KIDNEY DISEASE

FIELD OF THE INVENTION:

The present invention is in the field of medicine, in particular nephrology.

BACKGROUND OF THE INVENTION:

Diabetic kidney disease (DKD) is one of the most devastating microvascular complications of diabetes mellitus and amongst the most frequent causes for the development of end-stage renal disease (ESRD) (7). Current prevention and treatment options in DKD focus on lifestyle interventions, optimal regulation of blood glucose levels, and inhibition of the renin- angiotensin-aldosterone system (RAAS) to control blood pressure. Recently, SGLT2 inhibition was added to the therapeutic armamentarium to prevent of mitigate DKD (2-5). Of note, the prevalence of diabetes mellitus, and consequently DKD, is only anticipated to increase the upcoming years (6-8).

A key event in the pathogenesis of DKD is injury to the glomerular epithelial cell, or podocyte, leading to the development of proteinuria and eventually glomerulosclerosis (9-11). Various studies have highlighted that an important mechanism leading to podocyte injury in DKD is impaired podocyte autophagy (12-14). Autophagy is a crucial process of cells to generate energy and maintain cellular homeostasis during moments of cellular stress, for example via the recycling of proteins and cellular components like mitochondria. (15-17). Therefore, impaired podocyte autophagy leads to increased podocyte injury, whereas restoring podocyte autophagy is known to prevent podocyte injury and proteinuria in experimental DKD (18-20). Unfortunately, the molecular mechanisms underlying impaired podocyte autophagy during DKD remain largely elusive.

Two proteins potentially responsible for impaired podocyte autophagy during DKD are the calcium-dependent cysteine protease calpains and the calcium-permeable ion channel transient receptor potential channel C6 (TRPC6). TRPC6 expression and activity are increased in DKD and TRPC6 knock-out was shown to protect against the development of microvascular renal complications of diabetes mellitus in animal studies. Furthermore, TRPC6-mediated calcium (Ca ²⁺ ) influx has an inhibitory effect on podocyte autophagy (21, 22). Calpains are a family of 15 cysteine proteases activated by an increase in intracellular Ca ²⁺ concentration (25). Calpain has been recently shown as a non-conventional autophagy regulators, for example by degrading the autophagy-initiating protein autophagy related 5 (ATG5) (24, 25). Two members of the calpain superfamily, Calpain- 1 and calpain-2, have been extensively studied. They are ubiquitously expressed calpains that exist as heterodimers consisting of an isoform-specific catalytic domain encoded by Capnl and Capn2 genes, respectively, and a common regulatory domain encoded by Capnsl, a gene that is indispensable for calpain-l/-2 stability and activity. The mechanisms by which calpains are activated and identify their protein targets are complex and poorly understood. Calpain activity is regulated by a ubiquitous specific inhibitor, calpastatin (26). Importantly, TRPC6-mediated Ca ²⁺ -influx is known to activate calpains activity (27, 28). However, the interplay between TRPC6 and calpains activity in the context of impaired podocyte autophagy in DKD remains unknown.

SUMMARY OF THE INVENTION:

The present invention is defined by the claims. In particular, the present invention relates to the use of calpain inhibitors for the treatment of the diabetic kidney disease.

DETAILED DESCRIPTION OF THE INVENTION:

Diabetic kidney disease is associated with impaired podocyte autophagy and subsequent podocyte injury. However, the molecular mechanisms underlying the impaired autophagy in podocytes remain largely elusive. In the present invention, the inventors investigated how the calcium channel TRPC6 and the cysteine protease calpains impair podocyte autophagy in diabetic kidney disease. They demonstrated that TRPC6 knockdown in podocytes increased the autophagic flux as a result of decreased calpains activity. Diabetic kidney disease was mimicked in vivo by using both the streptozotocin-induced diabetes model upon unilateral nephrectomy and the BTBR ^ob/ob mouse model. In the in vitro experiments the inventors demonstrated that TRPC6 knockdown increased the autophagic flux in podocytes as a result of decreased calpain activity. Moreover, diabetes resulted in increased TRPC6 expression in podocytes in vivo together with a decreased podocyte autophagic flux. Transgenic overexpression of the endogenous calpain inhibitor calpastatin as well as pharmacological inhibition of calpain activity normalized podocyte autophagic flux, reduced nephrin loss and prevented the development of albuminuria in diabetic mice. In kidney biopsies from diabetic patients the inventors confirmed that TRPC6 overexpression in podocytes correlated with decreased calpastatin expression, autophagy blockade and podocyte injury. Taken together, the inventors discovered a new mechanism that connects TRPC6 and calpain activity to impaired podocyte autophagy, increased podocyte injury and development of proteinuria in the context of diabetic kidney disease. Therefore, targeting calpain might be a promising therapeutic strategy for the treatment of diabetic kidney disease by restoring podocyte autophagy.

The first object of the present invention thus relates to a method of treating diabetic kidney disease in a patient in need thereof comprising administering to the patient a therapeutically effective amount of a calpain inhibitor.

As used herein, the term “diabetic kidney disease” or “DKD” has its general meaning in the art and encompass the various lesions, involving all kidney structures that characterize protean kidney damage in patients with diabetes (Piccoli GB, Grassi G, Cabiddu G, Nazha M, Roggero S, Capizzi I, De Pascale A, Priola AM, Di Vico C, Maxia S, Loi V, Asunis AM, Pani A, Veltri A. Diabetic Kidney Disease: A Syndrome Rather Than a Single Disease. Rev Diabet Stud. 2015 Spring-Summer; 12(1-2) : 87-109). It thus refers to the kidney disease caused by diabetes, exacerbated by diabetes, or co-presenting with diabetes. It is a form of chronic kidney disease occurring in approximately 30% of patients with diabetes. It is defined as diabetes with the presence of albuminuria and/or impaired renal function (i.e. decreased glomerular filtration rate (See. de B, I, et al. Temporal trends in the prevalence of diabetic kidney disease in the United States. JAMA 2011 Jun. 22; 305(24):2532-2539). The term is also named “diabetic nephropathy”.

As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a patient having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a patient beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).

The calpain inhibitor of the present invention is particularly suitable for restoring podocyte autophagy. More particularly, the calpain inhibitor of the present invention is suitable for preventing or limiting podocyte injury. Even more particularly, the calpain inhibitor of the present invention is suitable for preventing or limiting proteinuria.

