Calpastatin Prevents Angiotensin II-mediated Podocyte Injury Through Maintenance Of Autophagy
Mar 17, 2022
for more information:ali.ma@wecistanche.com
Imane Bensaada1,3, Blaise Robin1,3, Joe¨lle Perez2, Yann Salemkour1, Anna Chipont1, Marine Camus1, Mathilde Lemoine1, Lea Guyonnet1, He´le`ne Lazareth1, Emmanuel Letavernier2, Carole He´nique1, Pierre-Louis Tharaux1 and Olivia Lenoir1
1Université de Paris, PARCC, Inserm, Paris, France; and 2Université Paris Descartes, Sorbonne Paris Cité, Paris, France
The strong predictive value of proteinuria in chronic glomerular pathies is firmly established as well as the pathogenic role of angiotensin II promoting the progression of glomerular disease with an altered glomerular fi-filtration barrier, podocyte injury and scarring of glomeruli. Here we found that chronic angiotensin II-induced hypertension inhibited autophagy efflux in mouse glomeruli. Deletion ofAtg5 (a gene encoding a protein involved autophagy)specifically in the podocyte resulted in accelerated dangio tensin II-induced podocytopathy, accentuated albuminuria, and glomerulosclerosis. This indicates that autophagy is a key protective mechanism in the podocyte in this condition. Angiotensin-II induced calpain activity in podocytes inhibits autophagy efflux. Podocytes from mice with transgenic expression of the endogenous calpain inhibitor calpastatin displayed higher podocyte autophagy at baseline that was resistant to angiotensin II-dependent inhibition. Also, sustained autophagy with calpastatin limited podocyte damage and albuminuria. These findings suggest that hypertension has pathogenic effects on the glomerular structure and function, in part through activation of calpains leading to blockade of podocyte autophagy. These findings uncover an original mechanism whereby angiotensin II-mediated hypertension inhibits autophagy via calcium-induced recruitment of calpain with pathogenic consequences in case of imbalance by calpastatin activity. Thus, preventing a calpain-mediated decrease in autophagy may be a promising new therapeutic strategy for nephropathies associated with high renin-angiotensin system activity.
KEYWORDS: angiotensin II; autophagy; calpastatin; hypertension; podocyte
Translational Statement
Given the crucial role of autophagy in the development of kidney diseases, pharmacological modulation of autophagy might be a promising strategy for the prevention and treatment of several kidney diseases. In parallel, overactivation of calpain activity in podocytes was found to play detrimental effects on podocyte function whereas its deleterious mechanisms of action were not identified. Here, we provide evidence that calpain links the deleterious action of angiotensin II to the detrimental blockade of autophagy in podocytes and suggest that calpain inhibition could be a promising therapeutic target for podocyte diseases partially through maintenance of podocyte autophagy.
Click to Cistanche deserticola ma for chronic kidney disease
Hypertension is second only to diabetes as a leading cause of progressive chronic kidney disease1–3 and even modest elevation in blood pressure is an independent risk factor for end-stage kidney disease.4 An increasing number of experimental studies have highlighted the importance of podocytes in the development of kidney injury. Progressive loss of podocytes and microvascular alterations appear early with the functional kidney decline in experimental hypertensive nephropathy.5 In patients, urinary excretion of viable podocytes was shown to be a sensitive and specific marker for preeclampsia,6,7 and patients with nephrosclerosis had a significantly lower density of glomerular podocytes than did kidney donors.8,9 Furthermore, the pathogenic role of angiotensin II (AngII) promoting the progression of glomerular disease is well established, not only in hypertensive conditions but also in several glomerular diseases.10–21
Glomerular hypertension results in glomerular capillary stretching, endothelial damage, and elevated glomerular protein filtration causing glomerular collapse and glomerulosclerosis. It also exerts a direct action on glomerular structures, causing signaling regulatory responses aimed to compensate. An activated systemic and local renin-angiotensin-aldosterone system (RAAS) fosters mesangial hyperplasia and synthesis of vascular permeability factors. Concomitantly, podocytes display calcium signaling22,23 and modify their shape upon AngII type 1 receptor (AT1)– dependent stimulation.24–28 These adaptive mechanisms become maladaptive in the long term, finally leading to glomerulosclerosis. AT1 mediates prominent RAAS involvement for blood pressure and salt and water homeostasis. Angiotensin-converting enzyme inhibitors and AT1 blockers are clinically used for the treatment of hypertension and heart failure in patients. Interestingly, both blockers also show a protective effect on kidney function.
Autophagy was demonstrated to be essential for the maintenance of cellular homeostasis, particularly in postmitotic cells29,30 and notably in podocytes.31–34 Autophagy is a lysosomal-associated degradation system for long-lived cytoplasmic proteins and dysfunctional organelles35,36 and involves sequestration of proteins and organelles in autophagosomes. The formation of autophagosomes is dependent on the induction of several genes including Map1lc3a/b, Beclin 1, and Tags. 37 There is growing evidence that dysregulation of the autophagic pathway is implicated in the pathogenesis of kidney aging and several kidney diseases such as acute kidney injury, polycystic kidney disease, aging, and diabetic nephropathy.31,32,38–41
Regulation of autophagy in podocytes, physiological and, above all, in a pathological context, is not well known. We recently demonstrated that podocyte autophagy is independent of the mechanistic target of rapamycin (mTOR) regulation in physiological conditions, making this cell type an exception.42 AT1 activation stimulates protein synthesis and protein turnover in cells. Thus, we reasoned that activation of the RAAS may also influence protein turnover stimulation and proteostasis.
In the present study, we focused on the role of AngIIsignaling in podocyte autophagy regulation. We identified the calcium-activated proteases calpains mediating a chronic blocking effect of AngII on podocyte autophagy. Further, we found that the endogenous calpain inhibitor calpastatin was able to prevent AngII-dependent autophagy inhibition and podocyte injury during hypertension.
These findings uncover an original mechanism wherebyAngII-mediated hypertension inhibits autophagy via calcium-induced recruitment of calpain with pathogenic consequences in case of imbalance by calpastatin activity.
METHODS
Animals
Calpastatin transgenic (CSTTg) mice were kindly provided by Dr. E. Letavernier.43 Mice with a podocyte-specific disruption of the Atg5 gene (Nphs2.cre Atg5lox/lox) were generated as previously described31 by crossing Nphs2.cre mice44 with Atg5lox/lox mice45 on the C57BL6/J background. Nphs2.cre Atg5lox/lox mice and control littermate males, aged 10 to 12 weeks, were used in this study. The hypertensive model was induced by s.c. infusion of AngII (Sigma-Aldrich, A9525) at a dose of 1 mg/kg/min for 4 to 6 weeks via osmotic minipumps (Alzet Corp, model 2006). Pumps were implanted s.c. on the back between the shoulder blades and hips. Mice received salt supplementation (3% NaCl) in food. Atg5lox/lox (wild-type [WT]) mice were used as controls in all studies. For deoxycorticosterone acetate (DOCA) salt with the nephron reduction model, adult male mice underwent unilateral left nephrectomy. Two weeks after nephrectomy, they received DOC pellets with the 21-day release (Innovative Research of America) implanted s.c. A second pellet was implanted 3 weeks after the first implant. All mice received 0.9% NaCl in drinking water ad libitum and were killed after 6 weeks of DOC administration.46 Experiments were conducted according to the French veterinary guidelines and those formulated by the European Community for experimental animal use (L358-86/609EEC) and were approved by the French Ministry of Research and local university research ethics committee (APAFIS-7646 and -22373).
