AMPK mediates regulation of glomerular volume and podocyte survival

Abstract

Herein, we report that Shroom3 knockdown, via Fyn inhibition, induced albuminuria with foot process effacement (FPE) without focal segmental glomerulosclerosis (FSGS) or podocytopenia. Interestingly, knockdown mice had reduced podocyte volumes. Human minimal change disease (MCD), where podocyte Fyn inactivation was reported, also showed lower glomerular volumes than FSGS. We hypothesized that lower glomerular volume prevented the progression to podocytopenia. To test this hypothesis, we utilized unilateral and 5/6th nephrectomy models in Shroom3-KD mice. Knockdown mice exhibited less glomerular and podocyte hypertrophy after nephrectomy. FYN-knockdown podocytes had similar reductions in podocyte volume, implying that Fyn was downstream of Shroom3. Using SHROOM3 or FYN knockdown, we confirmed reduced podocyte protein content, along with significantly increased phosphorylated AMPK, a negative regulator of anabolism. AMPK activation resulted from increased cytoplasmic redistribution of LKB1 in podocytes. Inhibition of AMPK abolished the reduction in glomerular volume and induced podocytopenia in mice with FPE, suggesting a protective role for AMPK activation. In agreement with this, treatment of glomerular injury models with AMPK activators restricted glomerular volume, podocytopenia, and progression to FSGS. Glomerular transcriptomes from MCD biopsies also showed significant enrichment of Fyn inactivation and Ampk activation versus FSGS glomeruli. In summary, we demonstrated the important role of AMPK in glomerular volume regulation and podocyte survival. Our data suggest that AMPK activation adaptively regulates glomerular volume to prevent podocytopenia in the context of podocyte injury.

Keywords: Cell Biology; Cytoskeleton; Mouse models; Nephrology; Structural biology.

Figure 1. Glomerular morphometry shows significantly lower Vglom in MCD versus FSGS cases.

Glomerular morphometry was performed using glomerular area profiles and Weibel-Gomez equation on scanned images of PAS-stained paraffin sections from NS biopsies at enrollment in a subset of the NEPTUNE cohort (n = 80 biopsies; 27 MCD, 38 FSGS, 15 membranous nephropathy). Dot plots compare (A) mean Vglom (μm³) for all FSGS (black solid circles), MCD (black hollow circles), and membranous nephropathy cases (black partly solid circles) in the NEPTUNE morphometry cohort and (B) mean Vglom (μm³) in pediatric cases (≤18 years) of FSGS (n = 13) and MCD (n = 18). Line and whiskers indicate mean ± SEM; Kruskal-Wallis test with Tukey’s post test (>2 groups) Mann-Whitney U test (2 groups); **P < 0.01, ***P < 0.001; PAS, periodic acid–Schiff.

Figure 2. Global or podocyte-specific Shroom3 knockdown reduced glomerular and podocyte volume.

(A–D) Global Shroom3-KD and control mice (~12 weeks) were DOX fed for 6 weeks (n = 8 each). Kidney tissues were embedded in plastic and 1 μm sections stained with Toluidine blue. Dot plots compare (A) mean Vglom (×1000 μm³) by Cavalieri method (10 glomeruli/mouse), (B) mean podocyte numbers per glomerulus per animal (fractionator-disector method), (C) ACR (mcg/mg) at 6 weeks, and (D) single kidney weights at euthanization (mg) in knockdown versus control groups. (E–I) To induce podocyte-specific Shroom3-KD mice and controls (~12 weeks old) were DOX fed for 6 weeks (n = 6 vs. 5). Dot plots compare (E) mean Vglom (×1000 μm³) by Cavalieri method (10 glomeruli/mouse), (F) mean podocyte numbers per glomerulus per animal (fractionator-disector method), (G) ACR (mcg/mg) at 6 weeks, and (H) single kidney weights at euthanization (mg) in podocyte-specific knockdown versus control groups. (I) Panel shows electron microscopic images of representative podocyte-specific Shroom3-KD mice and controls (n = 2 each) showing podocyte FPE in knockdown glomeruli. ACR, albumin/creatinine ratio. Line and whiskers indicate mean ± SEM; unpaired t test; *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3. Shroom3 knockdown restricted glomerular hypertrophy after unilateral nephrectomy.

Global Shroom3-KD and control mice (~12 weeks) were DOX fed for 6 weeks and then subjected to unilateral nephrectomy (n = 5 vs. 4). (A) Panel shows respective representative PAS-stained images (20×) of nephrectomized and remnant kidneys (at day 7 after nephrectomy) of knockdown and control mice. (B) Dot plots compare mean kidney weight of the remnant kidney (mg) in knockdown versus control groups. (C) Percentage change (or Δ) of mean Vglom and (D) Vglom components (podocyte, mesangial, and capillary + endothelium [Cap+Endo] components) of nephrectomized and remnant kidneys for each animal are shown in dot plots. (E) Dot plots show mean podocyte numbers per glomerulus of remnant kidney per animal in each group (fractionator-disector method, 10 glomeruli/animal). Line and whiskers indicate mean ± SEM; unpaired t test; *P < 0.05, **P < 0.01.