As used herein, the term “calpain” has its general meaning in the art and refers to a protein belonging to the family of calcium-dependent, non-lysosomal cysteine proteases (proteolytic enzymes) expressed ubiquitously in mammals and many other organisms. Calpains constitute the C2 family of protease clan CA in the MEROPS database. The calpain proteolytic system includes the calpain proteases, the small regulatory subunit CAPNS1, also known as CAPN4, and the endogenous calpain-specific inhibitor, calpastatin. The term “calpain” thus includes, but is not limited to, human calpain 1 or human calpain 2.

As used herein, the term “calpain inhibitor” refers to a biochemical or chemical compound which inhibits or reduces the activity of a calpain or the expression of a calpain gene. A “calpain inhibitor” can inhibit or reduce calpain activity and inhibits or reduce expression of a calpain gene. Calpains that can be inhibited by the methods and compositions described herein, include, but are not limited to a full-length calpain, a calpain homolog, a calpain variant, a calpain analog, a mutant calpain, a calpain fusion protein, or a calpain peptide mimetic. Exemplary calpain inhibitors include peptides, peptidomimetics, small molecules, compounds, agents, dominant negative mutants of calpain activity, ligand mimetics, antibodies (e.g., monoclonal, polyclonal or single chain Fv; intact or binding fragments thereof), or nucleic acids (e.g., RNA, DNA, antisense oligonucleotides, double stranded RNA oligonucleotides (RNAi) or DNA oligonucleotides (vectors) containing nucleotide sequences encoding for the transcription of shRNA molecules) or derivatives and analogs thereof. Further exemplary calpain inhibitors are disclosed herein.

The calpain inhibitors are well-known in the art as illustrated by Donkor IO. An update on the therapeutic potential of calpain inhibitors: a patent review. Expert Opin Ther Pat. 2020 Sep; 30(9) : 659-675 and Dokus LE, Yousef M, Bánóczi Z. Modulators of calpain activity: inhibitors and activators as potential drugs. Expert Opin Drug Discov. 2020 Apr; 15(4): 471- 486.. calpain inhibitors typically include calpastatin-based peptidomimetics; thalassospiramide lipopeptides; disulfide analogs of alpha-mercaptoacrylic acids; allosteric modulators; azoloimidazolidenones; and macrocyclic/non-macrocyclic carboxamides, calpain inhibitors also include calpastatin analogs.

Calpastatin is an endogenous protease inhibitor that acts specifically on calpain (a calcium- dependent cysteine protease). It consists of four repetitive sequences of 120 to 140 amino acid residues (domains I, II, III and IV), and an N-terminal non-homologous sequence (L). Thus, in some embodiments of the invention, the calpain inhibitor may be calpastatin or truncated forms of calpastatin such as CPIB, PCPIB, 7-mer-PCPlB, 11R-CS, CPlB-[4-23] as described in U.S. Pat. No. 6,015,787; U.S. Pat. No. 6,294,518; U.S. Pat. No. 6,867,186.

In some embodiments of the invention, the calpain inhibitor may be a peptidomimetic calpain inhibitor, a non-peptide calpain inhibitor. In some embodiments of the invention, the calpain inhibitor is selected from the group consisting of SJA-6017, BDA-410, SNJ-1757, SNJ-1945, A-705253, MDL-28170, SC488, NS-398, SC-560, AK275, E64, calpeptin, calpastatin, acetyl- calpastatin, leupeptin, AK295, AK275, N-acetyl-leucyl-leucylmethional (ALLM or calpain inhibitor II), N-acetyl- leucylleucyl-norleucinal (ALLN or calpain inhibitor 1), calpain inhibitor III (carbobenzoxy- valyl-phenylalanal; Z-Val-PheCHO), calpain inhibitor IV (Z-LLY-FMK; Z-LLY-CH.sub.2 F where Z=benzyloxycarbonyl), calpain inhibitor V (MuVal-HPh-FMK where Mu is morphlinoureidyl and Hph is homophenylalanyl), mimetics thereof and compounds disclosed in U.S. Pat. Nos. 12,921,366; U.S. Pat. Nos. 5,716,980; 5,714,471; 5,693,617; 5,691,368; 5,679, 680; 5,663,294,5,661,150; 5,658,906; 5,654,146; 5,639,783; 5,635,178; 5,629,165; 5,622,981; 5,622,967; 5,621,101; 5,554,767; 5,550,108; 5,541,290; 5,506,243; 5,498,728; 5,498,616; 5,461,146; 5,444,042; 5,424,325; 5,422,359; 5,416,117; 5,395,958; 5,340,922; 5,336,783; 5,328,909; 5,135,916.

In some embodiments of the invention, the calpain inhibitor is selected from synthetic calpain inhibitors such as PD- 150606 ((2Z)-3-(4-iodophenyl)-2-mercapto-2- Propenoic acid, 3-(4- iodophenyl)-2-mercapto-(Z)-2-propenoic acid) and alpha- mercaptoacrylic acid derivatives such as disclosed in Wang et al., 1996.

As used herein, the term "therapeutically effective amount" a sufficient amount of the calpain inhibitor of the present invention for treating or reducing the symptoms at reasonable benefit/risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination with the calpain inhibitor of the present inventions; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the calpain inhibitor of the present invention for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the calpain inhibitor of the present invention, typically from 1 mg to about 100 mg of the calpain inhibitor of the present invention. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.

Typically the calpain inhibitor of the present invention of the present invention is combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions. The term "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi- solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. In the pharmaceutical compositions of the present invention, the calpain inhibitor of the present invention can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1: Glomerular filtration function in diabetic mice with calpastatin overexpression. Evolution of the albumin-to-creatinine ratio in GFP-LC3 UniNx + STZ and GFP-LC3 CST ^Tg UniNx + STZ mice. Values are presented as individual plots and mean ± SEM. Two-way analysis of variance: Weeks, p= 0.0145; Genotype, p= 0.1098. Sidak’s multiple comparison test: ** p= 0.0026 for GFP-LC3 UniNx + STZ vs. GFP-LC3 CST ^Tg UniNx + STZ at week 6. Figure 2: Pharmacological calpain-1 inhibition with BDA-410 preserve the glomerular filtration function in type II diabetes model. Evolution of the albumin-to-creatinine ratio in BTBR ^ob/ob mice treated or not with BDA-410 for 6 weeks. Values are presented as individual plots and mean ± SEM. Two-way analysis of variance: Weeks, p=0.012; Treatment, p=.0.0062 Tukey’s multiple comparison test: p=0.037 for BTBR ^ob/ob and BTBR ^ob/ob + BDA-410 mice at week 12.

