Medications Activating Tubular Fatty Acid Oxidation Enhance the Protective Effects of Roux-en-Y Gastric Bypass Surgery in a Rat Model of Early Diabetic Kidney Disease

Figures

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  Figure 1. Overview of the study design. Created with BioRender.com. In the SHAM, RYGB, and RYGB-FMRR groups, glycaemic control was optimized for one week prior to surgery with daily subcutaneous injection of insulin degludec (Tresiba®, Novo Nordisk) to achieve a fasting plasma glucose below 12 mmol/L. When introducing medications in the RYGB-FMRR group, metformin monotherapy was introduced for the first two days to monitor for adverse responses, including anorexia. The remaining medications (fenofibrate, ramipril, and rosuvastatin) were commenced thereafter when no adverse response was observed. FMRR, fenofibrate, metformin, ramipril, and rosuvastatin; RYGB, Roux-en-Y gastric bypass; RYGB-FMRR, Roux-en-Y gastric bypass plus fenofibrate, metformin, ramipril, and rosuvastatin; SHAM, sham surgery (laparotomy); ZDSD, Zucker Diabetic Sprague Dawley.

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  Figure 2. Characterization of renal cortical transcriptomic changes across the experimental groups. (A) Principal component analysis of regularized log-transformed gene expression counts demonstrates renal cortical transcriptomic differences according to group assignment. (B) Dotplot of Reactome pathway over-representation analysis illustrating pathways commonly changed by both RYGB and RYGB-FMRR, and those uniquely changed by RYGB-FMRR (50). Dot colour represents the adjusted p-value for pathway enrichment while dot size is scaled by the gene ratio. Pathways pertaining to fatty acid metabolism are bolded. (C) Heatmap visualization of abundance estimates of eight immune and two stromal cell populations obtained using the Microenvironment Cell Populations-counter (MCP-counter) R package (54). Cell abundance estimates were centred and scaled by heatmap row. Each heatmap row displays abundance of a different cell population. Heatmap columns correspond to individual rats. Gaps between columns as well as the column annotations demarcate the four experimental groups from left to right: SD (blue), SHAM (grey), RYGB (green), and RYGB-FMRR (red). Colouring of heatmap cells reflects relative cell…

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  Figure 3. In silico deconvolution of the predicted cellular source of transcripts differentially expressed between the RYGB-FMRR and RYGB groups. (A) Transcripts identified in the RYGB-FMRR versus RYGB differential expression analysis were intersected with a human diabetic kidney single-nucleus RNA-sequencing dataset (56). Transcript expression levels in the human diabetic kidney, averaged across cell types by the Seurat function ‘AverageExpression’ and subsequently centred and scaled by row, are plotted on the heatmap. Heatmap rows display the differentially regulated transcripts whilst each column represents 1 of 12 identified renal cell types. Cell groupings of individual cell types in the heatmap columns are indicated in the legend key. An increasingly light colour in a specific cell type reflects an increased relative expression therein relative to other cell types. (B) Violin plots of cell-specific expression in the human diabetic kidney of three PPARα-responsive transcripts upregulated by RYGB-FMRR. Kidney cell types are presented on the x-axis with relative transcript expression levels in the human diabetic kidney on the y-axis. Each dot represents a single cell in the human…

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  Figure 4. In silico deconvolution of medication-specific and PPAR isotype-specific transcriptomic effects following RYGB-FMRR. (A, D) Genes responsive to FMRR medications (A) and PPAR isotypes (D) were retrieved from IPA, intersected with transcripts identified in the RYGB-FMRR versus RYGB differential expression analysis, and presented on doughnut plots with counts and percentages of transcripts in each category outlined. The inner doughnut layers outline all transcripts known to be responsive to each medication (A) or PPAR isotype (D). The outer layers stratify transcripts by presence in or absence from the RYGB-FMRR vs RYGB DEG list. (B, E) Venn diagram enumeration of the overlap and separation in transcripts present in the RYGB-FMRR vs RYGB DEG list and responsive to one or more of the FMRR medications (B) or one or more of the PPAR isotypes (E). These transcripts are labelled as ‘In DEG list’ in the outer doughnut layers in (A, D), respectively. (C, F) Subsets of genes present in the RYGB-FMRR vs RYGB DEG list and found to be responsive to FMRR medications (C) or PPAR isotypes (F) are presented on doughnut plots with counts and percentages of transcripts in each category outlined.…

