Compositions and methods for treating kidney diseases in pets

Abstract

Methods of culturing renal cells of pets are provided. Also provided are methods of producing spheroids and organoids from pet renal cells and uses thereof in regenerating renal function.

Description

[0001] COMPOSITIONS AND METHODS FOR TREATING KIDNEY DISEASES IN PETS

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit of priority from U.S. Provisional Patent Application No. 63 / 762,765 filed on February 25, 2025, which is fully incorporated herein by reference in its entirety.

[0004] FIELD AND BACKGROUND OF THE INVENTION

[0005] The present invention, in some embodiments thereof, relates to compositions and methods for treating kidney diseases in pets.

[0006] Chronic kidney disease (CKD) is a prevalent condition affecting both dogs and cats, with increasing incidence in aging populations. The estimated prevalence of CKD is 0.5- 1.0 % in dogs and 1.0-3.0 % in cats, rising significantly in older animals. Studies report that up to 10 % of dogs and up to 80 % of cats over 15 years old may develop CKD. The disease in dogs and cats is also irreversible and progressive in nature, and the end result is similar to human patients; interstitial nephritis and fibrosis resulting in loss of functional renal parenchyma and progression to end stage kidney disease (ESKD). Treatment strategies for CKD in companion animals are multifaceted and aim to manage symptoms, slow disease progression, and improve quality of life. CKD in dogs and cats is known to progress rapidly, animal with advanced disease have a life expectancy as short as several months.

[0007] In pursuing enhancing the lives of dogs facing kidney disease, stem cell therapy emerges as a beacon of hope and innovation. There are some products which are available on the market or are in advance stages of development such as VetStem™ and allogeneic adipose-derived mesenchymal stromal cell (aASC), see e.g., Milistetd et al. Arq. Bras. Med. Vet. Zootec., v.74, n.2, p.310-318, 2022.

[0008] Organoids are three-dimensional, multicellular structures that mimic the morphology and physiology of the tissue they originate from, whether normal or diseased. They can be generated from two main sources: pluripotent stem cells (PSCs) or adult stem cells (ASCs), each with distinct characteristics and applications in kidney research.

[0009] One potential limitation of patient-derived organoid cultures, including tubuloids, is the availability of fresh tissue. However, recent studies have shown that urine can serve as a source for kidney epithelial cells, offering a simpler, less invasive method to obtain patient material for useful spheroid cultures (WO2022 / 084998), however unlike organoids they are a much more primitive structure with no ability to proliferate or respond to drugs ex vivo. Also, no results were presented for treatment of pets.Additional Related Background Art:

[0010] Schutgens, F., Rookmaaker, M.B., Margaritis, T. el al. Tubuloids derived from human adult kidney and urine for personalized disease modeling. 2017 Nat Biotechnol 37, 303-313;

[0011] Guo H, Deng N, Dou L, Ding H, Criswell T, Atala A, Furdui CM, Zhang Y. 3-D Human Renal Tubular Organoids Generated from Urine-Derived Stem Cells for Nephrotoxicity Screening. ACS Biomater Sei Eng. 2020 Dec 14;6(12):6701-6709; or

[0012] Sun G, Ding B, Wan M, Chen L, Jackson J, Atala A. Formation and optimization of three-dimensional organoids generated from urine-derived stem cells for renal function in vitro. Stem Cell Res Ther. 2020 Jul 22; 1 l(l):309.

[0013] SUMMARY OF THE INVENTION

[0014] According to an aspect of some embodiments of the present invention there is provided a method of culturing kidney epithelial cells of a pet, the method comprising:

[0015] (a) isolating kidney epithelial cells from a biological sample of the pet;

[0016] (b) culturing the kidney epithelial cells under adherent conditions, thereby culturing kidney epithelial cells.

[0017] According to some embodiments of the invention, the pet is selected from the group consisting of a dog, a cat, a rabbit, a guinea pig and a bird.

[0018] According to some embodiments of the invention, the pet is selected from the group consisting of a dog and a cat.

[0019] According to some embodiments of the invention, the biological sample comprises urine. According to some embodiments of the invention, the urine is bladder urine.

[0020] According to some embodiments of the invention, the urine is voided urine.

[0021] According to some embodiments of the invention, the biological sample comprises a kidney biopsy.

[0022] According to some embodiments of the invention, the culturing comprises passaging for at least one passage.

[0023] According to some embodiments of the invention, the at least one passage is P0.

[0024] According to some embodiments of the invention, the at least one passage is Pl.

[0025] According to some embodiments of the invention, the culturing is performed in the presence of serum.

[0026] According to some embodiments of the invention, the culturing is performed in RE:MC.According to some embodiments of the invention, the culturing is performed in the presence of CD40 ligand (CD40L) and optionally at least one of epidermal growth factor (EFG), platelet-derived growth factor (PDGF)-AB-IO and fibroblast growth factor (FGF) basic.

[0027] According to some embodiments, at least one of the growth factors is of the pet’s origin. For instance, when the cells are of cat’s cells, the growth factor (e.g., recombinant) would be also of cat.

[0028] According to some embodiments, at least one of the growth factors is of human origin. According to some embodiments of the invention, the isolating is by centrifugation.

[0029] According to some embodiments of the invention, the adherent conditions comprise gelatin coating.

[0030] According to some embodiments of the invention, the biological sample is urine and the passaging is performed to enrich for urine derived epithelial cells (UD-EpC) and deplete squamous epithelial cells of vagina and / or bladder origin.

[0031] According to some embodiments of the invention, the pet is healthy.

[0032] According to some embodiments of the invention, the pet is diagnosed with a kidney disease.

[0033] According to an aspect of some embodiments of the present invention there is provided a cell culture comprising the renal epithelial cells obtainable according to the method as described herein.

[0034] According to an aspect of some embodiments of the present invention there is provided a method of producing a nephrospheroid, the method comprising culturing the renal epithelial cells obtainable according to the method as described herein under low-adherence conditions, thereby generating the nephrospheroid.

[0035] According to some embodiments of the invention, the low adherence conditions comprise polyHEMA.

[0036] According to some embodiments of the invention, the nephrospheroid is Pan-Cytokeratin+ / EpCAM+ / LRP2+ at the protein level.

[0037] According to some embodiments of the invention, the pet is a cat, the passaging is until Pl and the biological sample is bladder urine.

[0038] According to some embodiments of the invention, the pet is a dog, the passaging is until Pl, and wherein the biological sample is a kidney.

[0039] According to an aspect of some embodiments of the present invention there is provided a nephrospheroid obtainable according to the method as described herein.According to some embodiments of the invention, the nephro spheroid has an anti-fibrotic activity.

[0040] According to an aspect of some embodiments of the present invention there is provided a nephro spheroid comprising epithelial cells of a pet, the nephro spheroid is capable of forming a tubular nephric tissue upon transplantation and / or having an anti-fibrotic activity.

[0041] According to an aspect of some embodiments of the present invention there is provided a method of producing a kidney organoid, the method comprising:

[0042] (a) culturing the renal epithelial cells obtainable according to the method as described herein under conditions which allow formation of kidney organoids (ORGs); and

[0043] (b) passaging the ORGs.

[0044] According to some embodiments of the invention, the conditions which allow formation of kidney organoids (uORGs) comprise at least one of N-acetylcysteine (NAC) or an analog thereof, ROCK inhibitor, epidermal growth factor (EGF), fibroblast growth factor 10 (FGF-10), B27 and a Wnt activator.

[0045] According to some embodiments of the invention, the culturing the renal epithelial cells is by encapsulating the renal epithelial cells within an extracellular matrix (ECM).

[0046] According to some embodiments of the invention, the ECM comprises basement membrane extract (BME).

[0047] According to some embodiments of the invention, the culturing is in the absence of serum. According to some embodiments of the invention, the passaging is for at least 10 passages. According to some embodiments of the invention, the method further comprises isolating cells from the kidney organoid and / or analyzing the expanded organoids or cells thereof.

[0048] According to some embodiments of the invention, the pet is a dog, the renal epithelial cells are P0, and the organoid is ECAD+ and AQP1+ as determined at the protein level.

[0049] According to an aspect of some embodiments of the present invention there is provided an isolated kidney organoid (uORG) ex vivo produced from kidney epithelial cells of a pet and comprising a cystic interior, wherein the organoid is proliferative and a polarized tubule structure prior to transplantation.

[0050] According to an aspect of some embodiments of the present invention there is provided an isolated kidney organoid (ORG) obtainable according to the method as described herein.

[0051] According to some embodiments of the invention, the organoid comprises cells that are ECAD+ and AQP1+ as determined at the protein level.

[0052] According to some embodiments of the invention, the pet is selected from the group consisting of a dog, a cat, a rabbit, a guinea pig and a bird.According to some embodiments of the invention, the pet is a dog.

[0053] According to an aspect of some embodiments of the present invention there is provided a secretome of the nephro spheroid or organoid as described herein.

[0054] According to an aspect of some embodiments of the present invention there is provided a nephro spheroid or organoid or secretome of each for use in the treatment of a kidney disease;

[0055] According to some embodiments of the invention, the kidney disease is a chronic kidney disease (CKD).

[0056] According to some embodiments of the invention, the kidney disease is an acute kidney disease.

