mTOR-mediated podocyte hypertrophy regulates glomerular integrity in mice and humans

Abstract

The cellular origins of glomerulosclerosis involve activation of parietal epithelial cells (PECs) and progressive podocyte depletion. While mammalian target of rapamycin-mediated (mTOR-mediated) podocyte hypertrophy is recognized as an important signaling pathway in the context of glomerular disease, the role of podocyte hypertrophy as a compensatory mechanism preventing PEC activation and glomerulosclerosis remains poorly understood. In this study, we show that glomerular mTOR and PEC activation-related genes were both upregulated and intercorrelated in biopsies from patients with focal segmental glomerulosclerosis (FSGS) and diabetic nephropathy, suggesting both compensatory and pathological roles. Advanced morphometric analyses in murine and human tissues identified podocyte hypertrophy as a compensatory mechanism aiming to regulate glomerular functional integrity in response to somatic growth, podocyte depletion, and even glomerulosclerosis - all of this in the absence of detectable podocyte regeneration. In mice, pharmacological inhibition of mTOR signaling during acute podocyte loss impaired hypertrophy of remaining podocytes, resulting in unexpected albuminuria, PEC activation, and glomerulosclerosis. Exacerbated and persistent podocyte hypertrophy enabled a vicious cycle of podocyte loss and PEC activation, suggesting a limit to its beneficial effects. In summary, our data highlight a critical protective role of mTOR-mediated podocyte hypertrophy following podocyte loss in order to preserve glomerular integrity, preventing PEC activation and glomerulosclerosis.

Keywords: Cell Biology; Chronic kidney disease; Nephrology.

Figure 1. mTOR-mediated podocyte hypertrophy as a compensatory response to human somatic growth and FSGS.

(A) Transcriptional regulation of mTOR signaling in glomerular extracts from human biopsies. Comparison between FSGS (n = 23) and living donors (n = 42), showing fold-change of each respective gene (q < 0.05 in all genes except those with NS), and representative confocal image from indirect immunofluorescence showing a podocyte specific marker (Wilms’ Tumor 1; WT-1) and a downstream target of mTORC1 (phosphorylated ribosomal protein S6; p-rp-S6) in a glomerulus from a patient with primary FSGS. Scale bar: 10 μm. (B) Transcriptional regulation of PEC activation–related genes in glomerular extracts from human biopsies. Comparison between FSGS (n = 23) and living donors (n = 42), showing fold-change of each respective gene (q < 0.05 in all genes except those with no significance [NS]) and representative confocal image of PEC activation marker CD44, and histological staining (periodic acid–Schiff; PAS) showing sequential physical sections of a glomerulus with a segmental lesion (arrowhead). Scale bars: 100 μm. (C) Correlation analysis of regulated mTOR signaling and PEC activation genes. Each box represents an independent association (spearman rank coefficient; R). Every association was statistically significant unless labeled with NS. (D) Comparison of podocyte depletion indices (podocyte number, PN; podocyte density, PD) between glomeruli from children, adults, and adults with FSGS. (E) Association between podocyte number and glomerular volume (using Spearman’s rank coefficients) among children (blue), adults (gray), and adults with FSGS (red). Solid lines represent the regression line, and dotted lines the 95% CI. (F) Comparison of podocyte size indices (total podocyte volume divided by podocyte number, TPV:PN ratio; and total podocyte volume divided by podocyte density, TPV:PD ratio) between children, adults, and adults with FSGS. ****P < 0.0001; ***P < 0.001. In violin plots, red lines represent medians and blue lines represent IQRs; every gray dot represents 1 glomerulus. Kruskal-Wallis with Dunn’s multiple comparisons tests were used.

Figure 2. mTOR-mediated podocyte hypertrophy during acute podocyte loss.