Primary podocyte culture experiment
Differentiated primary podocytes were cultured as previously described.47,48 Briefly, the freshly isolated renal cortex was mixed and digested by collagenase I (Gibco, 17100-017) in Roswell Park Memorial Institute 1640 (Life Technologies, 61870-044). Tissues were then passed through 70 mm and 40 mm cell strainers (BD Falcon, 352340 and 352350). Glomeruli, which adhere to the 40 mm cell strainer, were removed with phosphate-buffered saline (PBS; Life Technologies, 10010023) þ 0.5% bovine serum albumin (Eurobio, HALALB07-65) injected under pressure and were then washed twice in PBS. Freshly isolated glomeruli were plated in 6-well dishes in Roswell Park Memorial Institute 1640 (Gibco, 61870036) supplemented with 10% fetal calf serum and 1% penicillin/streptomycin (Life Technologies, 15140122) to allow podocytes to exit from glomeruli and grow. Podocyte enrichment was verified by Western blot analysis as previously described31,48,49 (Supplementary Figure S1). Podocytes were cultured in the absence or presence of bafilomycin A1 (100 nmol/l, Sigma-Aldrich, B1793) for 4 hours. For immunofluorescence experiments, primary podocytes were plated on 4 dishes labels (Dutcher, 055071). Podocytes were then fixed in paraformaldehyde 4% for 10 minutes and processed for immunofluorescence.
Calpain activity assay
Intracellular calpain activity was determined in primary podocytes, as previously described.50–52 A total of 100,000 cells were cultured in 24-well tissue culture dishes in Roswell Park Memorial Institute 1640 supplemented with 10% fetal calf serum and 1% penicillin/streptomycin. After the indicated culture period, the medium was replaced with Krebs-Ringer HEPES bicarbonate (KRH) solution (pH 7.4) containing 4 mM CaCl2, with or without 10 mM calpain inhibitor-1, and incubated for 10 minutes before the addition of 50 mM calpain substrate N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methyl coumarin (Sigma-Aldrich, S6510). After a 90-minute incubation period, calpain activity was determined as the difference between FL fluorescence (measured at 360 nm excitation and 430 nm emission) with and without calpain inhibitor-1.
Western blot
Primary podocytes were scratched with 80 ml of radioimmunoprecipitation assay buffer containing phosphatase and protease inhibitor. Protein concentration was measured with the BCA Protein Assay Kit (Merck Biochemistry, 71285). Twenty micrograms of proteins were electrophoresed on Criterion XT precast gel (12%Bis-Tris, Bio-Rad, 3450124). Proteins were transferred to polyvinylidene difluoride membrane (Thermo Fischer Scientific, 88518). After blocking in 5% milk in Tris Buffer Saline 0.1% Tween (TBS-T), membranes were incubated with rabbit polyclonal anti-LC3 (1:1000, Cell Signaling Technology, 2575), rabbit polyclonal anti-ATG5(1:2000, Cell Signaling Technology, 2630), guinea pig polyclonal anti–Sequestosome 1 (SQSTM1)/P62 (1:10,000, PROGEN, GP62), rabbit polyclonal anti–calpain 1 domain IV (1:1000, Abcam, ab39170), rabbit polyclonal anti–calpain 2 amino-terminal end of domain I (1:1000, Abcam, ab39165), mouse monoclonal IgG1 anti–calpain 4 (1:1000, Santa Cruz Biotechnology, sc-32325), rabbit antipodocin (1:1000, Abcam, ab50339), guinea pig anti-nephrin (1:500, PROGEN, GP-N2), and rat monoclonal anti-tubulin (1:5000, Abcam,ab6160) antibody. After washing, membranes were incubated with horseradish peroxidase–linked antibody (1:2000, Cell Signaling Technology, 7074, 7076, 7077). The detection of specific signals was performed using the ECL Chemiluminescent Kit (Bio-Rad, 170-5070) on LAS 4000 device (Fuji). Densitometry analysis with ImageJ software(National Institutes of Health) was used for quantification.
Blood pressure measurements and physiological assessments
The systolic blood pressure of mice was recorded using the tail-cuff method (Visitech Systems Inc., BP-2000). Ten measurements from each mouse were taken, and then a mean value was determined. Systolic blood pressure was measured at baseline (12 weeks of age)and then weekly until the end of the treatment period. All mice were placed in metabolic cages with free access to water for a 6-hour urine collection. Urinary creatinine and plasma urea concentrations were analyzed spectrophotometrically by using a colorimetric method(Olympus, AU400). Urinary albumin excretion was measured using a specific enzyme-linked immunosorbent assay for the quantitative determination of albumin in mouse urine (Crystal Chem, 80630).
Histology
Kidneys were harvested and fixed in 4% PBS-buffered formalin. Paraffin-embedded sections (3-mm thick) were stained by Masson’strichrome to evaluate kidney morphology. Abnormalities in kidneys were graded on the basis of the presence and severity of component abnormalities, including glomerulosclerosis, mesangial expansion, tubular atrophy or casts, and fibrosis. The proportion of sclerotic glomeruli was evaluated by a blind examination of at least 50glomeruli per kidney section.
Immunofluorescence staining of kidney sections and primary podocytes
Fixed primary podocytes were blocked in TBS-T 3% bovine serum albumin and incubated overnight at 4°C with primary anti-bodies guinea pig anti-SQSTM1/P62 (1:1000, PROGEN, GP-62C) and rabbit anti-green FL fluorescent protein (GFP; 1:500, Abcam, ab290). After TBS-T rinses, FL fluorophore-conjugated secondary antibodies donkey anti–guinea pig IgG AF594-conjugated antibody (JacksonImmunoResearch, 706-585-148) and donkey anti-rabbit IgG AF488-conjugated antibody (Invitrogen, A21206) was applied. Images were taken using a Zeiss 2 ft fluorescent microscope, an AxioCam HRccamera, and Axiovision 4.3 software.