[0057] According to an aspect of some embodiments of the present invention there is provided a method of producing a secretome, the method comprising:

[0058] (a) providing a culture of the nephro spheroid or organoid as described herein;

[0059] (b) collecting conditioned medium of the culture, the conditioned medium comprising the secretome.

[0060] According to some embodiments of the invention, the method further comprises isolating a portion from the conditioned medium following step (b).

[0061] According to an aspect of some embodiments of the present invention there is provided a method of regenerating renal function, the method comprising administering to a subject in need thereof nephro spheroid or organoid as described herein or secretome thereof or portion thereof, thereby regenerating renal function.

[0062] According to an aspect of some embodiments of the present invention there is provided a nephro spheroid or organoid or secretome thereof for use in the treatment of a kidney disease.

[0063] According to an aspect of some embodiments of the present invention there is provided a method of drug design, the method comprising determining an effect of a test drug on the nephro spheroid or organoid as described herein or secretome thereof.

[0064] According to an aspect of some embodiments of the present invention there is provided a method of selecting treatment to a pet diagnosed with a kidney disease, the method comprising: (a) contacting a therapeutic agent with a nephro spheroid or organoid as described herein of a pet diagnosed with the kidney disease; and

[0065] (b) determining an alleviation in a renal symptom evident in the nephrospheroid or organoid following said contacting, said alleviation being indicative of an efficacious therapy.

[0066] According to some embodiments of the invention, said effect is cyst size of the organoid. Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains.Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[0067] BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

[0068] Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

[0069] FIGs. 1A-D shows the generation of cat urine-derived spheroids (uSPH), according to some embodiments of the invention. (A) Schematic representation of the process for generating uSPH from cat: urine is collected directly from a cat’s bladder. Kidney cells are isolated by centrifugation, and then seeded on gelatin-coated plates. Upon reaching confluence, the cells are transferred to three dimensional (3D) culture conditions to form spheroids. (B) Bright- field microscopy images of urine-derived kidney epithelial cells cultured in 2D (left) and 3D spheroid conditions (right). (C) Immunofluorescence staining of cat uSPH showing expression of epithelial markers Pan-cytokeratin and EpCAM, as well as the proximal tubular marker LRP2. (D) Parallel process performed using dog urine: urine-derived epithelial cells (uEpC) in 2D culture (left) and spheroid formation in 3D conditions (right).

[0070] FIGs. 2A-D show a process for generating dog urine-derived organoids (uORG), according to some embodiments of the invention. (A) Schematic representation of a process for generating dog uORGs: urine is collected directly from the dog’s bladder. Kidney cells are isolated by centrifugation, and then seeded on gelatin-coated plates. Upon reaching confluence, the cells are transferred to 3D culture conditions to form organoids. The organoids were then injected into the capsular-cortical space of the kidney. (B) Bright-field microscopy images of urine-derived kidney epithelial cells cultured in 2D (top left) and 3D organoid conditions (top right). Immunofluorescence staining of dog uORG shows expression of the epithelial marker E-cadherin (down left) and the proximal tubular marker LRP2 (down right). (C) Images depicting the process of ultrasound-guided injection into the dog’s kidney. (D) Creatinine and urea levels of two treated dogs before and after cell injection. The black bar represents the life expectancy of the dogs at that stage of chronic kidney disease (CKD).FIGs. 3A-E show a process for generating secretome from dog uORG according to some embodiments of the invention. (A) Schematic representation of the process for generating secretome from dog uORG: the secretome is collected from established organoids (as previously described) and administered intravenously to the dog. (B-E) Creatinine and urea blood levels of four dogs monitored throughout their treatment.

[0071] FIGs. 4A-B show characterization and efficacy of secretome according to some embodiments of the invention. (A) Heat map of proteomic analysis of secretomes derived from EpC and SPH obtained from urine and kidney cultures, demonstrating similarities between kidneyorigin and urine-origin secretomes. (B) In vitro fibrosis potency assay testing the effect of secretomes from different sources: AK-KOSEC (from human kidney tissue), KOSECOOl (from urine), and DOG-KOSEC (from dog kidney tissue).

[0072] FIGs. 5A-D show the generation of organoids and spheroids from kidney biopsy of cats. (A) Schematic representation of cat kidney-derived organoid generation from primary kidney tissue. (B) Representative phase-contrast images of cat kidney organoids. (C) Immunofluorescence staining for the epithelial marker E-cadherin and F-actin (phalloidin). (D) Immunofluorescence staining for the epithelial marker EpCAM.

[0073] FIGs. 6A-D show the generation of organoids and spheroids from kidney biopsy of cats. (A) Schematic representation of dog kidney-derived organoid generation from primary kidney tissue. (B) Representative phase-contrast images of dog kidney organoids. (C) Immunofluorescence staining for the epithelial marker E-cadherin and F-actin (phalloidin). (D) Immunofluorescence staining for kidney epithelial markers EpCAM and EMA.

[0074] FIGs. 7A-H show characterization and efficacy of secretome of rat kidney organoids according to some embodiments of the invention. A) Schematic representation of rat kidney-derived spheroid formation and secretome generation from primary kidney tissue. (B) Representative phase-contrast images of rat kidney-derived cells cultured in two-dimensional (2D) monolayer and three-dimensional (3D) spheroid conditions. (C) qPCR analysis of fibrosis-associated markers in TGF-P-activated fibroblasts, showing reduced expression following treatment with rat kidney secretome. (D) Schematic representation of the unilateral nephrectomy plus ischemia-reperfusion injury (uNx + IRI) rat model of chronic kidney disease (CKD). (E) Summary table of experimental groups, injury induction, and treatment regimens in the unilateral nephrectomy plus ischemia-reperfusion injury (uNx + IRI) rat model. (F) Rat KOSEC-treated animals exhibited significantly reduced urinary protein excretion, including albumin-to-creatinine ratio, total albumin, and fractional excretion of albumin, beginning at day 56. A dose-dependent effect was observed, with the higher rat KOSEC dose demonstrating greater efficacy. (G)Glomerular filtration rate (GFR) trajectory analysis revealed maladaptive hyperfiltration in vehicle-treated rats at day 56, coinciding with the onset of significant proteinuria. In contrast, KOSEC-treated rats maintained stable GFR levels. (H) Analysis of GFR slopes across days 56-91 and 70-91 demonstrated significantly less negative trajectories in KOSEC-treated rats, indicating preserved filtration stability and protection against progressive renal function decline.

[0075] DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

[0076] The present invention, in some embodiments thereof, relates to compositions and methods for treating kidney diseases in pets.

[0077] Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

[0078] Chronic kidney disease (CKD) is a prevalent and progressively worsening condition in pets, particularly in aging dogs and cats. It is characterized by the gradual loss of kidney function, which can lead to a variety of severe symptoms, including weight loss, lethargy, poor appetite, and increased thirst and urination. Currently, the management of CKD in pets primarily involves supportive treatments, including dietary modifications (e.g., kidney- specific diets), fluid therapy, and medications aimed at controlling symptoms like high blood pressure and proteinuria. While these interventions can help slow the progression of the disease, they do not cure CKD, and their effectiveness is often limited. The primary shortcomings of existing treatments include the lack of methods to reverse kidney damage, the complexity of managing fluid and electrolyte balance, and the frequent need for ongoing, expensive veterinary care. Additionally, many pets with CKD may experience a reduced quality of life due to the chronic nature of the disease and the side effects of current treatments, highlighting the need for more effective therapeutic solutions.

[0079] Kidney transplantation in pets, particularly dogs and cats, is technically feasible but rarely performed and comes with significant challenges. While kidney transplants have been successfully done in some cases, they are not common due to several practical, financial, and medical limitations.

[0080] Whilst conceiving embodiments of the invention and reducing them to practice, the present inventors were able for the first time, to produce regenerative kidney structures such as organoids and nephrospheroids from cultured kidney epithelial cells obtained from urine and kidney biopsies of pets (see Figures 1A-D and 4A-B). Organoids generated accordingly, were implanted in dogs suffering from chronic kidney disease (CKD). As shown in Figures 2A-D, their survival far exceeded historicalcontrols diagnosed with similar CKD stages. Moreover, kidney function parameters were mostly stabilized over the period of treatment, substantiating the potential of engrafted ORG to deliver beneficial signals to chronically injured dog kidneys. Similar results were obtained when treating with organoid secretome (see Figures 3A-E). The present inventors have also produced nephrospheroids and organoids from kidney biopsies of cats, dogs and rats and showed that they express tubular markers (Figures 5A-D, 6A-D and 7A-C). Organoids secretome had a therapeutic activity as shown in Figures 7F-H.

[0081] Thus, according to an aspect of the invention there is provided a method of culturing kidney epithelial cells of a pet, the method comprising:

[0082] (a) isolating kidney epithelial cells from a biological sample of the pet; and

[0083] (b) culturing the kidney epithelial cells under adherent conditions, thereby culturing kidney epithelial cells.

[0084] As used herein “pets” refers to a domesticated animal kept by humans for companionship, amusement, or other non-working purposes. This includes, but is not limited to, commonly kept animals such as dogs, cats, rabbits, birds, guinea pigs, and other small mammals (e.g., rats) or nonhuman species that are typically housed in a domestic environment. Pets may vary in species, breed, size, and care requirements, but they are generally not used for agricultural, commercial, or service-related tasks.