Figure 4. Shroom3 knockdown reduces cellular protein content and RNA biogenesis in vitro and in vivo mediated via FYN.

Puromycin-selectable, stable Shroom3 and Fyn knockdown podocytes were generated using lentiviral shRNA infection (Scramble-1 and Scramble-2 are respective scramble-sequence infected controls). Stable podocytes were differentiated (>7 days) in collagen-coated plates. (A) Representative panels show forward scatter (FSC) MFI of Scramble-1, Scramble-2, Shroom3-, and FYN-shRNA podocytes; (B) shows corresponding dot plots. Arbitrary gate (circle) shows compact clustering of knockdown cells versus scramble (n = 3 sets each). (C) Dot plots compare protein/DNA ratios of Scramble-1, Scramble-2, Shroom3-, and FYN-shRNA podocytes. Cycloheximide (CHX) was used as a positive control to inhibit protein synthesis. Dot plots show mean fold-changes of (D) 18S rRNA copies and (E) ribosomal protein S26 (RPS26) transcripts in Scramble-1, Scramble-2, Shroom3-, and FYN-shRNA (normalized to actin; n = 4 sets); (F) compares 18S rRNA copies in glomerular and tubular fractions of control versus Shroom3-KD mice. Line and whiskers indicate mean ± SEM; unpaired t test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; D and E compared by paired t test; RQ, relative quantity.

Figure 5. Shroom3 or Fyn knockdown increases cellular AMPK activation.

(A) WBs of lysates from Scramble-1, SHROOM3-shRNA (top panel), and Scramble-2 FYN-shRNA podocytes (top panel) probed for SHROOM3 or FYN and phosphorylated and total MTOR, phosphorylated and total AMPK, and actin. Representative WBs of lysates from Scramble-1, SHROOM3-shRNA (bottom panel), and Scramble-2 FYN-shRNA podocytes (bottom panel) probed for SHROOM3 or FYN, phosphorylated and total EF2, phosphorylated and total ULK1, LC3-I and II, and actin. (B) Box plots show respective relative band intensity (normalized to actin) (n ≥ 3 sets). (C) Representative immunofluorescence images (left panels = 20×) of SHROOM3-shRNA and Scramble-1 cells stained for LC3 (TRITC). Right panels show confocal images obtained after 24 hours of bafilomycin treatment. (D) Dot plots quantify LC3-positive vacuoles per podocyte (n = 2 sets). (E) Representative immunofluorescence images show glomerular staining for phosphorylated AMPK (top row) and merge with DAPI (bottom row) in Shroom3-KD and control mice. (F) Box plots quantify intensity of phosphorylated AMPK/per glomerular outline per group (30 glomeruli/animal; n = 5 each). (G) Representative WBs of glomerular lysates from Shroom3-KD mice and controls probed for Shroom3, phosphorylated and total Ampk, phosphorylated and total Ef2 and actin, and (H) respective relative band intensities are shown (normalized to actin; n > 4 each). WB, Western blot. In the box-and-whisker plot, the box represents the middle quartiles, the lines indicate the median, and the whiskers denote the 5th–9th percentile. Line and whiskers indicate mean ± SEM; unpaired t test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 6. AMPK activation reduces Vglom and mitigates podocytopenia in aged Shroom3-KD mice with podocyte FPE.

Shroom3-KD and control mice were age 1 year or older and DOX fed for 6 weeks (n = 5 per group). (A) Representative WBs of kidney lysates probed for total and phosphorylated Ampk, Turbo-Gfp, and actin of young controls, aged controls, and aged Shroom3-KD mice. Dot plots show (B) blood urea nitrogen (mg/dl) in aged control and Shroom3-KD mice. (C) Dot plots show podocytes/glomerulus/animal, and box plots (line at median) show distribution of podocytes/glomerulus/group. (D) Vglom per group (×1000 μm³) between aged control and Shroom3-KD groups. (E) Representative PAS images of 2 glomeruli (63×) showing mesangial expansion in aged Shroom3-KD mice (bottom row) versus aged controls (top row). In subsequent experiments, metformin-water (MF) was added at week 2 of DOX to aged control/Shroom3-KD mice. (F) Dot plots compare albumin/creatinine ratio (μg/mg) at 6 weeks DOX in aged control versus aged Shroom3-KD mice, both with and without MF treatment. (G) Dot plots compare podocytes/glomerulus/animal and box plots (line at median) show distribution of podocytes/glomerulus/group and (H) Vglom (×1000 μm³) per group between aged control+MF and Shroom3-KD+MF groups. In the box-and-whisker plot, the box represents the middle quartiles, the lines indicate the median, and the whiskers denote the 5th–9th percentile. Line and whiskers indicate mean ± SEM; unpaired t test; *P < 0.05, **P < 0.01, ***P < 0.001; WB, Western blot; PAS, periodic acid–Schiff; WT1, Wilms’ tumor 1 protein; 30 glomerular profiles/animal were used for WT1 immunofluorescence; 10 glomeruli/animal were measured for Vglom).

