Autophagy influences glomerular disease susceptibility and maintains podocyte homeostasis in aging mice

Abstract

Injury and loss of podocytes are leading factors of glomerular disease and renal failure. The postmitotic podocyte is the primary glomerular target for toxic, immune, metabolic, and oxidant stress, but little is known about how this cell type copes with stress. Recently, autophagy has been identified as a major pathway that delivers damaged proteins and organelles to lysosomes in order to maintain cellular homeostasis. Here we report that podocytes exhibit an unusually high level of constitutive autophagy. Podocyte-specific deletion of autophagy-related 5 (Atg5) led to a glomerulopathy in aging mice that was accompanied by an accumulation of oxidized and ubiquitinated proteins, ER stress, and proteinuria. These changes resulted ultimately in podocyte loss and late-onset glomerulosclerosis. Analysis of pathophysiological conditions indicated that autophagy was substantially increased in glomeruli from mice with induced proteinuria and in glomeruli from patients with acquired proteinuric diseases. Further, mice lacking Atg5 in podocytes exhibited strongly increased susceptibility to models of glomerular disease. These findings highlight the importance of induced autophagy as a key homeostatic mechanism to maintain podocyte integrity. We postulate that constitutive and induced autophagy is a major protective mechanism against podocyte aging and glomerular injury, representing a putative target to ameliorate human glomerular disease and aging-related loss of renal function.

Figure 1. Glomerular podocytes exert high levels of autophagy under basal conditions.

(A) In vivo analysis of autophagy using GFP-LC3 transgenic mice (arrow indicates autophagosome within the podocyte; nidogen served as a marker for the basement membrane). (B) Autophagosome (arrow, left) and autolysosome (arrow, right) on electron micrographs in podocytes of wild-type mice. (C) Podocytes displayed high levels of autophagy under basal conditions compared with tubular cells (n = 3 GFP-LC3 transgenic mice, 30 tubules and glomeruli of each mouse were analyzed). (D) Inhibition of autophagosomal degradation with chloroquine in differentiated podocytes induced a rapid accumulation of GFP-LC3–positive autophagosomes in GFP-LC3–transgenic podocytes. (E and F) Comparison of LC3-II accumulation in IMCD cells and differentiated podocytes in the presence of the lysosomal inhibitor chloroquine. CD2AP served as a loading control. Densitometric analysis indicated the significantly faster accumulation of LC3-II in differentiated podocytes (**P < 0.01, 2-tailed Student’s t test; 3 independent experiments). Scale bars: 20 μm (A, left, and D), 5 μm (A, middle and right); 500 nm (B), 250 nm (B, insets); 10 μm (D, insets).

Figure 2. Atg5-dependent autophagy is dispensable for glomerular development.

(A) Immunofluorescence staining of kidney sections derived from newborn GFP-LC3–transgenic mice identified autophagosomes during late podocyte differentiation in the late capillary loop stage. WT1 served as a marker for podocyte nuclei. Podocin served as a marker for podocyte foot processes. Arrows indicate GFP-LC3–positive autophagosomes. (B) Western blot analysis confirmed Atg5 deficiency in kidneys from Atg5^–/– mice by demonstrating the absence of Atg5 and the lack of LC3 conversion. (C and D) No morphological abnormalities of glomerular differentiation were detected in histology (C) or electron microscopy (D) of samples derived from fetal kidneys of Atg5^–/– mice. (E) Western blot analysis of isolated glomeruli from Atg5^Δpodocyte mice confirmed the absence of Atg5 and displayed the abrogated conversion of LC3-I. (F) Atg5^Δpodocyte mice were crossed with GFP-LC3–transgenic mice to confirm the functional ablation of autophagy. In these triple-transgenic mice, glomerular GFP-LC3–positive vesicles were completely absent and GFP-LC3 was diffusely distributed in the cytoplasm (arrows indicate cytosolic GFP signal). (G) No differences in the total number of glomeruli were detected in kidneys of Atg5^Δpodocyte mice or control littermates (P = 0.78, 2-tailed Student’s t test, n = 5 for each condition). Scale bars: 5 μm (A and F, middle and right), 20 μm (C and F, left), 2 μm (D, top), 500 nm (D, bottom).