[0085] According to a specific embodiment, the pet is selected from the group consisting of a dog, a cat, a rabbit, a guinea pig and a bird.

[0086] According to a specific embodiment, the pet is selected from the group consisting of a dog and a cat.

[0087] According to a specific embodiment, the pet is purebred.

[0088] According to a specific embodiment, the pet is mixed-breed.

[0089] According to a specific embodiment, the pet is a healthy pet, e.g., not suffering from CKD. This embodiment is of specific significance when the cells are used as donor cells to generate nephrospheroids or organoids or secretome thereof for therapy.

[0090] According to a specific embodiment, the pet is diagnosed with CKD. This embodiment is of specific significance for drug screening or autologous transplantations.

[0091] According to a specific embodiment, the pet is of any age or gender (male or female).

[0092] While the present specification refers to pets more emphasis is done on cats and dogs, however the skilled artisan in the art of veterinary medicine would know how to apply these teachings on other pets.According to a specific embodiment, the pet is at a senior stage of life (7 years or older in the case of cats and dogs), when CKD becomes more common, especially in cats and larger dog breeds.

[0093] As used herein “kidney epithelial cells” are epithelial cells which are derived from the renal tissue. The cells may be characterized by Pax8+p63-, which typically identify kidney epithelial cells (see for example, Peat TJ, Edmondson EF, Miller MA, DuSold DM, Ramos-Vara JA. Pax8, Napsin A, and CD 10 as Immunohistochemical Markers of Canine Renal Cell Carcinoma. Veterinary Pathology. 2017;54(4):588-594).

[0094] The phrase “urine derived epithelial cells” abbreviated as UD-EpCs, refers to epithelial cells which are shed from the kidney to the urine.

[0095] As used herein “culturing” refers to maintaining the number of cells isolated from the biological sample and ultimately increasing the number of cells by in vitro expansion (proliferation).

[0096] As used herein “biological sample” refers to a sample which comprises kidney epithelial cells. Such a sample can be obtained from anywhere in the urothelial system which comprises kidney epithelial cells.

[0097] According to a specific embodiment, the biological sample comprises urine.

[0098] Collection of urine samples from pets can be done in several ways which are well known in the art.

[0099] Urine samples such as from cats and dogs can be collected using methods which are known in the art. One common approach is free catch, where urine is collected as the animal urinates. This is also referred to herein as voided urine. For dogs, this is typically done during a walk, while for cats, it may involve collecting urine from the litter box using a sterile container. Cystocentesis may be employed to ensure sterility of the sample, where a veterinarian inserts a needle into the bladder to collect urine directly. Another method is catheterization, in which a catheter is inserted into the urethra to obtain urine, also requiring veterinary assistance.

[0100] According to a specific embodiment, the urine is bladder urine.

[0101] According to a specific embodiment, the urine is voided urine.

[0102] According to other embodiments, the biological sample comprises a kidney biopsy. Renal biopsy retrieval can be sone using methods which are well known in the art such as needle (percutaneous) biopsy, wedge biopsy (surgical biopsy), post-mortem (necropsy) biopsy) or tissue samples obtained from euthanized pets.

[0103] The tissue biopsy is typically washed, weighed, and minced. The dissected tissue is then subjected to enzymatic digestion to get isolated cells e.g., with collagenase IV. Followingdigestion, the tissue is filtered to obtain a single-cell suspension. The medium is then removed and the cells are suspended in growth medium (e.g., RE:MC). Once the sample is available, kidney epithelial cells are isolated therefrom.

[0104] Notably, urine samples comprise, aside from epithelial cells, also squamous cells (mostly observed in samples derived from women, likely of urethral origin), leukocytes and semen, in accordance with previous reports. However, these cell types typically do not adhere to the plate surface and gradually disappear following a few medium aspirations.

[0105] According to a specific embodiment, isolating urine cells is by centrifugation. Thus, according to a specific embodiment, to obtain kidney cells from the urine, urine samples are centrifuged (e.g., 400 x g), washed with buffer e.g., PBS and centrifuged, such as at higher, same or lower gravity e.g., at 200xg, than the first round. Then the cell pellet is seeded on an adherent substrate.

[0106] Thus, the cells are cultured under adherent conditions and in effect passaging is performed to enrich for UD-EpC and deplete squamous epithelial cells of vagina and / or bladder origin.

[0107] Therefore at P0 or Pl and certainly in later passages the culture is pure, i.e., more than 90 %, 95 % or 99 % cells are kidney epithelial cells.

[0108] As mentioned, passaging is also performed to achieve higher numbers of kidney or urine derived epithelial cells which is important in the process of producing nephrospheroids or organoids.

[0109] According to a specific embodiment “expanding” is by at least 10 fold increase in epithelial cells number compared to the amount adhered to the container following isolation from the (e.g., urine) sample.

[0110] According to a specific embodiment, the adherent conditions comprise gelatin coating, though other adherent natural or synthetic matrices may be used. Examples include, but are not limited to Poly-L-lysine (PLL) and Poly-D-lysine (PDL), Poly-ornithine (PLO), Dendritic polyglycerol amine (dPGA), SARSTEDT Cell+ plates and Coming® CellBIND® surface.

[0111] The phrase “adherent conditions” refers to conditions in which the cells attach to the surface of a container in which they are cultured such that a substantial portion of the cells cannot be removed from the surface of the container by mechanical manipulations that do not cause significant damage to the cells.

[0112] In general, where possible reagents are used which are derived from the pet or close homologs (evolutionary close) or synthetic reagents to avoid xeno-contaminants.

[0113] The conditions for culturing (expanding) urine epithelial cells further include serumcontaining medium.According to a specific embodiment, RE:MC or KSFM media are used for establishing UD-EpC or kidney cells cultures.

[0114] RE:MC: (renal growth medium: Mesenchymal Cell proliferation at a 1:1 ratio) (e.g., REGM Renal Growth BulletKit: REBM™ Basal Medium with Additives: hEGF, Insulin, Hydrocortisone, FBS, Transferrin, Triiodothyronine, Epinephrine Lonza, Cat No. CC-3190) and mesenchymal cell media, MC (Dulbecco's Modified Eagle Medium (DMEM) High Glucose, 1 % L-glutamine solution, 1 % Penicillin- Streptomycin Solution, Human PDGF-AB-10 (10-25ng / ml), Animal Free Human EGF (10-50 ng / ml), Human FGF-basic (10-50 ng / ml) and 5-10 % FBS] supplemented with CD40L (2-10 ng / ml).

[0115] KSFM:PR (keratinocyte serum free medium: progenitor cell medium at a 1: 1 ratio) KSFM (Keratinocyte-SFM Medium with Additives: Glutamine, Phenol Red, EGF and Bovine Pituitary Extract, ThermoFisher Cat No. 17005075), PR (DMEM-F12, hydrocortisone, cholera toxin, insulin, transferrin, Animal Free Human EGF and 10 % FBS).

[0116] According to a specific embodiment, the conditions for expansion in 2D culture comprise presence of CD40 ligand [CD40L (2-10 ng / ml)] and optionally at least one of platelet-derived growth factor [(PDGF)-AB-IO (10-25 ng / ml)], epidermal growth factor [(EGF) (10-50ng / ml)] and basic fibroblast growth factor [FGF-basic. bFGF, FGF2 (10-50 ng / ml)].

[0117] According to a specific embodiment, the medium is supplemented with CD40L.

[0118] According to a specific embodiment, the 2D culture medium for kidney epithelial cells or Ud-EpC cells comprise RE:MC: (renal growth medium: Mesenchymal Cell proliferation at a 1:1 ratio) (e.g., REGM Renal Growth BulletKit: REBM™ Basal Medium with Additives: hEGF, Insulin, Hydrocortisone, FBS, Transferrin, Triiodothyronine, Epinephrine Lonza, Cat No. CC-3190) and mesenchymal cell media, MC [Dulbecco's Modified Eagle Medium (DMEM) High Glucose, 1 % L-glutamine solution, 1 % Penicillin- Streptomycin solution, Human PDGF-AB-10 (10-25 ng / ml), Animal Free Human EGF (10-50ng / ml), Human FGF-basic (10-50 ng / ml) and 5-10% FBS] supplemented with CD40L (2-10 ng / ml).

[0119] As used herein, “serum” refers to naturally occurring serum or serum replacement. The use of serum replacement may be beneficial to avoid xeno-contamination.

[0120] According to a specific embodiment, the serum is fetal bovine serum (FBS), e.g., 5-10 %. Other sera and sera replacements include but are not limited to platelet lysate (PL) and plasma. Animal-derived alternatives encompass bovine ocular fluid, fish serum, and serum from bovine adults. Plant-derived alternatives feature sericin protein and plant-based protein hydrolysates. Chemically defined alternatives consist of serum-free media with definedcomponents, recombinant growth factors, and synthetic supplements such as lipids, vitamins, and trace elements.

[0121] When cells reach confluence (P0) e.g., 80-100 %, they are harvested such as by the use of a protease, e.g., trypsin and seeded to the next passage (Pl) or subjected to conditions which allow the formation of organoids (ORGs) or spheroids.

[0122] According to a specific embodiment, expanding is until P0.

[0123] According to a specific embodiment, expanding is until no later than Pl.