EXAMPLE:

Methods

Animals

All mice were bred and housed in a specific pathogen free animal facility and were given free access to water and standard chow.

Type I diabetes model green fluorescent protein (GFP)- light-chain 3 (LC3) calpastatin trangenic (CST ^Tg ) mice were obtained by crossing CST ^Tg mice (55) with GFP-LC3 mice (52). GFP-LC3 CST ^Tg mice and their respective control littermates (GFP-LC3) were used between 8 to 10 weeks of age. Only males were used in this study. GFP-LC3 and GFP-LC3 CST ^Tg mice were submitted to unilateral left nephrectomy. During surgical procedures, mice were anesthetized with isoflurane inhalation (2,5%). Pre-surgery and post-surgery analgesia were provided by buprenorphine (0.1 mg/kg) subcutaneous injection. Drugs were diluted in sterile phosphate buffer or saline. After 1 week of post-surgery recovery, mice were injected with Streptozocin (STZ) at a dose of 50mg/kg for 2 days (Sigma- Aldrich, S0130-50MG) to induce type I diabetes mellitus. Blood glycaemia was monitored every week on a OneTouch Verio glucometer by tail blood collection. Urine was collected in metabolic cages once a week. Mice were sacrificed 6 weeks after STZ injections for blood and organs collection.

Type II diabetes model. BTBR ^ob/ob mice were obtained by crossing 2 heterozygous BTBR ^ob/WT mice purchased from The Jackson Laboratory (004824). Males BTBR ^ob/ob mice and their wild- type littermates (BTBR ^WT/WT ) were used at 6 weeks of age for experiments. They were treated with calpains pharmacological inhibitor BDA-410, kindly provided by Mitsubishi Tanabe Pharma Corporation, at a dose of 100mg/kg/day administrated per os. After 6 weeks of treatment mice were sacrificed for organ collection.

Construction and culture and of a stable TRPC6 knockdown podocyte cell line

Conditionally immortalized mouse podocytes (MPC-5) were cultured as described previously (54). Briefly, MPC-5 were grown at permissive conditions at 33°C with 10 units interferon gamma (IFNγ, Sigma-Aldrich, 4777) per mL. To induce differentiation, IFNy was removed from the medium and cells were grown at 37°C for 2 weeks. A podocyte cell line stably expressing a doxycycline-inducible TRPC6 silencing short hairpin RNA (shRNA) construct was created by transfecting a TRPC6 shRNA construct into undifferentiated MPC-5 podocytes using Lipofectamine 3000 (Thermofisher Scientific, L3000001). Transfected MPC-5 podocytes were maintained at 33°C in the presence of 400μg/mL G418 (Sigma- Aldrich, Al 720) to select successfully transfected cells. To validate the TRPC6 knockdown, single clones were exposed to 5μg/mL Doxycycline (MP Biomedicals, 11420455) for 5 days to activate the knockdown construct. Because uninjured MPC-5 podocytes are known to show low TRPC6 expression levels, single clones were cultured for the last 24hrs in the absence or presence of the podocyte injury-inducing compound Adriamycin (0.25μg/mL, Sigma-Aldrich, D1515) to induce podocyte injury and increase TRPC6 expression. TRPC6 mRNA expression was subsequently investigated in single clones to validate the TRPC6 knockdown. Validated MPC-5 TRPC6 knockdown (KD) podocytes were thereafter cultured with a maintenance dose of 100μg/mL G418.

In vitro autophagic flux assessment

To investigate the role of TRPC6 and calpain in the context of autophagy in vitro, wildtype (WT) and TRPC6 KD podocytes were exposed for 48hrs to the calpain inhibitor calpeptin (1μM, Sigma-Aldrich, C8999). After 44hrs, podocytes were exposed to the autophagy inhibitor Bafilomycin A (100nM, Enzo Life Sciences, BML-CM110-0100) for the last 4hrs. Five days prior and during calpeptin exposure, podocytes were exposed to 5μg/mL Doxycycline to activate the knockdown construct.

RNA isolation and quantitative PCR Analysis RNA was isolated from podocytes using Trizol (Thermofisher Scientific, 15596018). Subsequently, 1 pg RNA was reverse-transcribed into cDNA using the Transcription First Strand cDNA synthesis kit (Roche, 04897030001) according to manufacturer’s instructions. Quantitative TRPC6 expression levels were determined by quantitative PCR using SYBR Green (Roche, 04673484001) on a CFX 96 C1000 Thermal Cycler (Bio-rad). TRPC6 levels were normalized to glyceraldehyde-3 -phosphate dehydrogenase (GAPDH) levels using the delta-delta CT method. Three independent experiments were performed, and each experiment consisted of three samples. All samples were measured in duplicate.

Calpain Activity Assay

Calpain activity was determined via the Calpain activity assay (Abeam, ab65308). Cells were collected via trypsinization and resuspended in extraction buffer, after which samples were processed according to manufacturer’s instructions. Fluorescent intensity was measured with excitation/emission at 400nm/505nm using a Tecan, Infinite Pro2000 plate reader (Tecan). Afterwards, protein content was measured for every sample via the bicinchoninic acid assay (BCA, Sigma-Aldrich, 71285-3) to correct for differences in cell density. The experiment was performed in sextuplicate and samples were measured in duplicate.