For formalin-fixed paraffin-embedded (FFPE) kidneys, sections(3 mm) were deparaffinized and hydrated and antigen retrieval was performed in heated citrate buffer (pH 6). Sections were then permeabilized with Triton 0.1% (Euromedex) and blocked in TBS-T 3%bovine serum albumin before overnight antibody incubation at 4 C.We used goat anti-nephrin (1:100, PROGEN, GP-N2), guinea pig anti-SQSTM1/P62 (1:1000, PROGEN, GP-62C), rabbit anti-GFP(1:1000, Abcam, ab290), goat anti-Podocalyxin (PODXL; 1:1000, Bio-Techne, AF-1556), and rabbit anti–Wilmʼs Tumor 1 (WT1;1:100, Abcam, ab192) antibody. Secondary antibodies were Alexa488– and Alexa 568–conjugated antibodies from Invitrogen. Nuclei were stained in blue using Hoechst. Slides were mounted using a fluorescent mounting medium (Dako, S3023). Photomicrographs were was taken with a Zeiss Axiophot photomicroscope and Axiovisionsoftware. Semiautomatic quantifications on Fiji were used for quantification of nephrin-positive and PODXLþ areas per glomerular section on at least 30 glomeruli per mouse. Podocytenumber was counted as the number of WT1þ nuclei per glomerular section on at least 30 glomeruli per mouse.
Transmission electron microscopy procedure
Small pieces of the renal cortex (1 mm3) were fixed in 3% glutaraldehyde grade (Electron Microscopy Sciences) for 1 to 30 days and washed thrice in PBS. Samples were postfixed in 1% osmium tetroxide 0.1 M (Electron Microscopy Science) in 0.1 M PBS (pH 7.4)and washed in water. Samples were dehydrated in alcohol grades and100% propylene oxide (Electron Microscopy Science). Resin infiltration was performed as follows: mix Epikote 812 and propylene oxide in a ratio of 1:1 for 30 minutes followed by mix Epikote 812and propylene oxide in a ratio of 1:2 for overnight room temperature. Samples were embedded in 4 mm gelatin capsules in 100%Epikote 812 and polymerized in an oven heated to 60 C. Ultrathin sections were cut with a UFC7 ultramicrotome (Leica MicrosystemsGmbH) and deposed on Gilder Grids 200 mesh (Electron Microscopy Science). They were counterstained with uranyl acetate 7%(LFG Distribution) and Reynold’s lead citrate (LFG). Samples were examined in the JEM1011 transmission electron microscope (JEOL)with the Orius SC1000 CCD camera (Gatan), operated with Digital Micrograph software (Gatan) for acquisition.
Cistanche-kidney disease
Quantitative polymerase chain reaction array
Freshly isolated glomeruli were frozen in QIAzol Lysis reagent (Qiagen) at-80℃. Total RNA extraction using the phenol-based method was processed according to the manufacturer’s recommendations. cDNAs were synthesized using the RT2 First Strand Kit (Qiagen, 330401), and real-time polymerase chain reaction (PCR) was performed using a Custom RT2 Profiler PCR Array (Qiagen, CLAM36771C) with RT2 SYBR Green qPCR Mastermix (Qiagen, 330502). The quantitative PCR plates were run on an Applied Biosystems StepOnePlus cycler. Each array contains quality control for reverse transcription efficiency and genomic DNA contamination. Quantitative PCR analysis was performed using the 2-DDCT method with the help of the GeneGlobe Data Analysis Center (www. Qiagen. com/shop/genes-and-pathways/data-analysis-center-overview-page) and expressed as the Log2 fold change in gene expression.
In silico proteomic analysis
In silico prediction of the calpain cleavage site was done with DeepCalpain (http://deepcalpain.cancerbio.info/help.php), GPS-CCD (http://ccd.biocuckoo.org), and CaMPDB (http://calpain.org/) online tools. Mouse protein amino acid sequence was obtained from Uniprot (https://www.uniprot.org). The results are resumed in Supplementary Tables S1 and S2.
Statistical analyses
All graphs represent individual values and mean±SEM. Statistical analyses were performed using GraphPad Prism software, version 9. Comparison between the 2 groups was performed using a parametricStudent t-test when samples passed the Anderson-Darling andD’Agostino normality tests and F test for equality of variance. Otherwise, a nonparametric Mann-Whitney test was used. Comparison between multiple groups was performed using 1-way or 2-way analysis of variance followed by a multiple comparison test with Simak's correction. Values of P < 0.05 were considered signifificant.*P < 0.05, **P < 0.01, ***P < 0.001.
Figure 1 | Angiotensin II (AngII) D high-salt diet (HSD)–induced hypertension inhibits glomerular autophagy. (a–c) Immunofluorescence of Podocalyxin (PODXL; red) and P62 (green) in glomeruli (a,a0 ) from wild-type (WT) mice, (b,b0 ) from WT mice after 6 weeks of AngII þ HSD, and (c,c0 ) from Nphs2.cre Atg5lox/lox mice showing the accumulation of P62 in podocytes during hypertension and in podocyte-specific ATG5-deficient mice. The arrowheads indicate P62þ dots in WT in (a0 ). Nuclei were counterstained with Hoechst (blue). Figure subparts with prime indicate higher magnification. Bars=50 mm. (d) Associated quantification of the P62þ area expressed as the percentage of the glomerular area. n=4 WT mice and n=5 WT with AngII þ HSD, and Nphs2.cre Atg5lox/lox mice. Values are presented as individual plots and mean±SEM. Mann-Whitney test: *P=0.0159. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.
RESULTS
AngII D high-salt diet-induced hypertension inhibited podocyte autophagy
Podocytes present a high level of autophagy in vivo, as shown by strong GFP expression in transgenic mice with GFP fusion to LC3 (GFP-LC3 mice), a key marker of autophagy (Supplementary Figure S2A). Autophagy is a dynamic process with the constant formation of autophagosomes and degradation of autophagolysosomes. Blocking autophagosomal degradation with chloroquine resulted in the accumulation of GFPþ dots, indicating high autophagic efflux in podocytes (Supplementary Figures S2A and B). Confirmation that GFPþ dots were autophagosomes was shown by double immuno- FL fluorescence for GFP and SQSTM1/P62, a chaperone protein degraded by autophagy (Supplementary Figure S2C and D). Again, chloroquine treatment induced the accumulation of GFPþ P62þ dots, demonstrating important autophagic efflux in podocytes. Finally, high autophagic efflux was conserved in vitro as shown by GFP and P62 expression in primary podocytes isolated from GFP-LC3 mice and strong accumulation of GFPþ and P62þ dots under bafilomycin A1 treatment, another blocker of autophagosomal degradation (Supplementary Figure S2E–H).