[0124] According to a specific embodiment, expanding is until no later than P2.

[0125] According to a specific embodiment, expanding is until no later than P3.

[0126] According to a specific embodiment, expanding is until no later than P4.

[0127] According to a specific embodiment, culturing comprises passaging for at least one passage. According to a specific embodiment, at least one passage is P0. According to some embodiments, feline cells are of passage P0.

[0128] According to a specific embodiment, at least one passage is Pl. According to some embodiments, canine cells are of passage Pl, P2 or P3.

[0129] According to a specific embodiment, at least one passage is P2.

[0130] According to a specific embodiment, the at least one passage is Pl for intended production of organoids.

[0131] According to a specific embodiment, the at least one passage is P2 for intended production of spheroids.

[0132] According to a specific embodiment, at least one passage is P3.

[0133] Thus, the present teachings contemplate a cell culture comprising renal epithelial cells obtainable according to the methods described herein.

[0134] These cells can be used in research and development of clinical and research products or subjected to further steps of culturing for the generation of nephro spheroids or organoids.

[0135] Thus, according to an aspect of the invention there is provided a method of producing a nephrospheroid. The method comprises culturing the renal epithelial cells obtainable as described herein under low-adherence conditions, thereby generating the nephrospheroid.

[0136] As used herein “nephrospheroid” or “spheroid” refers to an aggregate of kidney epithelial cells (such as urine derived). According to some embodiments, the aggregate is about 50-200 pm in diameter (as opposed to an organoid where the size may be 30-800 pm in diameter).

[0137] To form spheres, the kidney epithelial cells such as expanded as described herein are cultured under non-adherence or low-adherence conditions.The phrase “non-adherent conditions” or “low adherent conditions” refers to conditions in which the cells do not attach to the surface of a container in which they are cultured such that a substantial portion of the cells can be removed from the surface of the container by mechanical manipulations that do not cause significant damage to the cells.

[0138] Contemplated culture media include, but are not limited to IMDM (Invitrogen) or DMEM (Invitrogen).

[0139] According to another embodiment, the culture medium, when generating the nephrospheres, is devoid of serum.

[0140] According to a specific embodiment, the dish is coated with polyHEMA. Other reagents can be used to achieve such conditions as the use of commercial low-attachment flasks.

[0141] The nephro spheroid is not hollow and does not demonstrate a cystic structure ex vivo, rather it is a cell aggregate which at this stage is non-proliferative.

[0142] The nephro spheroid can be used in therapy as is.

[0143] The nephro spheroid can be used in therapy following dissociation.

[0144] The nephro spheroid can be used as a starting material (or a preceding step) in organoid formation.

[0145] Thus, according to a specific embodiment, the formation of spheroids is under low adherence conditions, followed by subjecting the spheroids to conditions which allow formation of ORGs in the presence of extracellular matrix (ECM) that encapsulates said spheroids.

[0146] According to some embodiments of the invention, the medium used for nephrospheroids formation comprise SFM: DMEM / F-12 [HAM] 1:1, 1% L-glutamine solution, 1% Penicillin-Streptomycin Solution (*10,000 units / ml), 1-10% Non-essential Amino acids (NEAA), 1-3% Sodium Pyruvate, 0.02% Chemically defined (CD), Lipid mix, 2% B27 supplement (X50), Heparin sodium Fresenius 5000IU / lml, Animal Free Human EGF (10-50 ng / ml), Human FGF-basic (10-50 ng / ml), 0.06-0.1 mg / ml Glucose, 0.01-1 mg / ml Transferrin, 2.5-10 ug / ml Insulin, 0.1 ug / ml Putrescine, 3 nM Sodium Selenite, 2 nM Progesterone).

[0147] According to some embodiments of the invention, formation of the nephro spheroid is for about 18-36 hours (e.g., 24 hours). However, it can be maintained in culture for 5-12 days.

[0148] According to some embodiments of the invention, the nephro spheroid is capable of forming a tubular nephric tissue upon transplantation.

[0149] According to some embodiments of the invention, the nephro spheroid exhibits proximal markers in vitro (e.g., LRP2 as shown in Figures 1A-D).

[0150] According to some embodiments of the invention, the nephro spheroid is Pan-Cytokeratin (e.g., CK7, CK18, CK19, CK20)+ / EpCAM+ / LRP2+ at the protein level.According to some embodiments of the invention, the pet is a cat, the passaging is until Pl and the biological sample is bladder urine.

[0151] According to some embodiments of the invention, the pet is a dog, the passaging is until Pl, and wherein the biological sample is a kidney.

[0152] According to an aspect of the invention, there is provided a nephro spheroid which can be obtained according to the method as described herein.

[0153] According to some embodiments, the nephrospheroids is endowed with anti-fibrotic activity.

[0154] This is of particular importance in kidney diseases which are characterized by fibrosis contributing to onset or progression of the disease.

[0155] Diseases relevant to pets that can lead to fibrosis in chronic kidney disease include diabetic nephropathy, which is common in diabetic cats and dogs, and hypertensive nephropathy, which can occur in older cats and dogs with high blood pressure. Focal segmental glomerulosclerosis (FSGS) is seen in dogs, especially in certain breeds, while chronic glomerulonephritis is a common cause of CKD in both dogs and cats. Chronic interstitial nephritis is frequently seen in cats, particularly older ones.

[0156] According to an embodiment, the nephro spheroid comprises epithelial cells of a pet, is capable of forming a tubular nephric tissue upon transplantation and / or has an anti-fibrotic activity.

[0157] The kidney epithelial cells or the nephrospheroids can be subjected to conditions which induce formation of kidney organoids (also referred to herein simply as “organoids”).

[0158] As used herein “kidney organoid” refers to a 3-dimensional structure of kidney cells, having a cystic (hollow) interior. The structure exhibits polarity with the same orientation as in the nephron tubule. Cells within the organoid are capable of proliferation. It can be maintained in culture (ex-vivo) for at least 10, 15, 20, 25 passages. The organoid comprises epithelial cells from various kidney segments (proximal, distal, loop). The organoid responds ex vivo to drugs which reduce cyst expansion and preserve kidney function. The term organoid, does not include a whole kidney or a fragment thereof, or a nephrosphere, which is an amorphous aggregate structure of nonproliferative nature in culture (described above).

[0159] Thus, according to an aspect of the invention, there is provided a method of producing a kidney organoid, the method comprising:

[0160] (a) culturing the renal epithelial cells obtainable according to the method as described herein under conditions which allow formation of kidney organoids (ORGs); and

[0161] (b) passaging said ORGs.Thus, the passaged cells of the 2D culture are encapsulated in extracellular matrix (ECM) such as 2000-8000 cells / pl, e.g., 4000 cells / pl. The cells are then seeded in domes (e.g., 15 pl-100 pl, e.g., 50 pl) which are allowed to solidify.

[0162] Examples of ECM which can be used include basement membrane extract (BME), VitroGel ORGANOID, Self-Assembling Peptide Hydrogels (PeptiGel Technologies), Gelatin Methacrylate (GelMA) Hydrogels (Advanced BioMatrix), Collagen Type I (Coming), and Sodium Alginate Hydrogel (Sigma- Aldrich). The vendors are just exemplary and should not be considered binding.

[0163] According to specific embodiments, conditions which allow formation of kidney organoids (such as urine cells derived organoids and referred to herein as “uORGs”) comprise at least one of N-acetylcysteine (NAC) or an analog thereof, ROCK inhibitor, epidermal growth factor (EGF), fibroblast growth factor 10 (FGF-10), B27, and a Wnt activator, e.g., R-spondin or R-spondin conditioned medium (others are listed below).

[0164] According to a specific embodiment, the ROCK inhibitor is Y-27632, though others can be used such as: fasudil, H-1152 and HA-1077 (a Fasudil analog).

[0165] Table 1 below provides some embodiments of such media. The functional annotation indicates that other ingredients can be used to mediate the designated function. Some substitutes are mentioned, e.g., in the case of ROCK inhibitor, each is category or ingredient is considered an individual embodiment.

[0166] Table 1

[0167]

[0168]

[0169] Culturing at this stage and passaging thereafter is being done in the absence of serum. Then the protocol proceeds to the next stage of ORGs formation as described above (encapsulation in BME).

[0170] Regardless of the protocol, whether it is the one step protocol (without an intermediate step of spheroids formation) or the two-step protocol (with an intermediate step of nephrospheroids formation), once ORGs are obtained they are passaged.

[0171] According to a specific embodiment, passaging is effected by mechanical disruption of the ORGs. The matrix is first depleted. For example, BME is dissolved in 4 °C (on ice) and then subjected to mechanical fractionation and split 2-20 (e.g., 2-10, 2-8). The passaging rate depends on the proliferation rate.

[0172] According to an embodiment of the invention, passaging is for at least 10, 20, 30, 50 or more passages e.g., up to 100 passages, e.g., 10-40 passages, 10-30 passages, 15-30 passages, 20-30 passages.At any step of the protocol the cells can be analyzed for any specific parameter, such as morphology, functionality, proliferation rate and / or marker expression, using methods which are well known in the art and rely (not exclusively) on molecular biology, single cell RNA sequencing, immunology, protein expression and cell biology (e.g., microscopy).

