Immunological Impact of a Gluten-Free Dairy-Free Diet in Children With Kidney Disease: A Feasibility Study

Abstract

Kidney disease affects 10% of the world population and is associated with increased mortality. Steroid-resistant nephrotic syndrome (SRNS) is a leading cause of end-stage kidney disease in children, often failing standard immunosuppression. Here, we report the results of a prospective study to investigate the immunological impact and safety of a gluten-free and dairy-free (GF/DF) diet in children with SRNS. The study was organized as a four-week summer camp implementing a strict GF/DF diet with prospective collection of blood, urine and stool in addition to whole exome sequencing WES of DNA of participants. Using flow cytometry, proteomic assays and microbiome metagenomics, we show that GF/DF diet had a major anti-inflammatory effect in all participants both at the protein and cellular level with 4-fold increase in T regulatory/T helper 17 cells ratio and the promotion of a favorable regulatory gut microbiota. Overall, GF/DF can have a significant anti-inflammatory effect in children with SRNS and further trials are warranted to investigate this potential dietary intervention in children with SRNS.

Keywords: dairy-free; diet; gluten-free; inflammation; steroid resistance nephrotic syndrome.

Figure 1

Study design and immunological analyses performed during the summer camp. (A) Blood, urine and stool were collected at the beginning of the summer camp. Participants underwent a GF/DF diet for 4 weeks. After that, samples were collected again and immune cells, proteins and microbiota were analyzed. (B-J) Changes in immune cell populations after GF/DF intervention. Flow cytometric analysis of fresh blood samples obtained from children at day 0 and day 26 during the camp, including eosinophils (B), CD4+ T cells (C), CD8+ T cells (D), effector T cells (E), regulatory T cells (F), Th1 cells (G), Th2 cells (H), Th17 cells (I) and Treg/Th17 ratio (J). Wilcoxon matched pair test was used to assess differences within immune subpopulations before and after gluten and dairy removal. Asterisks show significant values after correction for multiple testing by FDR<0.1. CM, central memory; Teff, T effector; T reg, T regulatory; *p<0.05; **p<0.01; ***p < 0.001.

Figure 2

Proteomic analysis of circulating biomarkers upon GF/DF intervention. (A) Heatmap of the top 17 proteins that changed in all participants during the camp, measured by SOMAscan. (B) Process network enrichment analysis of the top 17 proteins using Metacore. (C-F) Changes in serum biomarkers. Serum levels of cytokines and kidney biomarker were measured by Luminex, including GRO (C), TNFα (D), IL-8 (E) and KIM-1 levels (F). Wilcoxon matched pair test was performed to assess differences between before and after GF/DF intervention. Asterisks show significant values after correction for multiple testing by FDR<0.1. KIM, Kidney Injury Molecule; GRO, growth-regulated oncogene; *p<0.05; **p<0.01; ***unadjusted p<0.001.

Figure 3

Analysis of the microbiota in all participants before and after GF/DF intervention. (A) Taxa plots representing the relative frequency of bacteria at genus level in fecal samples collected before and after the camp. Each color represents a bacterial genus and each column represents a patient sample. (B) Difference in fecal relative abundance of genera Bacteroides and Lachnospira before and after the camp. (C) Fecal relative abundance of species Faecalibacterium prauznitzii before and after the camp. Wilcoxon matched pair test was performed to assess differences in relative abundance of operational taxonomic units (OTUs), asterisks show significant values after correction for multiple testing by FDR<0.1. *unadjusted p<0.05; **unadjusted p<0.01.

Figure 4

Zonulin levels before and after GF/DF diet implementation in all participants. Serum zonulin levels from serum samples obtained from children at day 0 and day 26 during the camp were determined by ELISA. Responders to the GF/DF intervention are shown in orange (patient 1) and blue (patient 6).