Contact: joanna.jia@wecistanche.com / WhatsApp: 008618081934791

Imane Bensaada, Blaise Robin3,Joeille Perez', Yann Salemkour, Anna Chipont, Marine Camus, Mathilde Lemoine',Lea Guyonnet', Helene Lazareth',Emmanuel Letavernier, Carole Henique', Pierre-Louis Tharaux' and Olivia Lenoir'

'Université de Paris, PARCC, Inserm, Paris, France; and Université Paris Descartes, Sorbonne Paris Cite, Paris, France

The strong predictive value of proteinuria in chronic glomerulopathies is firmly established as well as the pathogenic role of angiotensin ll promoting the progression of glomerular disease with an altered glomerular filtration barrier, podocyte injury and scarring of glomeruli. Here we found that chronic angiotensin Il-induced hypertension inhibited autophagy flux in mouse glomeruli. Deletion of Atg5 (a gene encoding a protein involved autophagy)specifically in the podocyte resulted in accelerated angiotensin I-induced podocytopathy, accentuated albuminuria, and glomerulosclerosis. This indicates that autophagy is a key protective mechanism in the podocyte in this condition. Angiotensin-Il-induced calpain activity in podocytes inhibits autophagy flux. Podocytes from mice with transgenic expression of the endogenous calpain inhibitor calpastatin displayed higher podocyte autophagy at baseline that was resistant to angiotensin Il-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 Il-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.

Cistanche deserticola prevents kidney disease

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 Il 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.

Hypertension is second only to diabetes as a leading cause of progressive chronic kidney disease-and even modest elevation in blood pressure is an independent risk factor for end-stage kidney disease. 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. In patients, urinary excretion of viable podocytes was shown to be a sensitive and specific marker for preeclampsia, and patients with nephrosclerosis had a significantly lower density of glomerular podocytes than did kidney donors. Furthermore, the pathogenic role of angiotensin II (Angel)promoting the progression of glomerular disease is well established, not only in hypertensive conditions but also in several glomerular diseases.

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 signaling and modify their shape upon AnglI type 1 receptor (AT1)-28These adaptive mechanisms dependent on stimulation.24-28 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 and notably in podocytes.1- Autophagy is a cll9olysosomal-associated degradation system for long-lived cytoplasmic proteins and dysfunctional organelles55 and involves sequestration of proteins and organelles in autophagosomes. The formation of autophagosomes is dependent on the induction of several genes including Mapllc3a/b, Berlin 1, and Atgs. 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.

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. 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 AnglI signaling in podocyte autophagy regulation. We identified the calcium-activated proteases calpains mediating a chronic blocking effect of AnglI on podocyte autophagy. Further, we found that the endogenous calpain inhibitor calpastatin was able to prevent AngI-dependent autophagy inhibition and podocyte injury during hypertension.

These findings uncover an original mechanism whereby AnglI-mediated hypertension inhibits autophagy via calcium-induced recruitment of calpain with pathogenic consequences in case of imbalance by calpastatin activity.

METHODS

Animals

Calpastatin transgenic(CST18) mice were kindly provided by Dr. E. Letavernier. Mice with a podocyte-specific disruption of the Atg5)were generated as previously described gene(Nphs2.cre Atg5'o by crossing Nphs2.cre mice with Ag5lxlomice5 on the C57BL6/J background. Nphs2.cre Atg5'oxlox 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 AnglI(Sigma-Aldrich, A9525)at a dose of 1 ug/kg/min for 4 to 6weeks 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. Atgsloxilox(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 DOCA 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.6 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./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 μm and 40 μm cell strainers （BD Falcon， 352340 and 352350). Glomeruli, which adhere to the 40 um cell strainer, were removed with phosphate-buffered saline(PBS; Life Technologies, 10010023)+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 (Supplementary Figure S1). Podocytes were cultured in the absence or presence of bafilomycin Al(100 nmol/, 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. 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 uM 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-methycoumarin (Sigma-Aldrich, S6510). After a 90-minute incubation period, cal-pain activity was determined as the difference between fluorescence (measured at 360 nm excitation and 430 nm emission)with and without calpain inhibitor-1.

Western blot

Primary podocytes were scratched with 80 ul of radio-immunoprecipitation 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 anti-podocin(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 a 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-um thick) were stained by Masson's trichrome 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 50 glomeruli 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 antibodies guinea pig anti-SQSTM1/P62(1:1000, PROGEN, GP-62C) and rabbit anti-green fluorescent protein(GFP;1:500, Abcam, ab290). After TBS-T rinses,fluorophore-conjugated secondary antibodies donkey anti-guinea pig IgG AF594-conjugated antibody(Jackson ImmunoResearch,706-585-148)and donkey anti-rabbit IgG AF488-conjugated antibody(Invitrogen, A21206) was applied. Images were taken using a Zeiss 2 fluorescent microscope, an AxioCam HRc camera, and Axiovision 4.3 software.

For formalin-fixed paraffin-embedded(FFPE) kidneys, sections (3 um) 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-T3%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-Wilm's Tumor 1(WT1;1:100, Abcam, ab192)antibody. Secondary antibodies were Alexa 488- 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 taken with a Zeiss Axiophot photomicroscope and Axiovision software. Semiautomatic quantifications on Fiji were used for quantifications of nephrin-positive and PODXL+ areas per glomerular section on at least 30 glomeruli per mouse. Podocyte number was counted as the number of WT1+ nuclei per glomerular section on at least 30 glomeruli per mouse.