Figure 7. AMPK inhibition reverses Vglom reduction and promotes podocytopenia in Shroom3-KD mice.

Shroom3-KD and control mice (~8 weeks old) were DOX fed for 8 weeks. AMPK-inhibitor Compound C was injected (20 mg/kg i.p. × 4 doses at week 5) and mice followed until week 8 when tissues were collected. (A) Dot plots show mean body weight (g) (day 0 vs. at week 8) in both groups. (B) Representative WBs of whole kidney lysates from control/Shroom3-KD mice confirmed inhibition of AMPK activation by Compound C. Dot plots show (C) blood urea nitrogen (BUN in mg/dl) and (D) morphometric quantification of Vglom (×1000 μm³) in Shroom3-KD and control mice treated with Compound C. (E) Representative immunofluorescence images (40×) of WT1 and DAPI stained glomerulus of Shroom3-KD and control mice receiving Compound C. (F) Dot plots show podocyte numbers/glomerulus/animal and box plots (line at median) show corresponding distribution of podocytes/glomerulus/group (30 glomerular profiles/mouse). In the box-and-whisker plot, the box represents the middle quartiles, the lines indicate the median, and the whiskers denote the 5th–9th percentile. Line and whiskers indicate mean ± SEM; unpaired t test; *P < 0.05, **P < 0.01, ***P < 0.001; WT1, Wilms’ tumor 1 protein.

Figure 8. AMPK activation reduces glomerular volume and preserves podocyte numbers in nephron loss–induced glomerular hypertrophy.

Adult BALB/c mice (near 8 weeks) underwent 2/3rd nephrectomy (2/3 Nx) followed by contralateral nephrectomy (Nx) 1 week later, and were followed for 6 more weeks when the remaining kidney tissue (1/6th remnant) was harvested. Experimental animals were gavaged with AMPK-activator PF06409577 at 3 doses/week; BALB/c (n = 6) versus BALB/c+PF (n = 5). (A) WBs of lysates from 2/3rd Nx and Nx kidneys showed AMPK activation in BALB/c+ PF versus BALB/c. Dot plots compare (B) albumin/creatinine ratio (μg/mg) and (C) blood urea nitrogen (mg/dl) at euthanization. (D) Box plots show the distribution of Vglom (×1000 μm³) in the 2/3rd Nx, Nx, and 1/6th remnants of BALB/c and BALB/c+PF animals (line at median; n = 10 glomeruli/sample). (E) Dot plots show intra-animal coefficients of variation of Vglom in 1/6th remnants expressed as percentage. (F) Representative images (60×) of 1/6th remnants of PAS- (top row) and MTS-stained sections (bottom row) show increased glomerulosclerosis in BALB/c group. (G) Percentage of glomeruli in 1/6th remnants with global sclerosis (on MTS stain) are shown. (H) Dot plot shows podocytes/glomerular profile/animal by WT1/DAPI stain, and box plots (line at median) show distribution of podocytes/glomerulus in each group (≥20 glomerular profiles per 1/6th remnant). In the box-and-whisker plot, the box represents the middle quartiles, the lines indicate the median, and the whiskers denote the 5th–9th percentile. Line and whiskers indicate mean ± SEM; unpaired t test; *P < 0.05, **P < 0.01, ***P < 0.001; WB, Western blot; PAS, periodic acid–Schiff; MT, Masson’s trichrome; glom, glomerulus; WT1, Wilms’ tumor 1 protein.

Figure 9. Role of AMPK signaling in glomerular volume regulation and podocyte survival in the context of podocyte injury.

Illustration depicts response of normal glomeruli to injury stimuli, i.e., nephron loss in 5/6th nephrectomy or podocyte FPE with Shroom3 knockdown (solid arcuate lines). Sustained hypertrophic stress or Ampk inhibition (Compound C or aging shown as red lines), along with podocyte FPE, led to glomerular volume dysregulation and podocytopenia. In these contexts, Ampk activation (green lines) with PF, MF, or by Shroom3 knockdown (in young mice) preserved glomerular volume regulation and protected against podocytopenia (arcuate dashed lines), thus promoting an MCD-like pathology in young Shroom3-KD mice.