WO2024018245A1 - Utilisation d'inhibiteurs de calpaïne pour le traitement de la maladie rénale diabétique - Google Patents

Utilisation d'inhibiteurs de calpaïne pour le traitement de la maladie rénale diabétique Download PDF

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WO2024018245A1
WO2024018245A1 PCT/IB2022/000413 IB2022000413W WO2024018245A1 WO 2024018245 A1 WO2024018245 A1 WO 2024018245A1 IB 2022000413 W IB2022000413 W IB 2022000413W WO 2024018245 A1 WO2024018245 A1 WO 2024018245A1
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podocyte
calpain
trpc6
autophagy
podocytes
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PCT/IB2022/000413
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Olivia Lenoir
Pierre-Louis Tharaux
Yann SALEMKOUR
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INSERM (Institut National de la Santé et de la Recherche Médicale)
Université Paris Cité
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/55—Protease inhibitors
    • A61K38/556—Angiotensin converting enzyme inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/63—Compounds containing para-N-benzenesulfonyl-N-groups, e.g. sulfanilamide, p-nitrobenzenesulfonyl hydrazide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/55—Protease inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/55—Protease inhibitors
    • A61K38/57—Protease inhibitors from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys

Definitions

  • the present invention is in the field of medicine, in particular nephrology.
  • Diabetic kidney disease 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.
  • RAAS renin- angiotensin-aldosterone system
  • SGLT2 inhibition was added to the therapeutic armamentarium to prevent of mitigate DKD (2-5).
  • 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.
  • TRPC6 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.
  • 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).
  • AGT5 autophagy-initiating protein autophagy related 5
  • Calpain- 1 and calpain-2 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.
  • the present invention is defined by the claims.
  • the present invention relates to the use of calpain inhibitors for the treatment of the diabetic kidney disease.
  • Diabetic kidney disease is associated with impaired podocyte autophagy and subsequent podocyte injury.
  • the molecular mechanisms underlying the impaired autophagy in podocytes remain largely elusive.
  • 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.
  • TRPC6 knockdown increased the autophagic flux in podocytes as a result of decreased calpain activity.
  • 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.
  • kidney biopsies from diabetic patients the inventors confirmed that TRPC6 overexpression in podocytes correlated with decreased calpastatin expression, autophagy blockade and podocyte injury.
  • 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.
  • kidney disease 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.
  • 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”.
  • treatment refers 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.
  • 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.
  • maintenance regimen 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.
  • 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.
  • 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).
  • 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.
  • the calpain inhibitor may be a peptidomimetic calpain inhibitor, a non-peptide calpain inhibitor.
  • 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 III (carbobenzoxy- valyl
  • 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.
  • 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.
  • 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.
  • the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day.
  • 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.
  • 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.
  • 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.
  • 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.
  • FIGURES are a diagrammatic representation of FIGURES.
  • Figure 2 Pharmacological calpain-1 inhibition with BDA-410 preserve the glomerular filtration function in type II diabetes model.
  • mice 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.
  • 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.
  • STZ Streptozocin
  • 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.
  • MPC-5 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 (IFN ⁇ , 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 400 ⁇ g/mL G418 (Sigma- Aldrich, Al 720) to select successfully transfected cells.
  • G418 Sigma- Aldrich, Al 720
  • single clones were exposed to 5 ⁇ g/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 (0.25 ⁇ g/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 100 ⁇ g/mL G418.
  • wildtype (WT) and TRPC6 KD podocytes were exposed for 48hrs to the calpain inhibitor calpeptin (1 ⁇ M, 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 5 ⁇ g/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.
  • GPDH glyceraldehyde-3 -phosphate dehydrogenase
  • 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.
  • BCA bicinchoninic acid assay
  • 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).
  • 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.
  • 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.
  • 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.
  • UCR urinary albumin-to-creatinine ratio
  • eGFR estimated glomerular filtration rate
  • 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.
  • 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).
  • JEOL JEM1011 transmission microscope
  • Single-cell RNAseq and Single-nuclei RNAseq data were extracted from public databases.
  • 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/.
  • Podocyte-specific TRPC6 overexpression is associated with autophagy blockade and podocyte dedifferentiation in murine diabetic models.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • WT1 Wilm's Tumor 1
  • 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).
  • uninjured podocytes show low TRPC6 expression levels, podocytes were exposed to Adriamycin to induce podocyte injury and increase TRPC6 expression.
  • 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.
  • TRPC6 KD calpain activity
  • 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/ ⁇ g in WT vs. 5.82 ⁇ 0.27 RFU/ ⁇ g in TRPC6 KD) (data not shown).
  • treatment with the calpain inhibitor calpeptin reduced calpain activity in WT podocytes (13.38 ⁇ 0.72 RFU/ ⁇ g in WT vs. 4.34 ⁇ 0.08 RFU/ ⁇ g in WT + calpeptin).
  • 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).
  • calpeptin treatment did not result in an altered LC3B-II/LC3B-I ratio for both WT and TRPC6 KD podocytes.
  • SQSTM1 expression was decreased in WT podocytes upon calpetin treatment (0.82 ⁇ 0.05 vs. 1 ⁇ 0.07).
  • 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.
  • 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).
  • LC3B punctae in TRPC6 KD cells compared to WT cells (1.365 ⁇ 0.145 vs. 1.037 ⁇ 0.071).
  • Calpastatin overexpression prevents podocyte injury and restores podocyte autophagic flux in type I diabetes model
  • 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).
  • the baseline albumin-to-creatinine ratio was identical between the two genotypes (data not shown).
  • 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.
  • 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).
  • TRPC6 overexpression, calpastatin decline and SQSTM1 accumulation correlate to glomerular injury in human kidneys from diabetic patients
  • 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).
  • SQSTM1 accumulated in podocyte from DKD patients compared to podocytes from control biopsies (data not shown).
  • GIG glomerular injury grade
  • CAST expression did not correlate to eGFR or UACR. (data not shown).
  • the mean TRPC6 expression still correlated positively to the GIG and negatively to the mean SYNPO expression (data not shown).
  • the mean SQSTM1 expression still correlated negatively to the mean SYNPO expression and positively to the mean GIG (data not shown).
  • 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.
  • 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).
  • 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.
  • 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).
  • calpain inhibitors 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.
  • TRPC6 is a slit diaphragm-associated Ca 2+ -channel, whose expression is known to be increased in DKD.
  • TRPC6 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.
  • TRPC6 increased podocyte-specific expression of TRPC6 is also observed in other glomerulopathies than DKD, such as focal segmental glomerulosclerosis (FSGS) (50).
  • FSGS focal segmental glomerulosclerosis
  • 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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Abstract