Western Blot

Protein samples were isolated using RIPA buffer (150mM NaCl, 50mM TrisHCl, 2mM EDTA, 0.5% Natrium deoxycholate, 0.2% sodium dodecyl sulfate (SDS) and 1% NP40 in MilliQ) supplemented with protease inhibitors (Roche, 11836170001) and phosphatase inhibitor (Roche, 4906845001). The protein content of cell extracts was determined using the BCA assay (Sigma-Aldrich, 71285-3) to ensure equal sample loading. The experiments were performed in sextuplicate. Twenty micrograms of proteins were loaded and run on NuPAGE pre cast gel (4- 12% Bis-Tris, Invitrogen, WG1401BX10). Proteins were subsequently transferred to polyvinylidene difluoride membrane using the iBlot system (Invitrogen, IB24001). Membranes were blocked in tris buffer saline (TBS) with 0.05% (v/v) Tween 20 (i.e. TBS-T) supplemented with 5% (w/v) milk overnight at 4°C. Primary antibodies were incubated for 4 hours at room temperature. The following antibodies were used: rabbit anti-LC3B (1 :1000, Cell Signaling Technology, 2630), guinea pig anti-SQSTMl (1 :10 000, Progen, GP-62C), and rat anti-Tubulin (1:5000, Abeam, Ab6160). Membranes were washed in TBS-T and incubated with horseradish peroxidase-conjugated antibodies (1 :2000, Cell Signaling Technology, 7074, 7076, 7077) for 2 hours. Protein bands were visualized using the ECL Chemilumiscent Kit (Bio-Rad, 170-5070) on a LAS 4000 device (FUJI). Quantification was performed on FIJI software (v2.3.0/1.53f).

Immunofluorescence on MPC-5 cells

Cells were fixed with ice-cold methanol at -20°C for 20 minutes. Subsequently, cells were washed three times with phosphate buffered saline (PBS) for 5 minutes at room temperature. Thereafter, cells were blocked for Ihr at room temperature with blocking solution consisting of TBS-T + 3% (w/v) bovine serum albumin (BSA, Sigma-Aldrich). Subsequently, cells were incubated overnight at 4°C with primary antibodies in blocking solution. Primary antibodies included guinea-pig anti-SQSTMl (1 :400, Progen, GP62-C) and rabbit anti-LC3B (1 :1000, Cell Signalling Technology, 43566S). The next day, cells were washed three times for 5 minutes with blocking solution at room temperature. Donkey anti-rabbit Alexa 488 and goat anti-guinea pig Alexa 594 (1 :500, all from Thermofisher Scientific) were subsequently incubated for 2hrs at RT in the dark. Then cells were washed three times for 5 minutes with blocking solution and cell nuclei were stained with 1 μg/mL Hoechst 33342 (Thermo fisher Scientific, H3570) for 10 minutes in the dark. Immediately afterwards, fluorescent images were captured with a Zeiss LSM900 confocal microscope using a 40x objective (Carl Zeiss Microscopy). The experiment was performed in sextuplicate and at least 5 images were captured per experiment. The percentage of cell surface area positive for SQSTM1 and LC3B was calculated using semi-automatic macros in FIJI (version 1.53c, National Institutes of Health). The percentage of SQSTM1 and LC3B positive areas of the total podocyte cell surface area was calculated as a measure of the podocyte autophagic flux.

Immunofluorescence staining of mouse kidney sections

Kidneys were formalin-fixed paraffin-embedded (FFPE), sectioned (3 pm), dewaxed and rehydrated before antigen retrieval in heated citrate buffer (pH=6). Sections were then permeabilized with TBS-T + Triton 0.1% and blocked in TBS-T + 3% BSA. Primary antibodies (Table 1) were incubated overnight at 4°C. After washing in TBS-T, secondary antibodies (Table 2) were incubated for 2 hours at RT. Nuclei were stained in blue using Hoechst. Slides were mounted using Fluoromount-G (Aviva Systems Biology, 0100-01). Images were acquired on a Zeiss AxioPhot microscope and ZEN software. FIJI (v2.3.0/1.53f) semi-automatic macros were used for quantifications of the percentage of NPHS1 -positive and PODXL-positive areas per glomerular section on at least 30 glomeruli per mouse. Podocytes number was counted as the number of WT1+ nuclei per glomerular section on at least 30 glomeruli per mouse. SQSTM1 and GFP puncta areas within glomerular PODXL area were measured on at least 30 glomeruli per mouse in order to monitor glomerular autophagic flux. Podocyte TRPC6 expression was quantified by measuring the TRPC6 positive area within the NPHS 1 positive area.

Analyses of human kidney biopsies

The Paris Cite Universite Nephropathology unit at Necker Hospital routinely receives kidney biopsies for first line histopathological diagnosis from the nephrology unit at Georges Pompidou European Hospital. Patients referred between December 19, 2019 and December 31, 2021 and with diagnosis of diabetes mellitus were identified for inclusion in this study. Control biopsies were identified as routine (month 3 or 12) post-kidney transplant biopsies. The study was limited to patients aged >18 years. Clinical and biological data were retrospectively collected from the patients’ file. The clinical data include: sex, age at the time of renal biopsy, and history of diabetes mellitus. Biological data at the time of renal biopsy include: urinary albumin-to-creatinine ratio (UACR) and estimated glomerular filtration rate (eGFR) using the Modification of Diet in Renal Disease equation. These data are synthetized in Supp Table 1.

Renal biopsies were fixed in formalin acetic acid and paraffin embedded (FAAFPE). 3 pm sections were stained with Masson trichrome. Biopsies from patients with diabetes mellitus with at least 2 glomeruli with class I-to-III diabetic glomerular lesions (55) were selected. Class 4 glomerular lesions were not analyzed. For control biopsies, only biopsies with no histological glomerular abnormalities were selected. For immunofluorescence, the staining protocol was similar to the protocol described for mouse samples. Images were acquired on a Leica SP8 confocal microscope and LASX software and on a Zeiss axiophot microscope and ZEN software. Quantifications were performed following the methodology previously described for mouse kidney sections analysis.

Transmission electron microscopy procedure

Mouse renal cortex samples were fixed in Trump's fixative (Electron Microscopy Sciences, 11750) at 4°C, processed for transmission electron microscopy as previously described (57) and were examined on a JEM1011 transmission microscope (JEOL) with a Orius SC 1000 CCD camera (Gatan).

Public databases

Single-cell RNAseq and Single-nuclei RNAseq data were extracted from public databases. For KPMP, the results are based upon data generated by KPMP: DK114886, DK114861, DK1 14866, DK114870, DK114908, DK114915, DK114926, DK114920, DK114923, DK1 14933, and DK114937. https://www.kpmp.org. Data was downloaded on April 19 ^th 2022. Others single-cell RNAseq data was extracted from the Kidney Interactive Transcriptomics. https://humphreyslab.com/SingleCell/. Bulk transcriptomics data were downloaded from Nephroseq public database: https://www.nephroseq.org/.