(A) Schematic representation of experimental design. (B) Urinary albumin to creatinine ratio (UACR); on the left, we show average per time point, and on the right, the evolution every 2 days per group. (C) Three-dimensional reconstruction of an intact mouse glomerulus after indirect immunofluorescence, solvent-based optical clearing, and confocal microscopy showing double labeling of podocytes with p57 (red) and synaptopodin (SNP, green). (D) Podocyte number. (E) Podocyte density. (F) Total podocyte volume per unit of podocyte number (TPV:PN ratio). (G) Total podocyte volume per unit of podocyte density (TPV:PD ratio). (H) Representative confocal image showing indirect immunofluorescence of a podocyte marker (SNP, red) and a downstream target of mTORC1 (phosphorylated ribosomal protein S6, p-rp-S6 in green) and quantification of the percentage of p-rp-S6–positive podocytes per group. (I) Quintile analysis of TPV:PD. ****P < 0.0001; ***P < 0.001; **P < 0.01. In B, bars represent means and error bars ± SEMs. In violin plots, red lines represent medians and blue lines represent IQRs; every gray dot represents 1 glomerulus. Kruskal-Wallis with Dunn’s multiple comparisons tests were used. Scale bars: (C) 30 μm, (H overview) 70 μm, and (H panels) 10 μm.

Figure 3. mTOR-mediated podocyte hypertrophy in a mouse model of FSGS.

(A) Schematic representation of experimental design. (B) Urinary albumin to creatinine ratio (UACR). (C) Periodic acid–Schiff (PAS) histological staining showing glomerulosclerosis in mice injected with high dose of DT and quantification of pathological glomeruli (%). (D) Representative confocal images after indirect immunofluorescence of a PEC-specific marker (Claudin-1; CLD1) and a PEC activation marker (CD44). (E) Podocyte number. (F) Total podocyte volume per unit of podocyte density (TPV:PD ratio). ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05. In B, bars represent means and error bars ± SEMs. Each dot represents 1 mouse. In violin plots, red lines represent medians and blue lines represent IQRs; every gray dot represents 1 glomerulus. Kruskal-Wallis with Dunn’s multiple comparisons tests were used. Scale bars: (C) 50 μm, (D) 30 μm.

Figure 4. Glomerular functional recovery is not associated with podocyte regeneration in a mouse model of FSGS.

(A) Schematic representation of experimental design, where a single diphtheria toxin (DT) injection is applied after oral doxycycline administration and a subsequent wash out (WO) period in order to genetically label podocytes with eGFP. (B) Representative images of glomerulosclerosis with quantitation of glomerulosclerosis scores. (C) De novo PEC activation indicated by upregulation of CD44 (red) in PECs using synaptopodin (SNP; green) as a podocyte marker. (D) Urinary albumin to creatinine ratio (UACR). (E) Progressive podocyte depletion and hypertrophy in glomeruli from transgenic mice injected with DT. (F) A combination of lineage tracing and immunolabeling allows FACS-based analysis of podocyte regeneration. (G) The percentage of GFP⁺ podocytes does not vary after induction of different levels of podocyte loss and development of FSGS. (H) Correlation between podocyte depletion and glomerulosclerosis scores. ****P < 0.0001; **P < 0.01; *P < 0.05. Bars represent means and error bars ± SEMs. Kruskal-Wallis with Dunn’s multiple comparisons tests were used. In correlation analyses (Spearman’s rank coefficients), solid lines represent the regression line and dotted lines the 95% CI. Scale bars: 30 μm.

Figure 5. mTOR-mediated podocyte hypertrophy in diabetic mice and humans.