[0173] Thus, according to a specific embodiment, the method comprises isolating cells from the 2D culture of epithelial cells, nephrospheroids, or ORGs and / or analyzing said expanded 2D culture of epithelial cells, nephrospheroids, or ORGs.

[0174] For example, EpCs and / or ORGs may be analyzed for markers of renal epithelial cells (e.g., tubular), morphology of renal cells (epithelial cells do not have a spindle shape for instance), confluence (e.g., 80-100 %); the ORG may be analyzed for markers such as described herein, as well as cyst shape, cyst size, effect of drugs or mutation analysis.

[0175] In pets, particularly dogs and cats, several types of genetic mutations have been linked to chronic kidney disease (CKD). One of the most prevalent is the PKD1 mutation, which is an autosomal dominant mutation in the PKD1 gene. This mutation is common in Persian cats and also seen in some dog breeds like the Cairn Terrier. It leads to the formation of cysts in the kidneys, which can cause progressive kidney damage. Another common mutation is related to focal segmental glomerulosclerosis (FSGS), with mutations in genes like the NPHS1 gene, which encodes for nephrin. This mutation is particularly prevalent in breeds such as the English Cocker Spaniel and Samoyed, and it leads to glomerular damage, scarring, and eventual kidney failure. These are just exemplary non-limiting embodiments since any type of mutation can be analyzed.

[0176] According to a specific embodiment, the pet is a dog, the renal epithelial cells are P0, and said organoid is ECAD+ and AQP1+ as determined at the protein level (e.g., in immunofluorescence staining).

[0177] The present teachings also contemplate the ORGs, secretome or fraction thereof derived from the methods described herein.

[0178] Also contemplated herein is the secretome or fraction thereof of nephrospheroids.

[0179] Thus, according to an aspect of the invention, there is provided an isolated kidney organoid (ORG) ex vivo produced from kidney epithelial cells of a pet, wherein the organoid is proliferative and comprises a polarized tubule structure prior to transplantation (see Figures 2A-D). It will be appreciated that a spheroid generated as described in WO2022 / 084998 is proliferative only following transplantation Cm vivo).

[0180] According to an additional or an alternative aspect there is provided an isolated kidney organoid (ORG) obtainable according to the method as described herein.ORGs described herein are endowed with a unique gene expression. Methods of determining gene expression are well known in the art. They can be determined at the level of RNA or protein. Global gene expression can be determined and alternatively or additionally they can be determined at the single cell level. Cell cluster data derived from single-cell RNA sequencing refers to the grouping of individual cells based on their gene expression profiles. These clusters represent distinct cell populations with similar molecular characteristics.

[0181] According to a specific embodiment, the pet is a dog, the renal epithelial cells are P0, and the organoid is ECAD+ and AQP1+ as determined at the protein level.

[0182] Strikingly, the present inventors identified that ORGs generated according to the present teachings are featured by all the tubular segments.

[0183] Thus according to an aspect of the invention there is provided a composition of matter comprising multiple ORGs from a single subject, said multiple ORGs comprising all tubular segments including a distal tubule, a proximal tubule, a collecting duct and a loop of Henle yet being devoid of glomerulus.

[0184] Such compositions can be collected and put in a bank in which is composition is indentified or indexed according to the donor.

[0185] Thus, according to an aspect there is provided a bank comprising multiple compositions (each being from a single subject) from multiple subjects.

[0186] As mentioned, also contemplated herein is the secretome of the ORGs described herein. As used herein “a secretome” refers to the set of secreted metabolites, proteins expressed by the ORGs and secreted into the environment, cell-derived particles such as exosomes, nucleic acid molecules (e.g., RNA), carbohydrates, lipids, small molecules which are secreted to the medium in which the ORGs are cultured. It can also be referred to as an ORG conditioned medium. It may include, for example, cytokines, growth factors, extracellular matrix proteins and regulators, and shed receptors. The secretome can be measured by mass spectrometry as well as other analytic methods.

[0187] Also contemplated is a method of producing a secretome, the method comprising:

[0188] (a) providing a culture of the ORGs as described herein; and

[0189] (b) collecting conditioned medium of the culture, the conditioned medium comprising the secretome.

[0190] According to a specific embodiment, the method further comprises isolating the secretome or a portion thereof from the conditioned medium following step (b).

[0191] Isolation of the secretome or fractions thereof typically comprises filtering, centrifugation, affinity chromatography, gel filtration chromatography, liquid chromatography or precipitationand for example protein isolation methods such as SDS-PAGE, Western blotting, ELISA, or mass spectrometry, the skilled artisan would know which methods to apply.

[0192] According to some embodiments, following expansion of organoids, secretome is collected every 36-72 hours (e.g., 48 hours), allowing for repeatable collection.

[0193] According to some embodiments, once spheroids are available, secretome is collected following 6-10 days in culture.

[0194] The ability of the organoids to form renal structures ex vivo and their response to renal drugs suggests that they can function in treatment of renal diseases.

[0195] Thus, according to an aspect of the invention there is provided a method of regenerating renal function, the method comprising administering to a subject in need thereof a therapeutically effective amount of the ORGs or nephrospheroids or secretome thereof, thereby regenerating renal function.

[0196] Thus, ORGs of the present invention can be used to treat any form of acute or chronic kidney disease. Examples include, but are not limited to, a glomerular disease (e.g., primary, secondary, genetic, acquired, immune-mediated among other), a tubulo-interstitial disease (e.g., primary, secondary, genetic, acquired, immune-mediated among other), combined glomerular and tubulo-interstitial disease, obstructive uropathy, nephrotoxicity (e.g., drug-mediated, toxin-mediated), acute kidney injury, acute tubular necrosis, cystic kidney disease, congenital anomalies of the kidney or urinary tract, acute kidney injury or acute tubular necrosis superimposed on chronic kidney disease.

[0197] The present teachings contemplate administration of the intact ORGs or spheroids though, partly dissociated structures or completely dissociated can also be used. Each of these should be considered as a separate independent embodiment of the invention.

[0198] The ORGs or spheroids in any form or secretome may be administered per se or as part of a pharmaceutical composition where they are mixed with a suitable carrier or excipient.

[0199] As used herein a "pharmaceutical composition" refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

[0200] Herein the term "active ingredient" refers to the cells in any form or secretome accountable for the biological effect.

[0201] Hereinafter, the phrases "physiologically acceptable carrier" and "pharmaceutically acceptable carrier" which may be interchangeably used refer to a carrier or a diluent that does notcause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under these phrases.

[0202] Herein the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

[0203] The cells in any form or secretome can be administered into a subject such as surgically or by infusion. For example, by ultrasound-guided transplantation of cells into capsule-cortex space or by intravenous (i.v.) infusion (such as in the case of secretome administration).

[0204] As mentioned, the ORGs or spheroids or secretome can be autologous or allogeneic. Allogeneic secretome may be of specific significance in the case of secretome (which is cell-free).

[0205] The regimen for transplantation is designed by the skilled artisan. Such a regimen is provided in the Examples section.

[0206] The ORGs in any form or secretome can be used to construct artificial kidney systems. Such a system can be based on a hollow fiber filtration system.

[0207] In one example of a filtration device, the ORGs or cells derived therefrom are grown on the interior of hollow fibers having relatively high hydraulic conductivity (i.e., ultrafiltration coefficient). The hollow fiber passes through a chamber that is provided with a filtrate outlet port. Arterial blood containing metabolic waste and other unwanted material is introduced into one end of the hollow fiber through an inlet port. Blood passed through the fiber and exits the other end of the fiber through an outlet port where it passed into the patient's vascular venous flow. As blood passes through the fiber, filtrate pass through the ORGs or cells derived therefrom lining the interior of the fiber and through the hollow fiber itself. This filtrate then passes out of the chamber containing the fiber through the filtrate outlet port. The device preferably includes many such hollow fibers each of which can be in its own chamber. Alternatively many, many hollow fibers (100-100,000 or even more) can be bundled together in a single chamber.

[0208] The ORGs or cells derived therefrom of the invention can be used to create a tubuleprocessing device. In such a device the ORGs or cells derived therefrom can be grown in a layer on the exterior of the semipermeable hollow fiber (i.e. a scaffold). The fiber is placed in a chamber that is provided with an inlet port and an outlet port. As ultrafiltrate from filtered blood flows through the chamber, reabsorbant passes through the cell layer and through the wall of the fiber into the lumen of the fiber from which it can be directed back into the patient's systemic circulation. Ultrafiltrate that is not reabsorbed passes through the outlet port of the chamber.In the devices described above, it can be desirable to coat the fiber surface that will bear the cell layer with extracellular matrix components. For example, the fiber can be coated with materials such as collagen (e.g., Type I collagen or Type IV collagen), proteoglycan, fibronectin, and laminin or combinations thereof. It can be desirable to combine various cell types on the inner or outer surface of the fibers. For example, it can be desirable to include endothelial cells and pericyte, vascular smooth muscle cells or mesangial cells or fibroblasts or combinations thereof. It can also be useful to provide a feeder layer of cells, e.g., irradiated fibroblasts or other cells that can provide soluble factors and structural support to cells they are indirectly or directly in contact with.

[0209] The above-described filtration system and the above-described tubule processing system can be combined to create an artificial kidney. Such systems are described in U.S. Pat. No. 6,150,164, hereby incorporated by reference. A number of suitable materials for forming the hollow fiber are described in U.S. Pat. No. 6,150,164, hereby incorporated by reference.