Transmission electron microscopy procedure

Small pieces of the renal cortex(1 mm)were fixed in 3% glutaraldehyde EM grade(Electron Microscopy Sciences) for 1 to 30 days and washed thrice in PBS. Samples were postfixed in 1% osmium te-troxide 0.1 M(Electron Microscopy Science) in 0.1 M PBS(pH 7.4)and washed in water. Samples were dehydrated in alcohol grades and 100% 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 812 and 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 Microsystems GmbH)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 Digi-talMicrograph software(Gatan)for acquisition.

Quantitative polymerase chain reaction array

Freshly isolated glomeruli were frozen in QIAzol Lysis reagent (Qiagen)at -80 °C. Total RNA extraction using the phenol-based method was processed according to the manufacturer's recommendations.cDNAs were synthesized using the RT²First Strand Kit (Qiagen, 330401), and real-time polymerase chain reaction(PCR)was performed using a Custom RT²Profiler PCR Array （Qiagen， CLAM36771C) with RT'SYBR Green qPCR Mastermix(Qiagen, 330502). The quantitative PCR plates were run on an Applied Bio-systems StepOnePlus cycler. Each array contains quality control for reverse transcription efficiency and genomic DNA contamination. Quantitative PCR analysis was performed using the 2-△△CT 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 2 groups was performed using a parametric Student t-test when samples passed the Anderson-Darling and D'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 Sidak's correction. Values of P<0.05 were considered significant. *P<0.05,**P<0.01,***P<0.001.

Figure 1| Angiotensin I(Angel)+ high-salt diet (HSD)-induced hypertension inhibits glomerular autophagy. (a-c)

Immunofluorescence of Podocalyxin (PODXL;red) and P62 (green) in glomeruli (a,a') from wild-type (WT) mice, (b,B) from WT mice after 6 weeks of Angel + HSD, and (c) from Nphs2.cre Atgsl/ox mice showing the accumulation of P62 in podocytes during hypertension and in podocyte-specific ATG5-deficient mice. The arrowheads indicate P62+ dots in WT in (a'). Nuclei were counterstained with Hoechst (blue). Figure subparts with prime indicate higher magnification. Bars =50 μm. （d）Associated quantification of the P62+ is expressed as the percentage of the glomerular area.n =4 WT mice and n=5 WT with Angel + HSD and Nphs2.cre Atgsoxlo mice. Values are presented as individual plots and mean ± SEM. Mann-Whitney test:*P= 0.0159.

Angel + 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 GFP+dots, indicating high autophagic flux in podocytes (Supplementary Figures S2A and B). Confirmation that GFP+dots were autophagosomes was shown by double immuno-fluorescence for GFP and SQSTM1/P62, a chaperone protein degraded by autophagy(Supplementary Figure S2C and D). Again, chloroquine treatment induced the accumulation of GFP+ P62+ dots, demonstrating important autophagic flux in podocytes. Finally, high autophagic flux was conserved in vitro as shown by GFP and P62 expression in primary podocytes isolated from GFP-LC3 mice and strong accumulation of GFP+ and P62+ dots under bafilomycin Al treatment, another blocker of autophagosomal degradation (Supplementary Figure S2E-H).

The capacity of hypertension to modulate podocyte autophagic responses were then assessed in mice infused with AngII with a high-salt diet(HSD) for 6 weeks and in non-hypertensive controls. As shown in Figure l, AnglI + HSD induced P62 accumulation in glomeruli with strong accumulation in podocytes, thus suggesting that Angl +HSD was responsible for podocyte autophagy blockade (Figure land). Interestingly, P62 accumulation in podocytes was similar to the one observed in mice deficient for podocyte autophagy(Nphs2.cre Atg5'olox mice).In another model of hypertension, the DOCA-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 Angel + HSD model

We then examined whether autophagy blockade only in podocytes(Nphs2.cre Atg5laxlox mice) affects glomerular injury in the AnglI + HSD model. We first confirmed that Nphs2. cre Atg5mice had normal blood pressure, normal kidney function, and no glomerular histological lesions until 10 months of age, as previously reported (Supplementary lox (WT) and Nphs2.cre Atgslox Figure S4).2 Then, Atg5'cl mice were infused with AngII with HSD for 6 weeks. Importantly, systolic blood pressure was similar in the 2 groups after AnglI 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. Angl infusion with HSD markedly increased urinary albumin-to-creatinine ratio in WT mice, and this effect was further significantly increased in mice(Figure 2b). Hypertensive Nphs2.cre Atg5lox/lox Nphs2.cre Atgsoxlox 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).

Podocyte number per glomerulus was significantly decreased in Nphs2.cre Atg5loxlox (Figure 2i-k).Podocyte injury in Nphs2.cre Atgsoxlo mice with AngII infusion + 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…mice. By contrast, few ultrahypertensive Nphs2. cre Atg5'oxlx structural 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 Angel + HSD-induced damage. Altogether, our results indicated that in the AnglIl + HSD model, autophagy inhibition aggravates podocyte injury and loss and induces subsequent focal and segmental glomerulosclerosis.

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 Il(Angel) infusion + high-salt diet (HSD). (a) Systolic blood pressure in Atgslo/b and Nphs2.cre Ataslo mice during 36 days of Angel +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) Angel +HSD (continued

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 ll (Angel; 100 nM) for 24 hours with or without calpeptin (10 uM), 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 Angll versus baseline," P=0.0056 for Angll + calpeptin versus Angll.(c) Calpain activity was measured on primary podocytes from wild-type (WT) or calpastatin transgenic (CST') mice treated or not treated with Angel (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 Angll versus baseline,*P=0.0420 for CST'9 Angll versus baseline, *P=0.0424 for WT versus CST9 Angll.

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