Figure 3. Functional cross-talk between the ubi­quitin proteasome pathway and autophagy in podocytes.

(A) Results from a 12-month follow-up of Atg5^Δpodocyte mice for proteinuria (2- to 4-month-old mice: n = 6 control mice, n = 8 Atg5^Δpodocyte mice; 8- to 12-month-old mice: n = 34 control mice, n = 33 Atg5^Δpodocyte mice; ***P < 0.0001 by 1-tailed Mann-Whitney U test, z = 4.16). (B) No obvious histological phenotype in 2- to 4-month-old mice or in 8- to 12-month-old mice. (C and D) Electron microscopy analysis identified significant changes including vacuolar degeneration and ER extension (arrows indicate vacuoles) in 8- to 12-month-old Atg5^Δpodocyte mice. (E) There was no accumulation of ubiquitinated proteins in glomerulus lysate of 8-month-old Atg5^Δpodocyte mice. (F) Significant increase of proteasome activity in glomerulus lysate of 8-month-old Atg5^Δpodocyte mice (***P < 0.001, by ANOVA/Scheffe test; glomeruli from n = 3 control mice and n = 3 Atg5^Δpodocyte mice). (G) Significant decrease of proteasome activity in total kidney lysate of wild-type mice 24 hours after intravenous injection with the proteasome inhibitor bortezomib compared with control mice injected with 0.9% NaCl (**P < 0.01, by 2-tailed Student’s t test, kidney lysates from n = 3 control and n = 6 bortezomib mice). (H) Significant albuminuria of Atg5^Δpodocyte mice 24 hours after bortezomib injection (*P < 0.05, by 2-tailed Student’s t test; n = 4 control and n = 3 Atg5^Δpodocyte mice). (I and J) Inhibition of the proteasome with MG132 in differentiated podocytes resulted in an increase of converted LC3-II protein and an accumulation of GFP-LC3–positive autophagosomes. Scale bars: 20 μm (B and J), 1 μm (C, top), 200 nm (C, bottom), 2 μm (D); 10 μm (J, insets).

Figure 4. Atg5 deficiency results in an age-dependent late-onset glomerulosclerosis.

(A) Results from a 24-month follow-up of Atg5^Δpodocyte mice for proteinuria (20- to 24-month-old mice; ***P < 0.001, by 1-tailed Mann-Whitney U test, z = 3.3, n = 22 control and n = 26 Atg5^Δpodocyte mice; **P = 0.004, by Fishers exact test). (B) On histology, 22-month-old Atg5^Δpodocyte mice displayed segmental and complete glomerulosclerosis and proteinaceous casts with tubular dilatation (arrows indicate glomerulosclerosis). (C) Statistical analysis of sclerosed glomeruli (**P = 0.0068, by 2-tailed Student’s t test; glomeruli of n = 5 control and n = 7 Atg5^Δpodocyte mice, 75 glomeruli of each mouse were analyzed). (D–G) Electron microscopy analysis revealed (D) extensive vacuolar degeneration of podocyte cell bodies and foot process fusion (arrows indicate vacuoles, arrowheads indicate foot process fusion), (E) damaged mitochondria (arrowheads indicate regular mitochondria, arrows indicate damaged mitochondria), and (F and G) lipofuscin accumulation (arrows indicate lipofuscin aggregates) (***P < 0.0001, by 2-tailed Student’s t test, podocytes of n = 3 control and n = 4 Atg5^Δpodocyte mice). Scale bars: 20 μm (B), 1 μm (D), 500 nm (E), 1 μm (F).

Figure 5. ER stress and the accumulation of oxidized and ubiquitinated protein aggregates result in a loss of podocytes in Atg5^Δpodocyte mice.