[0210] The present invention provides a method of using the cell populations of the present invention to characterize cellular responses to biologic or pharmacologic agents involving producing the cells as described herein and culturing with one or more biologic or pharmacologic agents, identifying one or more cellular responses to the one or more biologic or pharmacologic agents, and comparing the one or more cellular responses of the cultures. Tissue culture techniques known to those of skill in the art allow mass culture of hundreds of thousands of cell samples from different individuals, providing an opportunity to perform rapid screening of compounds suspected to be, for example, teratogenic or mutagenic.

[0211] Also provided is a method of drug design comprising determining an effect of a test drug on the ORG, nephro spheroid or secretome thereof.

[0212] The effect can be any selected by the skilled artisan, such as cyst size or cyst shape.

[0213] These teachings can be further harnessed towards tailored therapy against a disease.

[0214] Thus, according to an aspect of the invention, there is provided a method of selecting treatment for a subject diagnosed with a kidney disease, the method comprising:

[0215] (a) contacting the ORG, EpCs, nephrospheroids of a pet diagnosed with the kidney disease; and

[0216] (b) determining an alleviation in a renal symptom evident in the ORG, EpCs, nephrospheroids following the contacting, the alleviation being indicative of an efficacious therapy.

[0217] Several drugs are commonly used to manage chronic kidney disease (CKD) in pets, particularly dogs and cats, to slow the progression of the disease, manage symptoms, and improve quality of life. Some of the key drugs include:Angiotensin-converting enzyme (ACE) inhibitors: Drugs like enalapril or benazepril help reduce blood pressure and decrease the workload on the kidneys by dilating blood vessels, which can slow the progression of CKD.

[0218] Angiotensin II receptor blockers (ARBs): Telmisartan is used in cases where ACE inhibitors are not tolerated. It helps lower blood pressure and reduces proteinuria (protein in the urine), which is common in CKD.

[0219] Phosphate binders: Medications like aluminum hydroxide or calcium carbonate help control phosphate levels in the blood, which can become elevated in pets with CKD. High phosphate levels can worsen kidney damage.

[0220] Potassium supplements: In cases where potassium levels are low, potassium gluconate or similar supplements are given to maintain electrolyte balance.

[0221] Diuretics: Furosemide is often used to reduce fluid buildup (edema) that can occur in pets with kidney failure, particularly when there is fluid retention or pulmonary edema.

[0222] Anti-nausea and appetite stimulants: Medications like maropitant (Cerenia) and mirtazapine are commonly used to control nausea and stimulate appetite, both of which are common issues in pets with CKD.

[0223] Erythropoiesis-stimulating agents (ESA): Epoetin alfa or darbepoetin alfa may be prescribed to treat anemia, a common complication of CKD, by stimulating the production of red blood cells.

[0224] The specific treatment regimen will depend on the pet's condition, the stage of CKD, and any other underlying health issues. Regular veterinary monitoring is crucial to adjust treatments as needed.

[0225] An exemplary effect can be evident in a cyst size of the organoids.

[0226] As used herein the term “about” refers to ± 10 %.

[0227] The terms "comprises", "comprising", "includes", "including", “having” and their conjugates mean "including but not limited to".

[0228] The term “consisting of’ means “including and limited to”.

[0229] The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and / or parts, but only if the additional ingredients, steps and / or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

[0230] As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0231] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging / ranges between” a first indicate number and a second indicate number and “ranging / ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

[0232] As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

[0233] As used herein, the term “treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.

[0234] When reference is made to particular sequence listings, such reference is to be understood to also encompass sequences that substantially correspond to its complementary sequence as including minor sequence variations, resulting from, e.g., sequencing errors, cloning errors, or other alterations resulting in base substitution, base deletion or base addition, provided that the frequency of such variations is less than 1 in 50 nucleotides, alternatively, less than 1 in 100 nucleotides, alternatively, less than 1 in 200 nucleotides, alternatively, less than 1 in 500 nucleotides, alternatively, less than 1 in 1000 nucleotides, alternatively, less than 1 in 5,000 nucleotides, alternatively, less than 1 in 10,000 nucleotides.

[0235] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of asingle embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0236] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

[0237] EXAMPLES

[0238] Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion.

[0239] Materials and Methods

[0240] Harvesting cells from kidney

[0241] Dog, cat, and rat kidney samples were obtained from animals following euthanasia. Tissues were rinsed in phosphate-buffered saline (PBS), weighed, and minced into ~1 mm fragments using sterile surgical scalpels. Minced tissue was incubated for 2 h at 37 °C in Dulbecco’s Modified Eagle Medium (DMEM; Gibco) supplemented with 0.1% collagenase IV (Invitrogen). Following enzymatic digestion, the suspension was passed through 100-pm cell strainers to obtain a single-cell suspension. Cells were pelleted by centrifugation, resuspended in growth medium, and seeded into T175 culture flasks.

[0242] Establishment of primary culture from Urine samples - uEpC (urine derive epithelial cells)

[0243] Urine samples were centrifuged at 400xg, washed with PBS and centrifuged again at 200 x g. Then the cell pellet was seeded on two gelatin-coated wells in 24 well plate (0.1%, Biological Industries, 01-944-1B), with RE:MC growth medium. RE:MC: (renal growth medium: Mesenchymal Cell proliferation at a 1:1 ratio) (e.g., REGM Renal Growth BulletKit: REB M™ Basal Medium with Additives: hEGF, Insulin, Hydrocortisone, FBS (10 %), Transferrin, Triiodothyronine, Epinephrine Lonza, Cat No. CC-3190) and mesenchymal cell media, MC (Dulbecco’s Modified Eagle Medium (DMEM) High Glucose, 1 % L-glutamine solution, 1 % Penicillin-Streptomycin Solution, Human PDGF-AB-10 (10-25ng / ml), Animal Free Human EGF (10-50 ng / ml), Human FGF-basic (10-50 ng / ml) and 5-10 % FBS] supplemented with CD40L (2-10 ng / ml).uSPH / kSPH formation and dissociation

[0244] Upon reaching 80-100 % confluence, cells grown (refers to both urine and kidney, dependent in the example) as a monolayer were harvested and seeded on poly (2-hydroxyethylmethacrylate) (poly-HEMA; Sigma- Aldrich) pre-coated plates, in serum free media SFM: DMEM / F-12 [HAM] 1:1, 1% E-glutamine solution, 1% Penicillin-Streptomycin Solution (*10,000 units / ml), 1-10% Non-essential Amino acids (NEAA), 1-3% Sodium Pyruvate, 0.02% Chemically defined (CD), Eipid mix, 2 % B27 supplement (X50), Heparin sodium Fresenius 5000 lU / lml, Animal Free Human EGF (10-50 ng / ml), Human FGF-basic (10-50 ng / ml), 0.06-0.1 mg / ml Glucose, 0.01-1 mg / ml Transferrin, 2.5-10 ug / ml Insulin, 0.1 ug / ml Putrescine, 3 nM Sodium Selenite, 2 nM Progesterone), at a concentration of 5.5 - 13 x 104cells / mE. In order to dissociate the spheroids (SPH) into single cells, SPH were collected and incubated with TrypEE (GIBCO) For 10 min. SPH-derived cells were then counted for further tests.

[0245] Urine-derived organoids formation

[0246] 2D uEpC (cat or human) or dog urine derived epithelial cells (duEpC) were trypsinized and seeded with Cultrex BME (basement membrane extract, R&D Systems) at 4000 cells / pl, and plated on 24-well culture plates as 50-pl domes (concentration of 200,000 cells / 50ul BME). After incubation at 37 °C to allow the BME to solidify uORG- serum-free medium was added: The medium included R-spondin conditioned medium which was used at 10 % (v / v) concentration.) or recombinant protein Human R-Spondinl, (120-38-100ug; 250ng / ml) 1%NAC, lOng / ml FGF-10, 50ng / ml EGF, 5ug A83-O1, 1.5% B27 (X50), 1 % L-glutamine solution, 1 % Penicillin-Streptomycin Solution, XI NEAA, 10 uM Y-27632. uORG were mechanically disrupted with a bovine serum albumin (BSA)-treated 1,000-pl pipette tip and passaged at a 1:2 ratio every 2-3 weeks, depending on the proliferation rate

[0247] Direct Injection of duORG into a dog kidney

[0248] duORG were dissociated just prior to their use and by using an ultrasound guided approach 0.5X106 / Kilogram cells were implanted beneath the kidney capsule. One dog went through one injection and the second dog went through two injections, with a 250 days gap.

[0249] IV Injection of duORG derived secretome

[0250] KOSEC (kidney organo spheroid secretome) was collected from four wells of duORG culture (~2xl06cells), then the secretome was added into infusion bag containing saline. The dogs (mixed breed, Japanese spitz, pitbull, as in the above) got the infusion for an hour, and stayed another hour for observation. This procedure was done twice a week.