The capacity of hypertension to modulate podocyte autophagic responses was then assessed in mice infused with AngII with a high-salt diet (HSD) for 6 weeks and in nonhypertensive controls. As shown in Figure 1, AngII þ HSD induced P62 accumulation in glomeruli with strong accumulation in podocytes, thus suggesting that AngII þ HSD was responsible for podocyte autophagy blockade (Figure 1a–d). Interestingly, P62 accumulation in podocytes was similar to the one observed in mice deficient for podocyte autophagy (Nphs2.cre Atg5lox/lox mice). In another model of hypertension, the DOC-salt model, we also observed progressive P62 accumulation in podocytes along the time course of the disease (Supplementary Figure S3).
Deletion of Atg5 specifically in podocytes results in increased albuminuria, podocyte loss, and glomerular injury in the AngII D HSD model
We then examined whether autophagy blockade only in podocytes (Nphs2.cre Atg5lox/lox mice) affects glomerular injury in the AngII þ HSD model. We first confirmed that Nphs2. cre Atg5lox/lox mice had normal blood pressure, normal kidney function, and no glomerular histological lesions until 10 months of age, as previously reported (Supplementary Figure S4).32 Then, Atg5lox/lox (WT) and Nphs2. cre Atg5lox/ lox mice were infused with AngII with HSD for 6 weeks. Importantly, systolic blood pressure was similar in the 2 groups after AngII infusion during the course of the study (Figure 2a), although the tail-cuff method used to measure blood pressure might not have the ability to resolve small blood pressure differences. AngII infusion with HSD markedly increased urinary albumin-to-creatinine ratio in WT mice, and this effect was further significantly increased in Nphs2.cre Atg5lox/lox mice (Figure 2b). Hypertensive Nphs2.cre Atg5lox/lox mice also displayed significantly increased glomerular sclerosis when compared with WT littermates (Figure 2c–e). In agreement with the measured proteinuria, proteinaceous casts and tubular dilatation were significantly more prevalent in mice with podocyte deficiency in ATG5 (Figure 2f–h).
Figure 2 | Deletion of Atg5 specifically in podocytes results in a significant increase in albuminuria, kidney injury, and podocyte loss after 6 weeks of angiotensin II (AngII) infusion D high-salt diet (HSD). (a) Systolic blood pressure in Atg5lox/lox and Nphs2.cre Atg5lox/lox mice during 36 days of AngII þ HSD. n=9 mice per genotype. Values are presented as mean±SEM. Two-way analysis of variance (ANOVA): ns. In (b–m), n=10 mice per genotype. In (b,g,h,k), values are presented as individual plots and mean±SEM. (b) AngII þ HSD (continued)
Figure 2 | (continued) resulted in a dramatic increase of albuminuria in Nphs2. cre Atg5lox/lox mice compared with Atg5lox/lox control mice. Two-way ANOVA: genotype, P=0.013; time, P=0.0018. (c–f) Representative images of Masson’s trichrome–stained sections of glomeruli from Atg5lox/lox control and Nphs2.cre Atg5lox/lox mice after 6 weeks of AngII þ HSD. Bars=50 mm in (c,d). Bars=100 mm in (e,f). (g,h) Comparison (g) of the proportion of sclerotic glomeruli and (h) of the number of tubular casts per microscopic field. Mann-Whitney test: **P=0.0026 in (h), ***P=0.0003 in (g). (i,j) Representative immunofluorescence images of the expression of Wilmʼs Tumor 1 (WT1; red) and Podocalyxin (PODXL; green) in glomeruli from Atg5lox/lox control and Nphs2.cre Atg5lox/lox mice after AngII þ HSD for 6 weeks. Nuclei were stained with Hoechst (blue). Bars=50 mm. (k) Quantification of the number of WT1þ cells per glomerular section. Mann-Whitney test: **P=0.0029 in (l,m). Representative immunofluorescence images of the expression of CD44 (green) in glomeruli from Atg5lox/lox control and Nphs2.cre Atg5lox/lox mice after AngII þ HSD for 6 weeks. Nuclei were stained with Hoechst (blue). Bars=50 mm. (n,o) Representative transmission electron microscopy photomicrographs of sections of glomeruli from Atg5lox/lox control and Nphs2.cre Atg5lox/lox mice after AngII þ HSD for 6 weeks, showing podocyte foot process effacement (arrowheads) in hypertensive Nphs2. cre Atg5lox/lox mice. Bars=1 mm. n=3 mice per genotype. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.
Figure 3 | Calpain expression and activity in podocytes. (a) Western blot analysis of the expression of calpain-1, calpain-2, and calpain-4 in primary podocytes. Tubulin expression serves as normalization. (b) Calpain activity was measured on primary podocytes treated or not treated with angiotensin II (AngII; 100 nM) for 24 hours with or without calpeptin (10 mM). n=5 independent experiments. Values are presented as individual plots and mean±SEM. One-way analysis of variance (ANOVA): treatment, P=0.0035. Sidak’s multiple comparison test: *P=0.0128 for AngII versus baseline, ##P=0.0056 for AngII þ calpeptin versus AngII. (c) Calpain activity was measured on primary podocytes from wild-type (WT) or calpastatin transgenic (CSTTg) mice treated or not treated with AngII (100 nM) for 24 hours. n=7 independent experiments. Values are presented as individual plots and mean±SEM. Two-way ANOVA paired for treatment: genotype, P=0.0483. Sidak’s multiple comparison test: ***P=0.0009 for WT AngII versus baseline, *P=0.0420 for CSTTg AngII versus baseline, #P=0.0424 for WT versus CSTTg AngII. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.
Podocyte number per glomerulus was significantly decreased in Nphs2. cre Atg5lox/lox mice treated with AngII þ HSD (Figure 2i–k). Podocyte injury in Nphs2.cre Atg5lox/lox mice with AngII infusion þ HSD even progressed to focal and segmental glomerulosclerosis as shown by expression of the parietal epithelial cell (PEC) activation marker CD44 in glomeruli (Figure 2l and m). Electron microscopy analysis identified significant changes associated with ATG5 deficiency upon chronic AngII infusion with HSD, including foot process effacement in hypertensive Nphs2. cre Atg5lox/lox mice. By contrast, few ultrastructural defects were found in podocytes from WT mice even after 10 weeks of AngII infusion with HSD (Figure 2n and o), indicating that the autophagic activity of podocytes is required for their resistance to AngII þ HSD–induced damage. Altogether, our results indicated that in the AngII þ HSD model, autophagy inhibition aggravates podocyte injury and loss and induces subsequent focal and segmental glomerulosclerosis.
AngII D HSD activates calpain activity in podocytes that contributes to autophagy blockade
As calpain activity was found (i) to be activated by AngII in several cell types and (ii) to cleave several autophagy-related proteins,53 we wondered if AngII þ HSD– mediated autophagy blockade could be attributable to increased AngII-induced calpain activity. We first found that primary podocytes expressed the 3 ubiquitous forms of calpains (Figure 3a). We then showed that AngII stimulated calpain activity in primary podocytes. This rise in calpain activity was blocked by a selective calpain inhibitor (Figure 3b). We used a knock-in mouse with additional calpastatin transgene expression (leading to decreased calpain activity)43 to assess the role of calpains in AngII þ HSD–mediated kidney injury and autophagy blockade. Primary podocytes from CSTTg mice showed decreased calpain activity in response to AngII when compared with podocytes from control mice (Figure 3c). Thus, calpastatin overexpression in podocytes decreased AngII-mediated calpain activation.