Statistics

Data are expressed as individual dots and mean ± SEM. Statistical analyses were calculated with GraphPad Prism. Comparison between two groups was performed with a two-sided unpaired t test. A Welch’s correction was applied when the F test to compare variances was <0.05. Comparisons between multiple groups were performed with two-way analysis of variance (ANOVA) followed by a two-sided Tukey post hoc test. For multiple comparisons, we performed comparisons between rows and between columns only. For multivariate analysis, r correlation was computed using Pearson’s correlation. A P value < 0.05 was considered statistically significant.

Study approval

Animal experiments were conducted according to the French veterinary guidelines and those formulated by the European Community for experimental animal use (E358-86/609EEC), and were approved by the Institut National de la Sante et de la Recherche Medicale, local University Research Ethics Committee and French ministry of Research (APAFIS-#22373).

The part of the research involving human samples was approved after approval of the Ethics Evaluation Committee of Inserm (CEEI) (IRB00003888, FWA00005831). Archived samples were only used after obtaining the informed consent during routine clinical follow-up consultation in the HEGP hospital if not previously done at the time of the initial consultation. No sample was taken for the project itself, thus the project did not modify the medical care of the patients. Codes were used to ensure strict anonymity of patients. The study complies with the 2000 Declaration of Helsinki.

Results

Podocyte-specific TRPC6 overexpression is associated with autophagy blockade and podocyte dedifferentiation in murine diabetic models.

First, we investigated the link between impaired podocyte autophagy and TRPC6 activation in DKD. To this end, we analyzed the expression of TRPC6, LC3B and the autophagic cargo protein sequestosome 1 (SQSTM1, also called P62) in podocytes in two murine diabetic models: (i) a type 1 diabetes mellitus model of accelerated DKD combining streptozotocin (STZ)-induced diabetes with unilateral nephrectomy (29) and (ii) a type 2 diabetes model of DKD with the use of BTBR ^ob/ob mice. BTBR ^ob/ob mice express a truncated and dysfunctional form of the satiety hormone leptin, which induces obesity and hyperglycemia (30, 3 J). GFP- LC3 reporter mice were used in the type 1 diabetes mellitus model to monitor the autophagic flux in vivo (32). The GFP-LC3 transgene is known to have no deleterious or beneficial effect on the phenotype of the mice. In both DKD models, we demonstrated increased podocyte- specific TRPC6 expression, calculated as the percentage of podocyte cell surface area positive for TRPC6 (6.72±0.65% for GFP-LC3 UniNx vs. 22.10±0.62% for GFP-LC3 UniNx + STZ and 15.56+1.07% for BTBR ^WT/WT VS. 19.44+1.3% for BTBR ^ob/ob ) (data not shown). Unilateral nephrectomy alone did not change TRPC6 expression in GFP-LC3 mice when compared to non-nephrectomized mice (data not shown).

Subsequently, we investigated the effect of DKD on podocyte autophagic flux by measuring the GFP-LC3 puncta number, representing the number of autophagosomes, and SQSTM1 expression. The number of GFP-LC3 puncta in podocytes was identical in STZ-treated and non-STZ treated uni-nephrectomized mice (data not shown). On its own, GFP-LC3 punctae number per cell was not a relevant indicator of autophagic flux in conditions were the autolysosomal degradation was not artificially blocked. Indeed, autophagy is a dynamic process and changes in the flux may go unnoticed if the autophagosomal origins and degradation rates change similarly. However, in association with SQSTM1 expression it may point towards a blockade or an increase of the autophagic flux. SQSTM1 expression was increased in podocytes in both DKD models (0.063±0.006% for GFP-LC3 UniNx vs. 0.118±0.012% for GFP-LC3 UniNx + STZ and 0.057±0.013% for BTBR ^WT/WT VS. 0.173±0.033% for BTBR ^ob/ob ) (data not shown). These data suggested a decrease podocyte autophagic flux in the pathogenesis of DKD. In the type 1 diabetes model, we confirmed that TRPC6 expression correlated positively with SQTM1 expression (Pearson r= 0.626, p=0.009) and negatively with both NPHS1 expression (Pearson r= -0.736, p=0.001) and the number of podocytes (quantified as Wilm's Tumor 1 (WT1) positive (+) cells per glomerulus)(Pearson r= -0.601, p=0.013) (data not shown). Furthermore, SQTM1 expression correlated negatively with the number of WT1+ cells per glomerulus(Pearson r= -0.613, p=0.011) and NPHS1 expression (Pearson r= -0.481, p=0.059) (data not shown). In the type 2 diabetes model, we also confirmed that TRPC6 expression correlated positively with SQTM1 expression (Pearson r= 0.61, p=0.01); whereas SQTM1 correlated negatively with NPHS1 expression (Pearson r= -0.62, p=0.008) (data not shown). Therefore, our data indicate that podocyte TRPC6 overexpression in experimental DKD correlates with podocyte autophagic flux blockade and podocyte injury.

Regulation of autophagic flux in podocytes depends on TRPC6 activity.

Next, we studied whether TRPC6 regulates podocyte autophagy. First, we generated and validated a doxycycline-inducible TRPC6 knock-down (KD) podocyte cell-line. TRPC6 expression decreased by -50% when TRPC6 KD podocytes were treated with doxycycline (data not shown) (0.20±0.14 in WT control vs. 0.11±0.05 in TRPC6 KD control). As uninjured podocytes show low TRPC6 expression levels, podocytes were exposed to Adriamycin to induce podocyte injury and increase TRPC6 expression. When TRPC6 KD podocytes were treated with Adriamycin, TRPC6 expression increased 5 times in the absence of doxycycline (0.20±0.14 in WT control vs. 1.00 in WT Adriamycin, p<0.001). However, in the presence of doxycycline, Adriamycin treatment did no longer result in increased TRPC6 expression compared to uninjured podocytes, thereby validating the TRPC6 knockdown (0.11±0.05 in TRPC6 KD control vs. 0.19±0.10 in TRPC6 KD Adriamycin).