(A) Vacuolized podocytes stained positive for the ER marker calnexin (arrows indicate podocytes). (B) ER stress was confirmed by the increased detection of the ER stress marker GRP94 from lysates of isolated glomeruli. (C) The proteasomal activity of glomerular lysates of 22-month-old Atg5^Δpodocyte mice was significantly reduced compared with control mice (***P < 0.001, by ANOVA/Scheffe test: glomeruli from n = 3 control and n = 3 Atg5^Δpodocyte mice). (D) Short exposures of Western blots with glomerular lysates from 22-month-old control and Atg5^Δpodocyte mice showed a significant accumulation of poly-ubiquitinated proteins. (E and F) Accumulation of the ubiquitin-associated protein p62 in Atg5^Δpodocyte mice (arrows indicate podocytes). (G and H) Glomeruli from 22-month-old Atg5^Δpodocyte mice accumulated oxidized proteins (*P = 0.0186, by 2-tailed Student’s t test, glomeruli from n = 3 control and n = 3 Atg5^Δpodocyte mice) and significantly upregulated the podocyte stress marker UCH-L1. (I) Loss of podocytes was reflected by reduced podocyte numbers per glomeruli in non-sclerosed glomeruli of 22-month-old Atg5^Δpodocyte mice compared with control mice (**P = 0.0026, by 2-tailed Student’s t test, glomeruli from n = 3 control and n = 3 Atg5^Δpodocyte mice; 30 glomeruli for each mouse were analyzed). Scale bars: 5 μm (A), 5 μm (E).

Figure 6. Upregulation of autophagy in proteinuric diseases.

(A and B) Experimental models of proteinuria (here, BSA overload) induced an approximately 3-fold increase of GFP-LC3–positive autophagosomes in GFP-LC3–transgenic mice (arrows indicate autophagosomes) (**P = 0.0012, by 2-tailed Student’s t test, n = 3 control and n = 4 BSA-injected mice, 10 glomeruli each mouse). (C) Quantitative rt-PCR of microdissected glomeruli from human renal biopsies of patients with acquired proteinuric diseases: focal segmental glomerulosclerosis (FSGS; n = 13), membranous glomerulonephritis (MGN; n = 27), minimal change disease (MCD; n = 10), and controls (pretransplant allograft biopsies; n = 9). *P = 0.0468, ***P < 0.001, by 2-tailed Student’s t test. (D) Staining of patient biopsy samples for the autophagosome marker LC3 showed an upregulation of autophagosomes in podocytes in membranous glomerulonephritis compared with controls (pretransplant allograft biopsies). Arrows indicate autophagosomes. Podocin served as a podocyte marker. Scale bars: 5 μm.

Figure 7. Autophagy is critically involved in podocyte stress adaptation.

(A) Injection of PAN resulted in a dramatic increase of albuminuria in non-proteinuric 6-month-old Atg5^Δpodocyte mice compared with control mice (n = 6 for control and for Atg5^Δpodocyte mice, *P < 0.05, by 2-tailed Student’s t test). (B–D) PAN injection in Atg5^Δpodocyte mice resulted in (B) glomerulosclerosis in PAS staining (arrow indicates a sclerosed glomerulus), (C) foot process fusion in electron microscopy (arrows indicate fused foot processes), and (D) loss of podocytes (***P < 0.001, by 2-tailed Student’s t test, glomeruli from n = 4 control and n = 4 Atg5^Δpodocyte mice; 30 glomeruli for each mouse were analyzed). (E–H) Injection of Adriamycin resulted in (E) a significant increase of albuminuria in non-proteinuric 6-month-old Atg5^Δpodocyte mice compared with control littermates (n = 6 for control and for Atg5^Δpodocyte mice, *P < 0.05, by 2-tailed Student’s t test), and in (F) glomerulosclerosis (PAS staining, arrows mark segmental sclerosed glomeruli), (G) foot process fusion in electron microscopy (arrows indicate fused foot processes), and (H) loss of podocytes (***P < 0.001, by 2-tailed Student’s t test, glomeruli from n = 3 control and n = 3 Atg5^Δpodocyte mice; 30 glomeruli for each mouse were analyzed). Scale bars: 20 μm (B and F), 1 μm (C and G).