We next assessed autophagy levels in podocytes from CSTTg mice. We generated CSTTg mice with the GFP-LC3 transgene. LC3-GFP reporter allowed counting of autophagosomes as GFPþ/P62þ dots. P62 will accumulate in aggregates in cells when autophagic efflux is blocked. At the basal state, we counted fewer GFPþ P62þ dots (Figure 4a, b, and e) and less P62 accumulation (Figure 4c, d, and f) in podocytes of CSTTg GFP-LC3 mice than in podocytes of normal GFP-LC3 mice, suggesting increased autophagic efflux in podocytes of mice with high calpastatin abundance.
Figure 4 | Evaluation of autophagic efflux in podocytes of calpastatin transgenic (CSTTg) mice. (a,b) Representative immunofluorescence images of the expression of green FL fluorescent protein (GFP) (green) and P62 (red) in glomeruli from 12-week-old GFP-LC3 and CSTTg GFP-LC3 mice. The arrowheads indicate GFPþ P62þ autophagosomes. (c,d) Representative immunofluorescence images of the expression of P62 (green) and Podocalyxin (PODXL; red) in glomeruli from 12-week-old GFP-LC3 and CSTTg GFP-LC3 mice. Figure subparts with prime indicate higher magnification. Nuclei were stained with Hoechst (blue). Bars=50 mm. (e) Quantification of the number of LC3þ P62þ dots per podocyte. Mann-Whitney test: **P= 0.0065. (f) Quantification of P62þ area per glomerular section. Mann-Whitney test: *P=0.0420. In (e,f), n=5 GFP-LC3 mice and n=8 CSTTg GFP-LC3 mice. Values are presented as individual plots and mean±SEM. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.
Autophagic efflux can be monitored by the measurement of the conversion of the cytoplasmic form of LC3, LC3-I, to the autophagosomal cleaved and phosphatidylethanolamine– coupled form of LC3, LC3-II, on the Western blot. Podocytes from CSTTg mice showed increased LC3-I to LC3-II conversion and decreased P62 expression, both in the presence and in the absence of bafilomycin A1 (Figure 5a and b), indicating increased autophagic efflux in podocytes with calpastatin overexpression. Finally, the accumulation of GFPþ P62þ dots in podocytes after chloroquine administration was more important in CSTTg GFP-LC3 mice than in GFP-LC3 mice, thus confirming that calpastatin overexpression induces autophagic efflux in podocytes in vivo (Figure 5c–e). Altogether, our data suggest that AngII stimulates calpain activity in podocytes, that autophagy is inhibited by calpain in podocytes, and that inhibition of the endogenous calpain activity by calpastatin overexpression is sufficient to stimulate autophagic efflux in podocytes.
CSTTg mice are protected from AngII D HSD–induced podocyte injury
CSTTg mice did not show any kidney alteration until at least 12 months of age (Supplementary Figure S5). We analyzed podocyte injury in CSTTg mice during AngII þ HSD treatment. Although WT mice developed mild glomerulosclerosis and podocyte injury after 4 weeks of hypertension, as shown by abnormal expression of podocalyxin and nephrin, CSTTg mice presented fewer sclerotic glomerular lesions (Figure 6a and b) and preserved podocalyxin and nephrin expression (Figure 6c–h). Such differences in podocyte phenotype were observed at a stage where the density of podocyte nuclei was not different between WT and hypertensive CSTTg mice (Figure 6i–k).
Figure 5 | Blocking autophagosomal degradation confirmed increased podocyte autophagic efflux in calpastatin transgenic (CSTTg) mice. (a) Western blot analysis of the expression of LC3, Sequestosome 1 (SQSTM1)/P62, and ATG5 in primary podocytes from wild-type (WT) or CSTTg mice. Tubulin expression serves as normalization. Podocytes were treated or not treated with bafilomycin A1 (BafA1; 100 nM) for 4 hours before culture arrest. (b) Quantification of the LC3-II/tubulin and P62/tubulin ratios. n=10 WT mice and n=8 CSTTg mice. Values are presented as individual plots and mean±SEM. Two-way analysis of variance paired for treatment: for LC3-II/tubulin: genotype, P=0.0008; treatment, P < 0.0001; for P62/tubulin: genotype, P=0.0884; treatment, P < 0.0001. Sidak’s multiple comparison test: for LC3-II/tubulin: ***P < 0.0001 for WT+BafA1 versus WT þ BafA1, ***P < 0.0001 for CSTTg+BafA1 versus CSTTg þ BafA1, ##P=0.0028 for WT þ BafA1 versus CSTTg þ BafA1; for P62/tubulin: ***P < 0.0001 for WT+BafA1 versus WT þ BafA1, ***P < 0.0001 for CSTTg+BafA1 versus CSTTg þ BafA1. (c,d) Representative immunofluorescence images of the expression of green FL fluorescent protein (GFP; green) and P62 (red) in glomeruli from 12- week-old GFP-LC3 and CSTTg GFP-LC3 mice. Figure subparts with prime indicate higher magnification. Nuclei were stained with Hoechst (blue). Bars=50 mm. Mice were treated with chloroquine (CQ; 80 mg/kg) 4 hours before killing. The arrowheads indicate GFPþ P62þ autophagosomes. (e) Quantification of the number of LC3þ P62þ dots per podocyte. n=5 mice per genotype. Values are presented as individual plots and mean±SEM. Unpaired t-test with equal SD: *P=0.0302. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.
Figure 6 | Calpastatin overexpression prevents angiotensin II (AngII) D high-salt diet (HSD)–mediated podocyte injury. (a,b) Representative images of Masson’s trichrome–stained sections of glomeruli from wild-type (WT) and calpastatin transgenic (CSTTg) mice after 6 weeks of AngII þ HSD. Bars=50 mm. (c,d,f,g) Representative immunofluorescence images of the expression of (c,d) Podocalyxin (PODXL) and (f,g) nephrin (NPHS1) in WT and CSTTg mice after 6 weeks of AngII þ HSD and (e,h) associated quantifications. Bars=50 mm. (i,j) Representative immunofluorescence images of the expression of Wilmʼs Tumor 1 (WT1; green) and PODXL (red) in glomeruli from WT and CSTTg mice after 6 weeks of AngII þ HSD and (k) associated quantification of the number of WT1þ cells per glomerular section. Nuclei were stained with Hoechst (blue). Bars=50 mm. In (e,h,k), values are presented as individual plots and mean±SEM. n=9 WT mice and n=7 CSTTg mice. Unpaired t-test with equal SD: **P=0.0095 in (e), *P=0.0249 in (h), P=0.7891 in (k). To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.