Upon validating the TRPC6 KD podocyte cell line, we investigated the effect of TRPC6 KD on calpain activity, as TRPC6-mediated Ca ²⁺ -influx is known to regulate calpain activity (27). Calpain activity was decreased in TRPC6 KD podocytes in comparison to WT podocytes (13.38±0.72 RFU/μg in WT vs. 5.82±0.27 RFU/μg in TRPC6 KD) (data not shown). Moreover, treatment with the calpain inhibitor calpeptin reduced calpain activity in WT podocytes (13.38±0.72 RFU/μg in WT vs. 4.34±0.08 RFU/μg in WT + calpeptin). However, no effect of calpeptin treatment in TRPC6 KD was observed on calpain activity (5.82±0.27 RFU/μg in TRPC6 KD vs. 5.11±0.15 RFU/μg in TRPC6 KD + calpeptin) (data not shown). Of note, calpain activity in WT podocytes treated with calpeptin was similar to TRPC6 KD podocytes without calpeptin treatment (4.34±0.08 RFU/μg protein in WT + calpeptin vs. 5.82±0.27 RFU/μg protein in TRPC6 KD), suggesting calpeptin treatment and TRPC6 KD lead to a similar order of magnitude effect on calpain activity.

Upon validating that TRPC6 regulates calpain activity in podocytes, we investigated the effect of TRPC6 knockdown and calpain inhibition on the autophagic flux in podocytes. Autophagic flux was first assessed by Western Blot analysis through measurement of SQSTM1 and LC3B expressions. Of note, LC3B exists in two forms: the cytosolic form LC3B-I, and the phosphatidylethanolamine-conjugated autophagosomal form LC3B-II. Quantification of LC3B-II/LC3B-I ratio is used to assess the autophagic flux. The LC3B-II/LC3B-I ratio did not differ between WT and TRPC6 KD podocytes (data not shown). In addition, calpeptin treatment did not result in an altered LC3B-II/LC3B-I ratio for both WT and TRPC6 KD podocytes. On the contrary, SQSTM1 expression was decreased in WT podocytes upon calpetin treatment (0.82±0.05 vs. 1±0.07). In addition, TRPC6 KD podocytes showed lower SQSTM1 expression compared to WT podocytes (0.59±0.04 vs. 1±0.07), and calpeptin treatment further enhanced this drop in SQSTM1 level (0.45±0.04 vs. 1±0.07) (data not shown). Immunofluorescence analysis confirmed the Western blot analysis. Indeed, quantification of LC3B punctiform expression (i.e autophagosomes) demonstrated a significant increase in autophagosomes in podocytes treated with calpeptin (0.098±0.013 vs. 0.195±0.045). Surprisingly, in TRPC6 KD cells we observed less LC3B punctae than in WT podocytes (0.035±0.007 vs. 0.098±0.013) (data not shown). These findings suggested that calpain inhibition and TRPC6 KD stimulate the autophagic flux in podocytes.

Because autophagy is a highly dynamic process, we subsequently investigated the effect of calpain inhibition and TRPC6 KD on autophagy in the presence of Bafilomycin Al treatment. Bafilomycin Al inhibits the vacuolar ATPase H ⁺ pump and thereby prevents auto lysosome degradation. Calpeptin treatment increased LC3B and SQTM1 accumulation in WT podocytes after bafilomycin Al treatment (LC3B: 1.365±0.145 vs. 1.893±0.106; SQSTM1 : 0.622±0.085 vs. 0.890±0.095) (data not shown H). In contrast, calpeptin treatment had no effect on LC3B and SQTM1 expression in TRPC6 KD podocytes (LC3B: 1.037±0.071 vs. 0.940 ±0.019; SQSTM1 : 0.473±0.087 vs. 0.363±0.063) (data not shown). Moreover, we found lower levels of LC3B punctae in TRPC6 KD cells compared to WT cells (1.365±0.145 vs. 1.037 ±0.071). In addition, we quantified the fold change in LC3B and SQSTM1 expression in bafilomycin Al -treated versus non-bafilomycin Al -treated cells. We demonstrated a greater increase in LC3B and SQSTM1 accumulation in TRPC6 KD podocytes in comparison with WT podocytes upon bafilomycin Al treatment. No potentiator effect was measured in TRPC6 KD podocytes treated with calpeptin (data not shown). Taken together, these results show that calpain inhibition by calpeptin enhanced the autophagic flux in WT podocytes. Moreover, the inhibitory effect of calpain on autophagy is TRPC6-dependent, resulting in increased autophagic flux in TRPC6 KD podocytes.

Calpastatin overexpression prevents podocyte injury and restores podocyte autophagic flux in type I diabetes model

Next, we studied if diabetes-induced glomerular injury we could be prevented by modulating calpain activity and subsequently autophagy in vivo. To study this, we developed mice which constitutively overexpressed the endogenous calpain inhibitor calpastatin and induced type I diabetes mellitus with accelerated DKD in these mice (GFP-LC3 CST ^Tg UniNx + STZ) and compared them to wild-type diabetic mice (GFP-LC3 UniNx + STZ). Transgenic overexpression of calpastatin did not alter glycemia levels or bodyweight loss after inducing type I diabetes mellitus (data not shown). Moreover, the baseline albumin-to-creatinine ratio was identical between the two genotypes (data not shown). However, transgenic overexpression of calpastatin prevented the development of microalbuminuria 6 weeks after inducing type I diabetes (7.19+2.2 vs. 1.66±0.51) (Figure 1). Masson trichrome did not reveal an effect of calpastatin overexpression on the development of glomerulosclerosis in diabetic mice (data not shown). 6 weeks after the STZ injections, podocyte injury and podocyte loss was observed in GFP-LC3 UniNx + STZ mice as evidenced by reduced NPHS1 expression and decreased number of WT1+ cells, respectively. Calpastatin overexpression could not prevent podocyte loss upon inducing experimental type I diabetes (WT1+ cells: 9.5±0.4 in GFP-LC3 CST ^Tg UniNx + STZ mice vs. 9.0±0.2 in GFP-LC3 UniNx + STZ mice). However, calpastatin overexpression prevented NPHS1 loss (NPHS1 : 37.81+1.04% in GFP-LC3 CST ^Tg UniNx + STZ mice vs. 33.01±0.84% in GFP-LC3 UniNx + STZ mice) (data not shown). Moreover, ultrastructure analysis with transmission electron microscopy showed reduced foot process effacement in GFP-LC3 CST ^Tg UniNx + STZ mice than in GFP-LC3 UniNx + STZ mice (data not shown).