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  Figure 5. Network visualisation of medication-responsive genes which were differentially expressed between the RYGB-FMRR and RYGB groups. The network was constructed using the ‘My Pathways’ interface in IPA, and edited using ‘PathDesigner’ in IPA. The four medications administered to rats treated with RYGB-FMRR, and their corresponding drug targets, are bolded. The legend outlines node symbols corresponding to molecule types contained within the network. The directionality of gene expression change in the RYGB-FMRR vs RYGB DEG list is displayed by node colour (red = upregulated; green = downregulated). Relationships between medications and their gene targets are illustrated by edge colour (orange = predicted activation; blue = predicted inhibition). DEG, differentially expressed gene; IPA, Ingenuity Pathway Analysis; RYGB, Roux-en-Y gastric bypass; RYGB-FMRR, Roux-en-Y gastric bypass plus fenofibrate, metformin, ramipril, and rosuvastatin.

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  Figure 6. Characterization of urinary metabolomic changes following RYGB and RYGB-FMRR. (A, B) Principal component analysis biplots of PQN-normalized urinary 1H-NMR peaks obtained before and at 4 weeks after intervention (A, principal components 1-2; B, principal components 3-4). Baseline and follow-up samples from SD rats were considered together given the lack of change in metabolomic profiles evident in this group. Samples from SHAM rats at baseline and follow-up were considered alongside baseline samples from RYGB and RYGB-FMRR rats, and collectively designated as untreated ZDSD rats. Untreated ZDSD rats clustered based on disease severity into two subphenotypes, mild and severe, rather than by timing of sampling or by experimental group assignment. The definition of disease severity in this context was based on patterns of urinary metabolomic changes influencing subphenotypes observed after clustering of rats by principal component analysis. RYGB and RYGB-FMRR reflect post-intervention values in these two groups. Arrows indicate loading vectors of metabolites driving separation of the groups along principal components 1-4. (C) Dotplot of urinary 1H-NMR peaks ranked by importance to…

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  Figure 7. Multi-omic integration of the renal cortical transcriptome and urinary metabolome identifies a distinctive gene-metabolite signature following RYGB-FMRR. (A) A supervised, sparse partial least squares-discriminant analysis model was fit to regularized log-transformed gene expression counts and annotated, PQN-normalized urinary 1H-NMR peaks from 4 weeks after intervention using the DIABLO framework in the R package mixOmics (72, 73). An overview of the correlation structure between transcripts and metabolites selected by component 1 of the model, which separated RYGB-FMRR rats from the three other experimental groups, is presented. The left lower panel indicates the Pearson’s correlation coefficient between transcripts and metabolites, and the top right panel presents a scatterplot of the correlation structure between the two omics datasets for each sample. Each dot represents an individual rat, with colours indicating experimental group assignment. (B) Dimensionality reduction plot of the subspace spanned by the latent variables of the sparse DIABLO model indicating clustering of experimental groups along component 1 (x-axis) and component 2 (y-axis). The plot is facetted by…

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  Figure 8. Changes in glomerular volume and their relationship to renal cortical FAO transcripts and urinary nicotinamide and TCA cycle metabolites. (A) Representative images (20x, scale bar 100µm) of haematoxylin and eosin-stained kidney sections from the four experimental groups. (B) Glomerular volume (µm3) was assessed in 30 glomeruli from 6-8 animals per group. Data are plotted as violin plots with individual glomerular measurements superimposed. Within each group, each animal is identified as its own column of dots with a unique colour shade. Median group values are identified by the horizontal black line in each violin and printed on each violin. Statistical significance of between-group differences derived from multiplicity-corrected Wilcoxon rank-sum tests is presented. Statistical significance is denoted as follows: ns = not significant; * = p <0.05; ** = p <0.01; *** = p <0.001; **** = p <0.0001. (C) Correlation plots highlighting Pearson correlation r values for glomerular volume correlations with renal cortical transcripts and urinary metabolites. Regularized log-transformed gene expression counts were used for gene-structure correlations. Transcripts plotted are those which…