Similarly, after 6 weeks of hypertension, GFP-LC3 and CSTTg GFP-LC3 mice presented podocyte injury but lesions were more prominent in GFP-LC3 mice (Figure 7). The urinary albumin-to-creatinine ratio was higher in GFP-LC3 mice after 4 weeks of AngII þ HSD (Figure 7a). Podocyte number was not different between the 2 genotypes, but nephrin expression was significantly more decreased in GFP-LC3 (control) mice (Figure 7b–e). Correlating with calpastatin-mediated protection, the ultrastructural analysis showed mild and focal podocyte foot process effacement in GFP-LC3 mice treated with AngII þ HSD with better preservation of the foot process effacement in CSTTg mice, although the global quantification of the number of foot processes per glomerular basement membrane (GBM) length was not statistically different, most likely because podocyte injury is focal and segmental in our model (Figure 7f–h). Of note, macrophages and T-lymphocyte infiltration in kidneys were not different between the groups (Supplementary Figure S6). Taken together, these results demonstrated that calpastatin prevented AngII þ HSD–induced podocyte injury.
Calpastatin overexpression restores autophagic efflux in podocytes from mice after AngII D HSD treatment
Finally, we wondered whether calpastatin-mediated glomerular protection in the AngII þ HSD model implicated autophagy maintenance in podocytes. Interestingly, AngII þ HSD–induced P62 accumulation in podocytes was prevented in CSTTg and GFP-LC3 CSTTg mice (Figure 8a–d), indicating that calpastatin prevented blockade of podocyte autophagy. Of note, the number of GFPþ P62þ dots labeling of autophagosomes was similar in podocytes from hypertensive GFP-LC3 and GFP-LC3 CSTTg mice, thus suggesting that AngII þ HSD induced a slowdown of podocyte autophagic efflux rather than a complete arrest (Figure 8e–g).
In silico prediction of the calpain cleavage site in podocyte proteins
We used several in silico tools to predict potential calpain targets in podocytes (Supplementary Tables S1 and S2). Three online databases were compared: GPS-CCD,54 CaMPDB,55, and DeepCalpain.56 In silico analysis identified several podocyte proteins, nephrin and podocin among them, that could be cleaved by calpains. Thus, calpastatin-mediated glomerular protection could be linked to a reduction of calpain enzymatic activity, leading to reduced degradation of some podocyte proteins.
Furthermore, at least 3 autophagy-related proteins are direct targets of calpains. ATG5 is cleaved by calpains, leading to a disturbance in the ATG12-ATG5 complex formation.57,58 Administration of calpain inhibitors in vivo also prevented cleavage of the autophagy protein Beclin-1.59 In silico analysis of the putative cleavage site on autophagy-related proteins supports the hypothesis that calpain could regulate autophagy through the enzymatic cleavage of autophagy proteins.
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mRNA expression of endoplasmic reticulum (ER) and oxidative stress markers in glomeruli from mice treated with AngII + HSD
We evaluated endoplasmic reticulum (ER) stress and oxidative stress by quantitative PCR in glomeruli during AngII þ HSD treatment (Table 1). At baseline, we did not observe any change in mRNA expression of analyzed genes in glomeruli from WT and Nphs2.cre Atg5lox/lox mice (Supplementary Figure S7). After 6 weeks of hypertension, glomeruli from Nphs2. cre Atg5lox/lox mice showed different mRNA profile of genes of the ER stress and oxidative stress pathways with increased expression of Sod1, Prdx1, Atf4, Gpx1, and Hsp90b1 as compared with WT glomeruli, thus suggesting that autophagy depletion in podocytes favored AngII þ HSD–induced ER stress and oxidative stress. Conversely, glomeruli from CSTTg mice presented downregulation of several genes of the ER stress and oxidative stress pathways as well as decreased expression of some pro-apoptotic genes (Figure 9).
Taken together, these results indicate that calpastatin overexpression could prevent glomerular injury by reducing AngII þ HSD–induced ER and oxidative stress.
DISCUSSION
In the present study, we demonstrated that in AngII þ HSD–induced hypertension, podocyte autophagy is markedly downregulated. Furthermore, mice with podocyte-specific deletion of Atg5 were more prone to AngII þ HSD–induced glomerulosclerosis and podocyte loss, thus showing that autophagy in podocytes prevents the development of hypertensive nephropathy, highlighting the critical role of autophagy in AngII þ HSD–induced podocyte injury. Little is known about the extracellular stimuli that regulate cellular autophagy, and these results shed light on the pathophysiological regulation of podocyte autophagy by AngII.
In most human glomerulopathies, podocyte foot process effacement is a hallmark of glomerular injury leading to proteinuria. Autophagy is likely to play an essential role in maintaining podocyte function because these terminally differentiated cells display high rates of autophagy even in the absence of stress. A previous study showed that AngII promotes autophagy through the generation of reactive oxygen species in a conditionally immortalized murine podocyte cell line.60 Reactive oxygen species production is indeed a general inducer of autophagy in many cell types and the reasons for such discrepancy with our findings are unclear. Unlike this latter study, we used murine primary cultured podocytes retaining podocin and nephrin expression and in vivo approaches and not murine cell lines. We confirm previous reports that postmitotic podocytes exhibit an unusually high level of constitutive autophagy. Measurement of the increased amount of LC3-II after AngII stimulation in the presence or absence of lysosomal inhibitors is necessary to determine whether autophagic efflux is increased or blocked. Previous studies have neglected this phenomenon.60
Figure 7 | Calpastatin overexpression prevents angiotensin II (AngII) D high-salt diet (HSD)–mediated podocyte injury in green FL fluorescent protein (GFP)–LC3 mice. (a) AngII þ HSD resulted in a dramatic increase of albuminuria in GFP-LC3 mice compared with CSTTg (calpastatin transgenic) GFP-LC3 mice. n=8 to 13 mice per genotype. Values are presented as individual plots and mean±SEM. Two-way analysis of variance: genotype, P=0.0429; time, P=0.0165. Sidak’s multiple comparison test: *P=0.0453 for GFP-LC3 versus CSTTg GFP-LC3 mice at day 28. (b,c) Representative immunofluorescence images of the expression of Wilmʼs Tumor 1 (WT1; green) and nephrin (NPHS1) (red) in glomeruli from 18-week-old GFP-LC3 and CSTTg GFP-LC3 mice after 6 weeks of AngII infusion þ HSD. Nuclei were stained with Hoechst (blue). Bars=50 mm. Quantification of (d) NPHS1þ area per glomerular section and (e) the number of WT1þ cells per glomerular section in 18-week-old GFP-LC3 and CSTTg GFP-LC3 mice after 6 weeks of AngII infusion þ HSD. n=19 GFP-LC3 mice and n=25 CSTTg GFP-LC3 mice. Values are presented as individual plots and mean±SEM. Unpaired t-test with equal SD: **P=0.0010 in (d), P= 0.1158 in (e). (f,g) Transmission electron microscopy images of glomeruli from GFP-LC3 and CSTTg GFP-LC3 mice after 6 weeks of AngII þ HSD. The arrowheads indicate foot process effacement. Bars=1 mm. (h) Quantification of the number of foot processes per micrometer of glomerular basement membrane (GBM). n=3 mice per genotype. Values are presented as individual plots and mean SEM. Each plot represents the mean number of podocytes per micrometer of GBM on 1 continuous length of GBM. Unpaired t-test with equal SD: P=0.7512. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.