Finally, we also evaluated the effect of calpastatin overexpression on impaired podocyte autophagy in DKD. We could show that glomerular SQSTM1 expression was decreased in GFP-LC3 CST ^Tg UniNx + STZ mice compared to GFP-LC3 UniNx + STZ mice (SQSTM1 : 0.071±0.006 in GFP-LC3 CST ^Tg UniNx + STZ mice vs. 0.118±0.012 in GFP-LC3 UniNx + STZ mice) (data not shown) and that glomerular LC3 expression was increased (GFP-LC3 : 0.108±0.013 in GFP-LC3 CST ^Tg UniNx + STZ mice vs. 0.065±0.005 in GFP-LC3 UniNx + STZ mice) (data not shown). These results indicated that calpastatin overexpression prevented glomerular autophagic flux blockade in this experimental DKD model.

Calpains activity pharmacological inhibition with BDA-410 prevents podocyte injury and restores podocyte autophagic flux in type II diabetes model

BD4-410, a calpain inhibitor (55-55) was used to treat diabetic BTBR ^ob/ob mice. 6 weeks of daily administration of BDA-410 prevented the development of microalbuminuria in BTBRob/ob mice (Figure 2). WT1 and NPHS1 immunofluorescence demonstrated that pharmacological calpain inhibition partially prevented podocyte dedifferentation in BTBR ^ob/ob mice (NPHS1 area per glomerular area: 30.46±0.86% in BDA-410-treated mice vs. 26.66+1.10% in non-treated mice) but not podocyte loss (WT1+ cells: 9.19±0.46 in BDA-410- treated mice vs. 8.32±0.43 in non-treated mice) (data not shown). COL4 deposition was also reduced in BTBR ^ob/ob BDA-410-treated mice (13.34±0.70% in BDA-410-treated mice vs. 16.32±0.51% in non-treated mice) (data not shown). TEM ultrastructure analysis confirmed the mesangial expansion with collagen structure in BTBR ^ob/ob mice (data not shown). The capillary loops almost completely disappeared in BTBR ^ob/ob mice with focal complete podocyte foot process effacement. In BDA-410-treated mice, the capillary loops were expanded but maintained, glomerular endothelial cells body appeared properly flatten and podocyte foot process were more preserved overall (data not shown). The autophagic flux condition was assessed by SQSTM1 expression analysis. Whereas BTBR ^ob/ob mice presented glomerular accumulation of SQSTM1, BDA-410 treatment prevented SQSTM1 glomerular accumulation in BTBR ^ob/ob mice (0.09±0.01% in BDA-410- treated mice vs. 0.17±0.03% in non-treated mice), showing that the glomerular autophagic flux is maintained in BDA-410-treated mice (data not shown).

Together, these results demonstrated that the pharmacological calpain inhibition using BDA- 410 promotes glomerular autophagic flux maintenance and partially prevents glomerular injury by the preservation of the glomerular filtration barrier ultrastructure in BTBR ^ob/ob diabetic mice.

TRPC6 overexpression, calpastatin decline and SQSTM1 accumulation correlate to glomerular injury in human kidneys from diabetic patients

Next, we analyzed the expression of TRPC6, Calpastatin/CAST and SQSTM1 in renal biopsies from diabetic patients with DKD. Whereas TRPC6 expression was very low in the podocytes of control renal biopsies, TRPC6 expression was increased in podocytes from patients with DKD (data not shown). In addition, SQSTM1 accumulated in podocyte from DKD patients compared to podocytes from control biopsies (data not shown). Furthermore, a glomerulus- by-glomerulus Pearson's correlation analysis showed a negative correlation between podocyte- specific TRPC6 expression and expression of the podocyte-maturity marker Synaptopodin/SYNPO (Pearson r=-0.35, p=1.7e-8). TRPC6 expression also positively correlates to the glomerular injury grade (GIG) (Pearson r=0.32, p=2.5e-7). Moreover, SQSTM1 expression correlated negatively with SYNPO expression (Pearson r=-0.30, p=9.0e- 7), correlated positively with TRPC6 expression (Pearson r=0.48, p=3.0e-16) and correlated positively with the GIG (Pearson r=0.35, p=1.8e-8) (data not shown). Whereas CAST was predominantly expressed in podocytes in glomeruli from control patients, its expression decreased in glomeruli from patients with DKD (data not shown). Pearson's correlation analysis showed a positive correlation between CAST expression and SYNPO expression (Pearson r=0.15, p=0.007) (data not shown). CAST expression also correlated negatively with the GIG (Pearson r=-0.12, p=0.038) (data not shown). SQSTM1 did not correlate with CAST expression (Pearson r=-6.5e-4, p=0.991) (data not shown).

Finally, we also performed a correlation analysis patient-by-patient in order to determine if the mean glomerular staining per patient correlated to the biological parameters. As expected, the mean GIG correlated positively to the UACR and negatively to the eGFR. Conversely, SYNPO expression correlated negatively with the UACR and positively with the eGFR (data not shown). TRPC6 expression correlated negatively to the eGFR (Pearson r=-0.45) but did not correlate to the UACR (Pearson r=0.07). SQSTM1 expression correlated negatively to eGFR (Pearson r=-0.43) and correlate positively with the UACR (Pearson r=0.44) (data not shown). CAST expression did not correlate to eGFR or UACR. (data not shown). Of note, the mean TRPC6 expression still correlated positively to the GIG and negatively to the mean SYNPO expression (data not shown). Similarly, the mean SQSTM1 expression still correlated negatively to the mean SYNPO expression and positively to the mean GIG (data not shown).

Taken together, we showed that podocyte TRPC6 overexpression in human patients with DKD is associated with decreased Calpastatin expression, podocyte autophagic flux blockade and glomerular and podocyte injury. Podocyte TRPC6 overexpression and podocyte autophagy blockade were also correlated to the renal function, as evaluated by eGFR and UACR.

Discussion:

The current study elucidates the crucial involvement of the Ca ²⁺ channel TRPC6 and the cysteine protease calpain in impaired podocyte autophagy and subsequent podocyte injury in the context of DKD. Taken together, our data suggest that TRPC6-mediated Ca ²⁺ -influx results in elevated calpain activity, impaired podocyte autophagy, podocyte injury and development of proteinuria in DKD (not shown). Importantly, transgenic overexpression of the endogenous calpain inhibitor calpastatin, as well as pharmacological inhibition of calpains, normalized podocyte autophagic flux and prevented podocyte injury and proteinuria in experimental DKD.

