Microbiota and metabolome associated with immunoglobulin A nephropathy (IgAN)

Maria De Angelis, Eustacchio Montemurno, Maria Piccolo, Lucia Vannini, Gabriella Lauriero, Valentina Maranzano, Giorgia Gozzi, Diana Serrazanetti, Giuseppe Dalfino, Marco Gobbetti, Loreto Gesualdo, Maria De Angelis, Eustacchio Montemurno, Maria Piccolo, Lucia Vannini, Gabriella Lauriero, Valentina Maranzano, Giorgia Gozzi, Diana Serrazanetti, Giuseppe Dalfino, Marco Gobbetti, Loreto Gesualdo

Abstract

This study aimed at investigating the fecal microbiota, and the fecal and urinary metabolome of non progressor (NP) and progressor (P) patients with immunoglobulin A nephropathy (IgAN). Three groups of volunteers were included in the study: (i) sixteen IgAN NP patients; (ii) sixteen IgAN P patients; and (iii) sixteen healthy control (HC) subjects, without known diseases. Selective media were used to determine the main cultivable bacterial groups. Bacterial tag-encoded FLX-titanium amplicon pyrosequencing of the 16S rDNA and 16S rRNA was carried out to determine total and metabolically active bacteria, respectively. Biochrom 30 series amino acid analyzer and gas-chromatography mass spectrometry/solid-phase microextraction (GC-MS/SPME) analyses were mainly carried out for metabolomic analyses. As estimated by rarefaction, Chao and Shannon diversity index, the lowest microbial diversity was found in P patients. Firmicutes increased in the fecal samples of NP and, especially, P patients due to the higher percentages of some genera/species of Ruminococcaceae, Lachnospiraceae, Eubacteriaceae and Streptococcaeae. With a few exceptions, species of Clostridium, Enterococcus and Lactobacillus genera were found at the highest levels in HC. Bacteroidaceae, Porphyromonadaceae, Prevotellaceae and Rikenellaceae families differed among NP, P and HC subjects. Sutterellaceae and Enterobacteriaceae species were almost the highest in the fecal samples of NP and/or P patients. Compared to HC subjects, Bifidobacterium species decreased in the fecal samples of NP and P. As shown by multivariate statistical analyses, the levels of metabolites (free amino acids and organic volatile compounds) from fecal and urinary samples markedly differentiated NP and, especially, P patients.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Fecal cultivable bacteria of the…
Figure 1. Fecal cultivable bacteria of the main microbial groups.
Cultivable cells (log cfu/g) found in the fecal samples of immunoglobulin A nephropathy (IgAN) non progressor (NP) and progressor (P) patients, and healthy controls (HC). Data are the means of three independent experiments (n = 3). The top and bottom of the box represent the 75th and 25th percentile of the data, respectively. The top and bottom of the error bars represent the 5th and 95th percentile of the data, respectively. ○, Outliers data. Group student's t-test p-values were also shown.
Figure 2. Total and active bacteria found…
Figure 2. Total and active bacteria found in feces of subjects.
Relative abundance (%) of total (16S rDNA) and metabolically active (16S rRNA) bacteria, which were found at the phylum level in the fecal samples of immunoglobulin A nephropathy (IgAN) non progressor (NP) and progressor (P) patients, and healthy controls (HC).
Figure 3. Comparison of total and active…
Figure 3. Comparison of total and active bacterial phyla found in feces of subjects.
Bacterial phyla distribution (%) found in fecal samples of immunoglobulin A nephropathy (IgAN) non progressor (NP) and progressor (P) patients, and healthy controls (HC). The X-axis represents the proportion of phyla from total (16S rDNA) and active (16S rRNA) bacteria.
Figure 4. Principal component analysis (PCA) of…
Figure 4. Principal component analysis (PCA) of total bacterial genera found in feces of subjects.
Score plot of the three principal components (PC) after principal component analysis (PCA) of total bacterial genera (16S rDNA), which were found in the fecal samples of immunoglobulin A nephropathy (IgAN) non progressor (NP) and progressor (P) patients, and healthy controls (HC). 1–16, number of fecal samples for each group of subjects.
Figure 5. Principal component analysis (PCA) of…
Figure 5. Principal component analysis (PCA) of active bacterial genera found in feces of subjects.
Score plot of the three principal components (PC) after principal component analysis (PCA) of metabolically active bacterial genera (16S rRNA), which were found in the fecal samples of immunoglobulin A nephropathy (IgAN) non progressor (NP) and progressor (P) patients, and healthy controls (HC). 1–16, number of fecal samples for each group of subjects.
Figure 6. Fecal levels of free amino…
Figure 6. Fecal levels of free amino acids (FAA) in subjects.
Concentration (mg/kg) of individual free amino acids (FAA) found in the fecal samples of immunoglobulin A nephropathy (IgAN) non progressor (NP) and progressor (P) patients, and healthy controls (HC). Data are the means of three independent experiments and standard deviations, performed in duplicate (n = 6).
Figure 7. Principal component analysis (PCA) of…
Figure 7. Principal component analysis (PCA) of volatile organic metabolites found in feces of subjects.
Score plots of the two principal components (PC) after principal component analysis (PCA) of volatile organic metabolites of the fecal (A) and urine (B) samples of immunoglobulin A nephropathy (IgAN) non progressor (NP) and progressor (P) patients, and healthy controls (HC).

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