(A) Transcriptional regulation of mTOR signaling in glomerular extracts from human biopsies. Comparison between diabetic nephropathy (DN; n = 14) vs. living donors (n = 42), showing fold-change of each respective gene (q < 0.05 in all genes except those with NS), and representative confocal image from indirect immunofluorescence showing a podocyte specific marker (Wilms’ Tumor 1; WT-1) and a downstream target of mTORC1 (phosphorylated ribosomal protein S6; p-rp-S6) in a glomerulus from a patient with DN. Scale bar: 10 μm. (B) Transcriptional regulation of PEC activation–related genes in glomerular extracts from human biopsies. Comparison between DN (n = 14) vs. living donors (n = 42), showing fold-change of each respective gene (q < 0.05 in all genes except those with NS) and representative confocal image of PEC activation marker CD44 (green) and PEC specific marker Annexin 3 (ANXA3; red). Scale bars: 10 μm. (C) Correlation analysis of regulated mTOR signaling and PEC activation genes. Each box represents an independent association (spearman correlation; R). Every association was statistically significant unless labeled with NS. (D) Schematic representation of experimental design for a hyperglycemic model via streptozotocin (STZ) injection with subsequent injection of diphtheria toxin (DT) to induce selective podocyte loss. (E) Fasting plasma glucose. (F) Kidney weight. (G) Periodic acid–Schiff (PAS) stainings showing development of glomerular lesions only after DT injection. (H) De novo PEC activation (CD44 upregulation; green). (I) Podocyte number. (J) Representative confocal image showing upregulation of mTORC1 signaling in podocytes after podocyte loss. (K) Podocyte density. (L) Total podocyte volume by unit of podocyte density (TPV:PD ratio). ****P < 0.0001; ***P < 0.001; *P < 0.05. In E and F, bars represent means and error bars ± SEMs. Each dot represents 1 mouse. In violin plots, red lines represent medians and blue lines represent IQRs; every gray dot represents 1 glomerulus. Kruskal-Wallis with Dunn’s multiple comparisons tests were used. Scale bars: (G) 50 μm, and (H and J) 100 μm.

Figure 6. mTORC1 hyperactivation in podocytes leads to podocyte loss and PEC activation.

(A) Schematic representation of experimental design and validation of tuberous sclerosis complex 1–KO (TSC1-KO) in isolated podocytes. (B) Representative confocal images after indirect immunofluorescence for a podocyte specific marker (Synaptopodin, SNP) and a PEC activation marker (CD44) combined with the percentage of glomeruli with CD44⁺ PECs. (C) Glomerular volume. (D) Podocyte number. (E) Podocyte density. (F) Total podocyte volume per unit of podocyte density (TPV:PD ratio). (G) Associations between percentage of glomeruli with CD44⁺ PECs and podocyte number. (H) Associations between podocyte number and glomerular volume. (I) Associations between TPV:PD and glomerular volume. (J) Associations between TPV:PD and podocyte number. ****P < 0.0001; **P < 0.01; *P < 0.05. In B, bars represent means and error bars ± SEMs. Each dot represents 1 mouse. In violin plots, red lines represent medians and blue lines represent IQRs; every gray dot represents 1 glomerulus. Kruskal-Wallis with Dunn’s multiple comparisons tests were used. In correlation analyses (Spearman’s rank coefficients), solid lines represent the regression line and dotted lines the 95% CI. Scale bars: 80 μm.

Figure 7. Pharmacological inhibition of mTOR signaling impairs podocyte hypertrophy and leads to FSGS.

(A) Schematic representation of experimental design. (B) Representative confocal images after indirect immunofluorescence showing pharmacological inhibition of ribosomal protein S6 phosphorylation (p-rp-S6; green) in podocytes (Synaptopodin; SNP in red). (C) Urinary albumin to creatinine ratio (UACR). (D) Periodic acid–Schiff (PAS) histological stainings showing glomerulosclerosis in mTOR-treated mice during induction of podocyte loss. (E) Percentage of pathological glomeruli. (F) Representative confocal images after indirect immunofluorescence showing de novo PEC activation (CD44 upregulation; green) in mice treated with mTOR inhibitors during induction of podocyte loss via diphtheria toxin (DT) administration. (G) Podocyte number. (H) Total podocyte volume per unit of podocyte density (TPV:PD ratio). ****P < 0.0001; ***P < 0.001; **P < 0.01. In C and E, circles and bars represent means and error bars ± SEM. In violin plots, red lines represent medians and blue lines represent IQRs; every gray dot represents 1 glomerulus. Kruskal-Wallis with Dunn’s multiple comparisons tests were used. Scale bars: (B) 30 μm, (D) 60 μm, and (F) 50 μm.

Figure 8. The limits of podocyte loss and hypertrophy.

(A and B) Respective comparisons of different indices of podocyte depletion and hypertrophy (in percent relative to controls) in each experimental setting presented in this study. (C) Association between percentage of podocyte hypertrophy and percentage of podocyte depletion. In C, each dot represents the average of 1 experiment; interrupted line shows the threshold of podocyte depletion, after which PEC activation and glomerulosclerosis are detectable.