Calpains are abundant cytoplasmic proteases that can cleave many intracellular signaling and structural proteins. Normal calpain activation plays a role in key signaling processes: they regulate cell behavior, actin cytoskeletal dynamics, cell adhesion and motility, endoplasmic reticulum stress, apoptosis, and inflammation upon interaction with their multiple substrates. Conversely, abnormal calpain activation is responsible for the degradation of most of the cellular protein pool. Aberrantly increased calpain activity may drive kidney disease in rodent models and in humans by different mechanisms: sustaining the inflammation, or damaging the podocyte and disrupting the integrity of the glomerular filtration barrier (56).

Expression of CAPN 1 is not detected in renal cells in single-cell RNAseq data from the Kidney Interactive Transcriptomics and the Kidney Tissue Atlas public database; whereas CAPN2 and CAST appear to be highly enriched in podocytes (data not shown). Our data confirmed enrichment of CAST protein expression in podocytes in mouse and human kidneys. Using in silico analysis, we previously demonstrated that calpains may cleaved some constituents of the podocyte such as NPHS1, NPHS2, SYNPO, ACTN4 or PODXL (57), besides targeting cytoskeleton proteins (38, 39) and proteins of the autophagy machinery. High co-expression of CAPN2 and its inhibitor CAST within the same cells might therefore suggest that tight regulation of calpain activation/ inhibition is required to maintain podocyte homeostasis.

We observed strong decrease of CAST expression in podocytes in human kidney biopsies from diabetic patients. Analysis of Nephroseq database showed that glomerular changes in CAPN1 and CAST mRNA expression are associated to NPHS1 mRNA expression and GFR in DKD patients (data not shown). Interestingly, CAPNI expression increased in glomeruli from DKD patients concomitantly to CAST and CAPN2 decrease. These data support the notion that modulation of calpain activity may impact podocyte homeostasis in DKD. Future studies should elucidate the molecular mechanisms responsible for changes in calpains and calpastatin expression in DKD. Therefore, increasing calpastatin expression in diabetic patients might be an important mechanism to prevent impaired podocyte autophagy, subsequent podocyte injury and proteinuria formation.

BDA-410 is an orally active calpain inhibitor with relatively selective inhibition of calpain- 1 (Ki value of 130 nM) rather than calpain-2 (K _i value of 630 nM) (35, 40). BDA-410 has proven interesting beneficial in neurological disorders and aging. It improves the memory and synaptic transmission in mouse model of Alzheimer disease (40). It also alleviates neurodegeneration in a mouse model of inherited cerebellar ataxia (41). In aging-related disease models, BD A-410 ameliorates aging phenotype via increased lipogenesis and reduction of body weight (42) and enhancement of muscle force (43). It also allows recovery from age-related phenotype in a- klotho deficient mice, a model of accelerated aging (33). No significant side effects were reported in any of these studies, suggesting good tolerance of systemic administration of this calpain 1 inhibitor in mice, at least at the doses used and for the duration of treatment. In the present study, we showed that BDA-410 ameliorates diabetic-induced glomerular injury in a mouse model of type 2 diabetes mellitus, with preservation of the autophagic flux in glomeruli. Interestingly, BDA-410 treatment not only prevents the podocyte injury but also preserve the glomerular structure overall, suggesting that calpain- 1 inhibition may be beneficial for other ceils than podocytes in kidneys. Because physiological roles of calpains are generally auxiliary, their inhibition is usually well tolerated and supports the rationale for using conventional calpain inhibitors to treat diseases that are aggravated by increased calpain activity. BDA-410 is not currently implicated in a clinical trial but others calpain inhibitors are (44, 45). Therefore, targeting calpains might be a promising therapeutic strategy for the treatment of DKD by restoring podocyte autophagy.

We and others have previously demonstrated that maintenance of the autophagic flux is important to maintain podocyte homeostasis in DKD (19, 20). However, the molecular mechanisms underlying impaired podocyte autophagy in DKD remained largely elusive. Calpains have been found to participate in autophagic flux regulation through different actions (24, 25, 46). We demonstrated previously that genetic calpastatin constitutive overexpression in mice was able to stimulate the autophagic flux in podocytes and that calpastatin-mediated autophagic flux activation prevented podocyte injury in a model of hypertension-induced glomerular injury, limiting glomerular oxidative and ER stress (37). However, the regulation of calpains in DKD has not been explored. TRPC6 is a slit diaphragm-associated Ca ²⁺ -channel, whose expression is known to be increased in DKD. In podocytes, TRPC6 directly interacts with CAPN1/2 at the plasma membrane to regulate their activity, in a Ca ²⁺ -independent manner. This interaction is crucial to control cell motility and adhesion through regulation of Paxillin, FAK and Talin-1 (28). Previous studies showed that the interaction between TRPC6 and calpain also contributes to podocyte injury and podocyte death via aberrant cytoskeletal remodeling (27, 47). In the current study, we observed that TRPC6-mediated calpain activation also leads to podocyte injury via impaired podocyte autophagy. Importantly, we could support the clinical relevance of these findings in diabetic patients; the expression of both SQSTM1 and TRPC6 was enhanced in podocytes from diabetic patients and SQSTM1 expression showed a negative correlation with expression of the podocyte marker synaptopodin. These findings highlight that the interaction between TRPC6 and calpains is more complex and diverse than previously anticipated. The results of this study also support the use of TRPC6 inhibitors like BI 749327 or larixyl acetate for preventing podocyte injury in DKD by restoring autophagy (48, 49). The TRPC6 inhibitor BI 749327 was previously shown to reduce renal fibrosis upon inducing unilateral ureteral obstruction (UUO) (48). Of note, increased podocyte-specific expression of TRPC6 is also observed in other glomerulopathies than DKD, such as focal segmental glomerulosclerosis (FSGS) (50). Moreover, impaired podocyte autophagy is known to contribute to further disease progression of FSGS (51, 52). Therefore, it would be interesting to investigate in future studies if TRPC6 also contributes to impaired podocyte autophagy and further disease progression in the context of FSGS via aberrant calpain activation. An improved understanding of the interaction between TRPC6, calpains and autophagy might highlight the underlying molecular mechanisms of the pathogenesis of podocytopathies.

The data of this study sheds new light on the underlying pathogenic mechanisms of impaired podocyte autophagy in DKD and highlights the therapeutic potential of inhibiting calpain as treatment options for DKD.

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Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

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