Here, we used a hypertensive model based on AngII perfusion and HSD. Podocytes exhibit AT1 receptors and are exposed to freely filtered peptides such as AngII.13,16,26,61–65 It is assumed that the observed renoprotective effects of RAAS inhibition could be—at least partially—due to a blockade of this podocyte-specific RAAS. Likewise, in vivo studies confirmed the effect of AngII on podocyte injury and podocyte-specific gene targeting of AT1 demonstrated that activation of AT1 receptors in the glomerulus in experimental lupus nephritis is sufficient to accelerate kidney injury in the absence of hypertension.66,67 Calpain-mediated autophagy dysregulation in our model could be linked to direct AngII-AT1 signaling on podocytes or to be a consequence of hypertension. We may provide an early answer to this question. Indeed, in the DOC-salt model, we also found P62 accumulation in podocytes from hypertensive mice (Supplementary Figure S3). This suggests that autophagy blockade occurs in podocytes in this model, which is supposed to be independent of AngII.68 Further studies evaluating podocyte injury pertaining to calpain activity and autophagic efflux in mice deficient for AT1 in podocytes selectively would be required to delineate if such regulation of podocyte autophagy depends on direct or indirect effects of AngII.
Figure 8 | Calpastatin overexpression prevents angiotensin II (AngII) D high-salt diet (HSD)–induced autophagy downregulation in podocytes. (a,b) Representative immunofluorescence images of the expression of P62 (green) and Podocalyxin (PODXL; red) in glomeruli from green FL fluorescent protein (GFP)–LC3 and CSTTg (calpastatin transgenic) GFP-LC3 mice after 6 weeks of AngII þ HSD. Figure subparts with prime indicate higher magnification. Nuclei were stained with Hoechst (blue). Bars=50 mm. (c,d) Quantification of P62þ area per glomerular section. n=9 wild-types (WT) mice and n=7 CSTTg mice in (c), and n=16 GFP-LC3 mice and n=16 CSTTg GFP-LC3 mice in (d). Values are presented as individual plots and mean±SEM. Mann-Whitney test: **P=0.0033 in (c), **P=0.0011 in (d). (e,f) Representative immunofluorescence images of the expression of GFP (green) and P62 (red) in glomeruli from GFP-LC3 and CSTTg GFP-LC3 mice after 6 weeks of AngII þ HSD. Figure subparts with prime indicate higher magnification. The arrowheads indicate GFPþ P62þ autophagosomes. (g) Quantification of the number of LC3þ P62þ dots per podocyte. n=11 GFP-LC3 mice and n=16 CSTTg GFP-LC3 mice. Values are presented as individual plots and mean±SEM. Unpaired t-test with equal SD: P=0.1014. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.
Table 1 | Custom RT2 Profiler PCR Array
Calpain-1 and calpain-2 are ubiquitous pro-inflammatory proteases, whose activity is controlled by calpastatin, their specific inhibitor. Indeed, calpastatin selectively inhibits calpains and no other proteases to date. Calpain activation has been linked recently to kidney injury in several pathological contexts.69,70 The calcium channel transporter transient receptor potential channel C6 was found to activate calpain-1 in podocytes via Ca2þ/calcineurin activation. Kidneys of patients with focal and segmental glomerulosclerosis had increased transient receptor potential channel C6 expression, increased calpain and calcineurin activity, and reduced expression of the calpain target talin-1, which is critical for podocyte cytoskeletal stability.71 Transient receptor potential channel C6 also directly binds to calpain-1 and calpain-2. This interaction is crucial for the regulation of talin-1 cleavage and control motility of podocytes.72
Transgenic mice overexpressing calpastatin are protected against vascular remodeling and AngII-dependent inflflammation73; against inflammation in models of glomerulonephritis,43 sepsis,74 or allograft rejection75; and against age-related inflammation.76 Podocyte injury in these mice has not been explored. Peltier et al. showed that overexpression of calpastatin prevented AngII-dependent perivascular inflammation in kidneys.43 Thus, kidney protection in CSTTg mice could be mediated, at least partially, by an anti-inflammatory mechanism. We evaluate macrophages and lymphocytes infiltration in our model and found no significant difference in kidney inflammation between CSTTg and control mice when analyzing global kidney leukocyte infiltration (Supplementary Figure S6).
Calpain was involved recently in autophagy regulation (reviewed recently in Weber et al.53) with interesting and the bioprotective properties of calpain inhibition in diabetic context through the restoration of autophagy.77 Here, we reported that calpain inhibition through calpastatin overexpression (i) prevented podocyte injury during hypertension and (ii) restored autophagy in podocytes, thus highlighting a novel deleterious role of calpain activation during hypertension through the inhibition of autophagy.
Nearly all ATG proteins were shown to be cleaved by calpains in vitro. 78 Here we postulated that autophagy maintenance in hypertensive CSTTg mice was mediated by calpain inhibition. To support our hypothesis, we showed that podocytes from CSTTg have a decreased calpain activity when challenged with AngII in vitro (Figure 3c). We found increased ATG5 protein level in podocytes from CSTTg mice, which suggests that calpastatin overexpression prevented calpain-mediated ATG5 cleavage in this context (Figure 5a). In contrast, it was demonstrated that calpastatin-mediated calpain inhibition could be independent of the inhibition of their protease activity79; thus, we could not exclude regulation of autophagy by calpastatin independent to calpain enzymatic activity.
In summary, these findings revealed a previously unrecognized role of calpastatin in the regulation of podocyte autophagy and provided a lead for the investigation of novel therapeutic strategies to enhance podocyte survival during hypertensive nephropathies.
DISCLOSURE
All the authors declared no competing interests.