[0251] EpC from kidney tissue was obtained as previously described (arari-Steinberg O, Omer D, Gnatek Y, Pleniceanu O, Goldberg S, Cohen-Zontag O, Pri-Chen S, Kanter I, Ben Haim N, BeckerE, Ankawa R, Fuchs Y, Kalisky T, Dotan Z, Dekel B. Ex Vivo Expanded 3D Human Kidney Spheres Engraft Long Term and Repair Chronic Renal Injury in Mice. Cell Rep. 2020 Jan 21;30(3):852-869.e4. doi: 10.1016 / j.celrep.2019.12.047. PMID: 31968258.). The generation of SPH and secretome production followed the same procedure as for cells obtained from urine.

[0252] Fibrosis Induction:

[0253] Fibroblasts (NHDF-Ad - Human Dermal Fibroblasts) were seeded on 12 well plates with growth medium: DMEM, 10 % FBS, 1 %P / S, 1 % glutamine. After 72 hours the cells went through starvation using starvation medium for 24 hours: DMEM, 0.4 % FBS, 1 % P / S, 1 % glutamine. Then, fibrosis was induced by adding 50 ng / ml TGF-b recombinant protein for 48 hours. Subsequently, the cells were ready for secretome treatment. After treatment (48, 72 or 96 hours) the cells were collected for gene expression.

[0254] Agarose block preparation

[0255] SPH-derived cells were washed with PBS, fixed with 4 % PFA for 2 h, washed again with PBS and suspended in 200 pl of 2 % UltraPure low melting point agarose (Invitrogen) (in 5% sucrose). Agarose with cells was then molded (the wide edge of 1 ml tip was cut and used as a mold), fixed with 4 % PFA overnight and paraffin embedded.

[0256] Immunofluorescence staining (IF)

[0257] 5uM Paraffin sections were pre-treated with OmniPrep solution (pH 9.0) in 95°C for one hour in accordance with the manufacturer's protocol (Zytomed Systems). Blocking was done using Cas-Block solution (Invitrogen immunodetection, 00-8120) for one hour followed by one hour incubation with two of the following primary antibodies: Anti-Cytokeratin (WSS) (Rb; 1 / 200; DAKO, Z0622), anti-Aqpl (Rb; 1 / 1000; abeam, 168387), anti-LRP2, anti-ECAD, anti-EpCAM. Detection was done using alexa488 conjugated anti-rabbit and alexa555-conjugated anti-mouse secondary antibodies (Invitrogen) for 60 min. Mounting medium containing DAPI (Dapi Fluoromount- G; SouthernBiotech, 0100-20) was applied. Slides were analyzed using Olympus BX51 fluorescent microscope and Olympus DP72 camera, or confocal microscope ZEISS LSM700. Photo analysis was done using ZEN software.

[0258] EpC from kidney tissue was obtained as previously described (Arari- Steinberg et al. Ex Vivo Expanded 3D Human Kidney Spheres Engraft Long Term and Repair Chronic Renal Injury in Mice. Cell Rep. 2020 Jan 21;30(3):852-869.e4. doi: 10.1016 / j.celrep.2019.12.047. PMID: 31968258.). The generation of SPH and secretome production followed the same procedure as for cells obtained from urine.

[0259] In vivo evaluation of anti fibrotoc activity of rat kidney cells derived organoids: For in vivo evaluation, a rat chronic kidney disease (CKD) model was used, established usingischemia-reperfusion injury combined with unilateral nephrectomy (IRI + UNX), a well-validated model of progressive CKD characterized by sustained renal dysfunction, fibrosis, and long-term disease progression. Rats were treated twice weekly with either low-dose (2x) or high-dose (4x) rat-KOSEC and compared to vehicle-treated and sham groups (UNX only, without IRI).

[0260] EXAMPLE 1

[0261] Demonstrating Compassionate Treatment in Canine CKD through a Large Animal Model Study

[0262] Establishing primary cultures of urine-derived kidney cells

[0263] Urine was collected directly from the cat’ s bladder, and cells were isolated by centrifugation and washing (detailed in Materials and Methods). The cells were then seeded on gelatin-coated plates to allow proliferation and expansion under 2D adherent conditions using serum-containing media (RE:MC, detailed in Materials and Methods) for two passages (Figure 1 A). Once confluent, the cells were harvested and transferred to poly-HEMA-coated flasks to grow as spheroids under serum-free conditions (SFM) (Figure IB). The cat urine-derived spheroids (cat-uSpH) were positively stained for kidney epithelial markers, including Pan-cytokeratin, LRP2, and EpCAM (Figure 1C).

[0264] The same procedure was applied using dog urine. Following the same protocol, urine samples were collected directly from the dog’s bladder, and the isolated cells were seeded and expanded under 2D conditions, similar to cat urine-derived kidney cells. The cells were then transitioned to 3D culture conditions to form uSPH (Figure ID).

[0265] To form organoids, to culture the cells as organoids, the cells were taken to 3D immediately after the first 2D passage (P0). Once the cells reached confluence, they were transferred to a 3D culture to allow organoid generation and establish 3D dog kidney cultures (Figure 2A- scheme). The present inventors succeeded in robustly growing dog urine-derived kidney organoids (duORG) in BME (Figure 2B). duORG showed tubuloid morphology similar to human urine-derived organoids counterparts and positively stained for proximal and distal markers (AQP1 and E-CAD, respectively) (Figure 2B).

[0266] Example 2

[0267] Autologous transplantation of dog urine-derived kidney organoids into dogs harboring CKD

[0268] The present inventors then used duORG for autologous implantation in dogs characterized by CKD. Accordingly, duORG were dissociated just prior to their use and by using an ultrasound guided approach 0.5X106 / Kilogram cells were implanted beneath the kidney capsule (Figure 2C). Two dogs were monitored for over a year. Importantly, their survival far exceeded historicalcontrols harboring similar CKD stages (Figure 2D). Moreover, kidney function parameters were mostly stabilized over this period (Figure 2D), indicating potential of engrafted duORG to deliver beneficial signals to chronically injured dog kidneys.

[0269] Example 3

[0270] IV injection of secretome generated from dog urine-derived kidney organoids into dogs harboring CKD

[0271] To further investigate the effect of duORG-secreted factors generally termed secretome, the present inventors administered the secretome intravenously (i.v.) to dogs with advanced CKD (Figure 3A). Four dogs were treated; two with progressive CKD and two with an acute kidney injury superimposed on CKD. The first dog received six infusions of secretome over a period of 28 days (Figure 3B). The second dog received 10 kidney organospheroids secretome (KOSEC) infusions over a period of 72 days (Figure 3C). The third dog for 28 days, with twice a week infusion (Figure 3D) and the fourth dog for 100 days (Figure 3E), with the same protocol (twice a week). Importantly, all secretome infusions were well tolerated with no apparent SAEs, and all dogs were rapidly discharged following infusion. Secretome treatment can potentially induce an immune response, nevertheless, analysis of C-Reactive Protein showed it (CRP) mostly decreased even with multiple infusions or alternatively elevate following a bacterial infection and not related to secretome administration. This was also the case when donor secretome was prepared from a different dog and infused across strains. Concomitantly, it was found kidney function (with some evidence for Urea reduction) stabilized even in the acute on chronic kidney presentation and increased survival compared to expected historical outcomes in these specific clinical scenarios (Figure 3B -3E).

[0272] Example 4

[0273] Kidney tissue SPH-derived secretome

[0274] Unlike in humans, collecting urine from a dog or cat is an invasive procedure. Therefore, kidney epithelial cells cultures from kidney tissue were also investigated. Dog kidney samples were obtained from CKD dogs that had undergone euthanasia. The collected tissues were washed with PBS, weighed, and minced into ~1 mm slices using sterile surgical scalpels. The dissected tissue was then incubated for two hours at 37 °C in Iscove’s Modified Dulbecco’s Medium (IMDM) supplemented with 0.1 % collagenase IV. Following digestion, the tissue was filtered through 100 pm cell strainers to obtain a single-cell suspension. The medium was removed by centrifugation, and the cells were resuspended in RE:MC medium and plated on T175 flasks. This process was highly efficient, as the cells successfully expanded for two passages in 2D conditionsand subsequently formed 3D spheroids. After 8-10 days in 3D culture, the secretome was collected. A similar protein expression pattern was evidenced between samples derived from urine and those from tissue (Figure 4A). Therefore, it was suggested that secretome generated from dog (or cat) tissue-derived SPH could serve as a potential treatment for CKD in dogs or cats. To test this, the present inventors collected secretome from dog kidney-SPH and evaluated its effect in a fibrosis potency assay. Fibroblasts were activated using TGF-P and treated with the secretome. The results demonstrated that the secretome successfully downregulated fibrosis-related markers, including COL1A1, COL1A2, COL3A1, and PERIOSTIN, similar to secretome derived from human urine-SPH (KOSEC001) and adult human kidney-SPH (AK-KOSEC) (Figure 4B).

[0275] Example 5

[0276] Kidney tissue derived 2D cultures, spheroids and organoids from large animals Establishing primary cultures of feline tissue-derived kidney cells

[0277] Kidney tissue from euthanized cats was minced and digested with collagenase type IV to obtain a single-cell suspension. Cells were either seeded directly in a matrix to generate kidney organoids or cultured under 2D conditions to establish adherent kidney epithelial cell cultures. Upon reaching confluency, 2D cultures were transferred to 3D conditions, either embedded in matrix to form organoids or seeded under non-adherent conditions to generate spheroids (Figure 5A). The resulting organoids exhibited tubular morphology (Figure 5B). Immunostaining demonstrated positive expression of kidney epithelial markers, including E-cadherin and EpCAM (Figures 5C-D). The results shown are for organoids generated directly from tissue (without a 2D expansion step).