Une maladie rénale diabétique est associée à une autophagie podocytaire altérée et à une lésion podocytaire ultérieure. Dans la présente invention, les inventeurs ont étudié comment le canal calcique TRPC6 et les protéases à cystéine calpaïnes altèrent l'autophagie podocytaire dans une maladie rénale diabétique. Ils ont démontré que l'inactivation de TRPC6 dans des podocytes a augmenté le flux autophagique suite à une diminution de l'activité des calpaïnes. Une maladie rénale diabétique a été mimée in vivo en utilisant à la fois le modèle de diabète induit par la streptozotocine lors d'une néphrectomie unilatérale et le modèle de souris BTBRob/ob. Le diabète a résulté en une expression accrue de TRPC6 dans des podocytes in vivo conjointement avec un flux autophagique de podocyte réduit. La surexpression transgénique de la calpastatine inhibitrice de la calpaïne endogène ainsi que l'inhibition pharmacologique du flux autophagique de podocyte a normalisé l'activité de la calpaïne, a réduit la perte de néphrine et a prévenu le développement de l'albuminurie chez des souris diabétiques. Dans des biopsies rénales provenant de patients diabétiques, les inventeurs ont confirmé que la surexpression de TRPC6 dans des podocytes est corrélée à une expression de calpastatine réduite, un blocage de l'autophagie et une lésion des podocytes. Par conséquent, le ciblage de la calpaïne peut être une stratégie thérapeutique prometteuse pour le traitement d'une maladie rénale diabétique par restauration de l'autophagie podocytaire.
PCT/IB2022/000413 2022-07-22 2022-07-22 Utilisation d'inhibiteurs de calpaïne pour le traitement de la maladie rénale diabétique WO2024018245A1 (fr)

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