ACKNOWLEDGMENTS
This work was supported by the Institut National de la Santé Et de la recherche Médicale (Inserm) and Université de Paris. IB wassupported by a graduate fellowship from the Ministère de l’EducationNationale, de la Recherche et de la Technologie. OL was funded through a European Foundation for the Study of Diabetes (EFSD)award supported by the EFSD/Novo Nordisk Programme for DiabetesResearch in Europe and a grant from the Société Francophone duDiabète (SFD). BR and CH were funded by Starting Grant 107037 from European Research Council and the European Union (P-LT). YS was supported by a graduate fellowship from the Fondation de France.
We thank Elizabeth Huc, Nicolas Perez, Corina Suldac, and the ERIU970 team (Université de Paris, PARCC, Inserm, Paris, France) forassistance in animal care and handling, Nicolas Sorhaindo forbiochemical measurements (ICB-IFR2, Laboratoire de Biochimie,Hôpital Bichat, Paris, France), and Alain Schmitt and Jean-Marc Massefor transmission electron microscopy (Institut Cochin, Paris, France).We thank Morgane Le Gall (Cochin proteomic facility 3P5, Paris,France) for help in in silico analysis. We acknowledge administrativesupport from Véronique Oberweis, Bruno Pillard, and Cyrille Mahieux(Université de Paris, PARCC, Inserm, Paris, France).
SUPPLEMENTARY MATERIAL
Supplementary File (PDF)
Figure S1. Primary podocyte culture expresses podocyte markers. Western blot analysis of the expression of the podocyte markers NPHS1 and NPHS2 in primary podocyte culture from WT and CSTTg mice. Tubulin (TUBA) serves as a loading control. Representative of n=4 mice per genotype.
Figure S2. The high basal level of podocyte autophagy. (A, B) Representative immunofluorescence images of the expression of GFP (green) and NPHS1 (red) in glomeruli from GFP-LC3 mice treated or not with CQ (80mg/kg) 4 hours before killing. Arrowheads show GFPþ autophagosomes. (C,D) Representative immunofluorescence images of the expression of GFP (green) and P62 (red) in glomeruli from GFP-LC3 mice treated or not with CQ (80mg/kg) 4 hours before killing. Arrowheads show GFPþ P62þ autophagosomes. (A–D) (0 ) represent higher magnification. Nuclei were stained with Hoechst (blue). Bar=50 mm. N=4 mice per condition (E–H) Representative immunofluorescence images of the expression of GFP (green) and P62 (red) in primary podocytes from GFP-LC3 mice treated (F, H) or not (E, G) with Bafifilomycin A1 (100 nM) for 4 hours. N=5 mice per condition.
Figure S3. P62 accumulates in podocytes in the DOC-salt model of hypertension. (A–C) Representative immunofluorescence images of the expression of P62 (green) and PODXL (red) in glomeruli from WT mice after 2 to 6 weeks of DOC-salt model. (0 ) represent higher magnification. Nuclei were stained with Hoechst (blue). Bar ¼ 50 mm. (D) Quantification of P62 area per podocyte area (%). N=5–6 mice per condition. One-way analysis of variance: time P=0.0059, Sidak’s multiple comparison test: D42 versus D14 **P=0.0055, D28 versus D14 P=0.8590.
Figure S4. Podocyte autophagy is dispensable for podocyte development. (A) Systolic blood pressure, (B) urine albumin-to-creatinine ratio, and (C) blood urea nitrogen levels in Atg5lox/lox and Nphs2.cre Atg5lox/lox mice. N=5–6 mice per genotype. Values are presented as individual plots and means±SEM. Mann-Whitney test: P=0.7273 (A), P=0.4286 (B), and P=0.6623 (C). (D–E) Representative images of Masson’s trichrome–stained sections of glomeruli from Atg5lox/lox and Nphs2.cre Atg5lox/lox mice. (F–G) Representative immunofluorescence images of the expression of PODXL (green) and WT1 (red) in Atg5lox/lox and Nphs2.cre Atg5lox/lox mice. Nuclei were stained with Hoechst (blue). (D–G) Bar=50 mm. N ¼ 6 mice per genotype. (H–I) Representative photomicrographs of transmission electron microscopy sections of glomeruli from Atg5lox/lox and Nphs2.cre Atg5lox/lox mice. Bar=1 mm. N=3 mice per genotype.
Figure S5. Calpastatin overexpression does not influence kidney function at baseline. (A) blood urea nitrogen and (B) plasma albumin levels in 12-week-old GFP-LC3 and CSTTg GFP-LC3 mice. N=5 GFP-LC3 and N=6 CSTTg GFP-LC3. Values are presented as individual plots and means±SEM. Mann-Whitney test: P=0.6623 (A) and P=0.9307 (B). (C,D) Representative images of Masson’s trichrome–stained sections of glomeruli from GFP-LC3 and CSTTg GFP-LC3 mice. (E–H) Representative immunofluorescence images of the expression of PODXL (E, F) and NPHS1 (G, H) in GFP-LC3 and CSTTg GFP-LC3 mice. (C–H) Bar=50 mm. (I, J) Associated quantification of PODXL and NPHS1 area per glomerular section. N=5 GFP-LC3 and N=6 CSTTg GFP-LC3. Values are presented as individual plots and means±SEM. Mann-Whitney test: P=0.1898 (A) and P=0.8413 (B).
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Figure S6. Calpastatin overexpression does not influence global kidney inflammation. Representative immunohistochemistry of the expression of F4/80 (A, B) and CD3 (D–E) in GFP-LC3 and CSTTg GFP-LC3 mice after 6 weeks of Angiotensin II þHSD. Bar=200 mm. (C, F) Associated quantification of F4/80 and CD3 area per kidney section. N=7 CSTTg GFP-LC3 and N=8 GFP-LC3 mice. Values are presented as individual plots and means SEM. Mann-Whitney test: P=0.3969 (C) P= 0.3357 (F).
Figure S7. Podocyte autophagy deficiency does not induce ER stress or oxidative stress in young adults at baseline. qPCR analysis of the mRNA expression of genes of the ER stress, oxidative stress, and apoptosis pathway by Qiagen qPCR array in glomeruli from WT and Nphs2.cre Atg5lox/lox mice (A) and WT and CSTTg mice (B). N=4 mice per genotype. For Nox3, Ct >33.
Table S1. In silico prevision of calpain cleavage sites. Calpain cleavage sites were predicted in podocyte-related and autophagy-related proteins with GPS-CCD (http://ccd.biocuckoo.org), CaMPDB (HTTP:// calpain.org) and DeepCalpain Predict (http://deepcalpain.cancerbio. info/help.php). Supplementary File (Excel)
Supplementary Table S2. The full file of in silico prevision of calpain cleavage sites. Calpain cleavage sites were predicted in podocyte-related and autophagy-related proteins with GPS-CCD (http://ccd. biocuckoo.org), CaMPDB (http://calpain.org), and DeepCalpain Predict (http://deepcalpain.cancerbio.info/help.php). Prediction cleavage sites are resumed for each protein for GPS-CCD and DeepCalpain Predict.
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