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  Figure 9. Changes in glomerular ultrastructure and their relationship to renal cortical FAO transcripts and urinary nicotinamide and TCA cycle metabolites. (A) Representative images (9900x, scale bar 1µm) of transmission electron microscopy images of glomerular capillary loops from the four experimental groups. (B–D) Podocyte foot process frequency (per 8µm of glomerular basement membrane length), podocyte foot process diameter (nm), and glomerular basement membrane thickness (nm) were quantified using transmission electron microscopy images from 6 animals per group. Six determinations of podocyte foot process frequency were made per animal, while twenty-four determinations of podocyte foot process diameter and glomerular basement membrane thickness were made per animal. Data are plotted as violin plots with individual glomerular measurements superimposed. Within each group, each animal is identified as its own column of dots with a unique colour shade. Median group values are identified by the horizontal black line in each violin and printed on each violin. Statistical significance of between-group differences derived from multiplicity-corrected Wilcoxon rank-sum tests is presented.…

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  Figure 10. Changes in proximal tubular mitochondrial roundness and their relationship to renal cortical FAO transcripts and urinary nicotinamide and TCA cycle metabolites. (A, C) Representative images (16500x, scale bar 1µm) of transmission electron microscopy images of mitochondria in the pars convoluta (A) and pars recta (C) regions of the proximal tubule from the four experimental groups. (B, D) Mitochondrial roundness in the pars convoluta (B) and pars recta (D) was quantified using transmission electron microscopy images from 6 animals per group. Mitochondria were quantified in 15 non-overlapping images captured from 3 distinct pars convoluta and pars recta regions (5 images/region) for each animal. Data are plotted as violin plots with individual mitochondrial measurements superimposed. Within each group, each animal is identified as its own column of dots with a unique colour shade. Median group values are identified by the horizontal black line in each violin and printed on each violin. Statistical significance of between-group differences derived from multiplicity-corrected Wilcoxon rank-sum tests is presented. Statistical significance is denoted as follows: ns = not significant; *…

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  Figure 11. Comparison of renal cortical lipid metabolism parameters between RYGB and RYGB-FMRR rats matched for improvements in metabolic control and urinary albumin excretion. (A) One rat each from the RYGB and RYGB-FMRR groups, matched for body weight, plasma glucose, and albuminuria, was used for a comparative analysis of renal cortical lipid metabolism transcript expression and proximal tubular mitochondrial morphology. Body weight, plasma glucose, and log urinary albumin excretion rate values pre- and post-intervention are plotted, highlighting both similar baseline values and magnitude of improvement in these parameters for each rat from the RYGB and RYGB-FMRR groups. (B) Heatmap of renal cortical peroxisomal and mitochondrial lipid metabolism transcript expression for the matched RYGB and RYGB-FMRR rats, indicating increased relative expression of lipid metabolism transcripts in the RYGB-FMRR rat. Regularized log-transformed gene expression counts from RNA-sequencing, centred and scaled by row, are plotted. Transcripts plotted are those which resulted in enrichment of peroxisomal and mitochondrial lipid metabolism pathways in RYGB-FMRR rats (also highlighted in the circular network…

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  Figure 12. Overview schematic. Created with BioRender.com. 1H-NMR, proton nuclear magnetic resonance spectroscopy; FA, fatty acid; FAO, fatty acid oxidation; FMRR, fenofibrate, metformin, ramipril, and rosuvastatin; GBM, glomerular basement membrane; H&E, haematoxylin and eosin; PPARα, peroxisome proliferator-activated receptor-alpha; PT, proximal tubule; RNA-Seq, ribonucleic acid next-generation sequencing; RYGB, Roux-en-Y gastric bypass; RYGB-FMRR, Roux-en-Y gastric bypass plus fenofibrate, metformin, ramipril, and rosuvastatin; SD, Sprague Dawley; SHAM, sham surgery (laparotomy); TCA, tricarboxylic acid; TEM, transmission electron microscopy; UAER, urinary albumin excretion rate; VLCFA, very-long-chain fatty acid; ZDSD, Zucker Diabetic Sprague Dawley.