIN expression was significantly increased in the ONS-PL group at the 3-month intervention compared to baseline. The new oral nutritional supplement associated with probiotics increases the expression levels of miR-29a and miR-29b after 3 months of intervention, modifying the expression of target genes with anti-inflammatory and anti-fibrotic actions. This study highlights the potential benefit of this oral nutritional supplement, especially associated with probiotics, in malnourished patients with chronic renal disease on hemodialysis.

Keywords: chronic kidney disease; circulating miRNAs; gene expression; hemodialysis; inflammation; malnutrition; oral nutritional supplement; probiotics; renal fibrosis.

Figure 1

Study flowchart. ONS: Oral nutritional supplement.

Figure 2

MiR-29a and miR-29b expression levels. Examinations were carried out at baseline and after 3 and 6 months. All the values come from the logarithm of the correspondent parameter. miR-93 was used as the reference gene. Data are presented as mean ± standard deviation. * p < 0.05 versus baseline. + p < 0.05 versus control. ONS-PL = oral nutritional supplement with placebo; ONS-PR = oral nutritional supplement with probiotics. Note: the negative values in miRNAs’ expression result from the fact that the 2^−ΔCt value is lower than 1.

Figure 3

In silico analysis and schema recapitulating the interactions between miR-29a, miR-29b and TGs involved in inflammation, lipid metabolism and bone formation. (A,B) miRTarbase 9.0, Tarbase v.8 and targetScan 8.0 were used to identify possible TGs of miR-29a and miR-29b (yellow nodes). Panther and DAVID databases were used to identify TGs (green nodes) implicated in inflammation, lipid metabolism and bone formation. The interactions among miRNAs and TGs were visualized using Cytoscape v.3.10.1.

Figure 4

mRNA expression levels from human blood. Examinations were carried out at baseline and after 3 and 6 months. All the values come from the logarithm of the correspondent parameter. * p < 0.05 versus baseline. + p < 0.05 versus control. & p < 0.05 versus 3-month intervention. ONS-PL = oral nutritional supplement with placebo; ONS-PR = oral nutritional supplement with probiotics. PTEN: Phosphatase and tensin homolog, TGFB1: Transforming growth factor beta 1, RUNX2: Runt-related transcription factor 2, LEPTIN, TNFa: Tumor necrosis factor a.

Figure 5

Correlations between miR-29a and miR-29b expression levels and serum biomarkers. Examinations were carried out at baseline and after 3 months. Correlations were analyzed using Pearson’s correlation coefficient using the percentage change of the logarithm of the variables between 3 months and baseline in each group. (A) Correlation between miR-29a and IL-10. (B) Correlation between miR-29b and TNFalpha. (C) Correlation between miR-29b and IL-8. ONS-PL = oral nutritional supplement with placebo; ONS-PR = oral nutritional supplement with probiotics. * p < 0.05.