[0278] Establishing primary cultures of canine tissue-derived kidney cells

[0279] The same procedure was used to generate kidney organoids from canine kidney tissue (Figure 6A). Canine kidney organoids displayed well-defined tubular structures (Figure 6B) and stained positively for kidney epithelial markers, including E-cadherin, EpCAM, and EMA (Figures 6C-D). Marker localization indicated epithelial polarity, with basolateral staining patterns observed. Of note, Figure 6C upper panel - organoids generated from a 2D culture, while the lower panel shows organoids generated directly from tissue.

[0280] Establishing primary cultures of rat tissue-derived kidney cells

[0281] Rat kidney spheroids were generated using the same workflow: tissue digestion, expansion under 2D conditions, and subsequent transfer to 3D non-adherent culture (Figure 7A-B). After 7 days in 3D culture, conditioned medium (kidney organosphere secretome; KOSEC) was collected for evaluation of therapeutic potential in vitro and in vivo.Example 6

[0282] Kidney derived organoids are endowed with anti-fibrotic activity

[0283] To assess anti-fibrotic activity in vitro, activated fibroblasts were treated for 48 hours with rat-KOSEC, followed by analysis of fibrosis-associated gene expression. Treatment with rat-KOSEC resulted in downregulation of fibrotic markers, with effects comparable to the human counterpart (Figure 7C, as in the assay of Figure 4B).

[0284] For in vivo evaluation, a rat chronic kidney disease (CKD) model was used. The model is a well-validated model of progressive CKD characterized by sustained renal dysfunction, fibrosis, and long-term disease progression (Figure 7D). Rats were treated twice weekly with either low-dose (2x) or high-dose (4x) rat-KOSEC and compared to vehicle-treated and sham groups (UNX only, without IRI; Figure 7E).

[0285] Serial assessment of renal parameters demonstrated progressive albuminuria in vehicle-treated rats, consistent with maladaptive remodeling and CKD progression (Figure 7F). In contrast, rat-KOSEC-treated animals showed significantly reduced urinary protein excretion (albumin / creatinine ratio, fractional excretion of albumin, and total albumin) from day 56 onward. A clear dose-dependent effect was observed, with the higher rat-KOSEC dose achieving greater efficacy (Figure 7F).

[0286] Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

[0287] It is the intent of the Applicant(s) that all publications, patents and patent applications referred to in this specification are to be incorporated in their entirety by reference into the specification, as if each individual publication, patent or patent application was specifically and individually noted when referenced that it is to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting. In addition, any priority document(s) of this application is / are hereby incorporated herein by reference in its / their entirety.

Claims

WHAT IS CLAIMED IS:

1. A method of culturing kidney epithelial cells of a pet, the method comprising: (a) isolating kidney epithelial cells from a biological sample of the pet;(b) culturing the kidney epithelial cells under adherent conditions, thereby culturing kidney epithelial cells.

2. The method of claim 1, wherein the pet is selected from the group consisting of a dog, a cat, a rabbit, a guinea pig and a bird.

3. The method of claim 1, wherein the pet is selected from the group consisting of a dog and a cat.

4. The method of any one of claims 1-3, wherein said biological sample comprises urine.

5. The method of any one of claims 1-3, wherein said urine is bladder urine.

6. The method of any one of claims 1-3, wherein said urine is voided urine.

7. The method of any one of claims 1-3, wherein said biological sample comprises a kidney biopsy.

8. The method of any one of claims 1-7, wherein said culturing comprises passaging for at least one passage.

9. The method of claim 8, wherein said at least one passage is PO.

10. The method of claim 8, wherein said at least one passage is Pl.

11. The method of any one of claims 1-10, wherein said culturing is performed in the presence of serum.

12. The method of any one of claims 1-11, wherein said culturing is performed in RE:MC.

13. The method of any one of claims 1-12, wherein said culturing is performed in the presence of CD40 ligand (CD40L) and optionally at least one of epidermal growth factor (EFG), platelet-derived growth factor (PDGF)-AB-IO and fibroblast growth factor (FGF) basic.

14. The method of any one of claims 1-13, wherein said isolating is by centrifugation.

15. The method of any one of claims 1-14, wherein said adherent conditions comprise gelatin coating.

16. The method of any one of claims 4-6 and 8-15, wherein said biological sample is urine and said passaging is performed to enrich for urine derived epithelial cells (UD-EpC) and deplete squamous epithelial cells of vagina and / or bladder origin.

17. The method of any one of claims 1-16, wherein said pet is healthy.

18. The method of any one of claims 1-16, wherein said pet is diagnosed with a kidney disease.

19. A cell culture comprising the renal epithelial cells obtainable according to the method of any one of claims 1-18.

20. A method of producing a nephrospheroid, the method comprising culturing the renal epithelial cells obtainable according to the method of any one of claims 1-18 under low-adherence conditions, thereby generating the nephrospheroid.

21. The method of claim 20, wherein said low adherence conditions comprise polyHEMA.

22. The method of any one of claims 20-21, wherein said nephrospheroid is Pan-Cytokeratin+ / EpCAM+ / LRP2+ at the protein level.

23. The method of any one of claims 20-22, wherein said pet is a cat, said passaging is until Pl and said biological sample is bladder urine.

24. The method of any one of claims 20-22, wherein said pet is a dog, said passaging is until Pl, and wherein said biological sample is a kidney.

25. A nephro spheroid obtainable according to the method of any one of claims 20-24.

26. The nephro spheroid of claim 25 having an anti-fibrotic activity.

27. A nephro spheroid comprising epithelial cells of a pet, the nephro spheroid is capable of forming a tubular nephric tissue upon transplantation and / or having an anti-fibrotic activity.

28. A method of producing a kidney organoid, the method comprising:(a) culturing the renal epithelial cells obtainable according to the method of any one of claims 1-18 under conditions which allow formation of kidney organoids (ORGs); and(b) passaging said ORGs.

29. The method of claim 28, wherein said conditions which allow formation of kidney organoids (uORGs) comprise at least one of N-acetylcysteine (NAC) or an analog thereof, ROCK inhibitor, epidermal growth factor (EGF), fibroblast growth factor 10 (FGF-10), B27 and a Wnt activator.

30. The method of any one of claims 28-29, wherein said culturing said renal epithelial cells is by encapsulating said renal epithelial cells within an extracellular matrix (ECM).

31. The method of claim 30, wherein said ECM comprises basement membrane extract (BME).

32. The method of any one of claims 28-31, said culturing is in the absence of serum.

33. The method of any one of claims 28-32, wherein said passaging is for at least 10 passages.

34. The method of any one of claims 28-33 further comprising isolating cells from said kidney organoid and / or analyzing said expanded organoids or cells thereof.

35. The method of any one of claims 28-34, wherein said pet is a dog, said renal epithelial cells are PO, and said organoid is ECAD+ and AQP1+ as determined at the protein level.

36. An isolated kidney organoid (uORG) ex vivo produced from kidney epithelial cells of a pet and comprising a cystic interior, wherein the organoid is proliferative and a polarized tubule structure prior to transplantation.

37. An isolated kidney organoid (ORG) obtainable according to the method of any one of claims 28-35.

38. The organoid of claim 36 or 37, comprising cells that are ECAD+ and AQP1+ as determined at the protein level.

39. The organoid of any one of claims 36-38, wherein said pet is selected from the group consisting of a dog, a cat, a rabbit, a guinea pig and a bird.

40. The organoid of any one of claims 36-38, wherein said pet is a dog, a cat or a rat.

41. A secretome of the nephro spheroid or organoid of any one of claims 25-27 and 36-40.

42. The nephro spheroid or organoid of any one of claims 25-27 and 36-40 or secretome of each for use in the treatment of a kidney disease.

43. The nephro spheroid or organoid of claim 42, wherein said kidney disease is a chronic kidney disease (CKD).

44. The nephro spheroid or organoid of claim 42, wherein said kidney disease is an acute kidney disease.

45. A method of producing a secretome, the method comprising:(a) providing a culture of the nephro spheroid or organoid or any one of claims 25-27 and 36-40;(b) collecting conditioned medium of said culture, said conditioned medium comprising said secretome.

46. The method of claim 45, further comprising isolating a portion from said conditioned medium following step (b).

47. A method of regenerating renal function, the method comprising administering to a subject in need thereof nephro spheroid or organoid or any one of claims 25-27 and 36-40 or secretome thereof or portion thereof, thereby regenerating renal function.

48. The nephro spheroid or organoid of any one of claims 25-27 and 36-40 or secretome thereof for use in the treatment of a kidney disease.

49. A method of drug design, the method comprising determining an effect of a test drug on the nephrospheroid or organoid or any one of claims 25-27 and 36-40 or secretome thereof.

50. A method of selecting treatment to a pet diagnosed with a kidney disease, the method comprising:(a) contacting a therapeutic agent with a nephrospheroid or organoid of any one of claims 25-27 and 36-40 of a pet diagnosed with the kidney disease; and(b) determining an alleviation in a renal symptom evident in the nephrospheroid or organoid following said contacting, said alleviation being indicative of an efficacious therapy.

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