NOVEL RENAL DISEASE MODEL

FIELD OF THE INVENTION

The invention relates to a renal disease model comprising: a matrix which comprises glomerular endothelial cells, mesangial cells and podocytes; and an agent which induces renal disease morphology. This invention also relates to methods of making and using the same, and kits thereof.

BACKGROUND OF THE INVENTION

Glomerulosclerosis is the fibrotic glomerular scarring that occurs in many primary renal and systemic chronic kidney diseases, including idiopathic focal segmental glomerulosclerosis (FSGS) and the nephropathy that commonly accompanies diabetes. Progressive chronic kidney disease (CKD) leads to end stage renal failure (ESRF), which carries a high morbidity and mortality. In the US the prevalence of CKD over the age of 60 is 33.2%, and by the end of 2013 468,386 people were undergoing dialysis for ESRF (National Institutes of Health, Bethesda, MD. (2015) United States Renal Data System Annual Data Report). Renal replacement therapy with haemodialysis or peritoneal dialysis provides only 10-15% of normal renal function, and the relative risk of death on haemodialysis is 28.6 compared to the normal population at age 30-34 (NHS (2010) Kidney Disease: Key Facts And Figures). Transplantation is not available to all, carries risks associated with immunosuppression, and the current average lifespan of a renal graft is 10-15 years (Wolfe, R.A. ef al. (1999) N Engl J Med 341 , 1725-1730). Preventing or reversing glomerulosclerosis would dramatically improve the health and life expectancy of a large population of patients, and provide a major financial saving to healthcare providers. However, at present the mechanisms of

glomerulosclerosis are poorly understood and therapeutic strategies are limited.

Numerous animal models have been used to study glomerulosclerosis (Yang, H.C. ef al. (2010) Drug Discov Today Dis Models 7, 13-19), but these use a range of insults that often do not reflect the aetiology in human disease, take weeks or months to evolve, and have been poor predictors of therapeutic responses in humans. Streptozotocin is commonly used to induce diabetes and diabetic nephropathy in rats. However the histological changes of glomerulosclerosis, including Kimmelsteil-Wilson nodules, are much less severe than seen in humans and take weeks to months to develop (Becker, G.J., and Hewitson, T.D. (2013) Nephrol Dial Transplant 28, 2432-2438). In addition streptozotocin is toxic to renal cells independent of a diabetic effect (Becker, G.J., and Hewitson, T.D. (2013) supra).

Furthermore, mice are much more resistant to streptozotocin (Rees, D.A., and Alcolado, J.C. (2005) Diabet Med 22, 359-370). The 5/6 nephrectomy model produces widespread glomerulosclerosis at 12 weeks, but its utility is hampered by a high rate of mortality from this point due to uraemia (Yang, H.C., Zuo, Y., and Fogo, A.B. (2010) Drug Discov Today Dis Models 7, 13-19). The unilateral urethral obstruction model of CKD is not a usual cause of CKD, and only replicates the tubule-interstitial changes and not glomerulosclerosis (Chevalier, R.L., Forbes, M.S., and Thornhill, B.A (2009) Kidney Int 75, 1145-1152). Cell culture studies have been of limited value in modelling glomerulosclerosis because the development of lesions is dependent on complex cellular networks and matrix interactions that are not established in typical cell culture models, often involving a single cell type in monolayer culture.

There is therefore a need to provide an improved model of glomerulosclerosis that is able to overcome the problems associated with currently available methods.

SUMMARY OF THE INVENTION

According to a first aspect of the invention, there is provided a renal disease model comprising

(a) a matrix;

(b) glomerular endothelial cells, mesangial cells and podocytes; and

(c) an agent which induces renal disease morphology.

According to a further aspect of the invention, there is provided the use of a kit, comprising the matrix as defined herein and an agent which induces renal disease, as a renal disease model. According to a further aspect of the invention, there is provided a method of identifying an agent for the treatment of renal disease comprising contacting a test compound with the model as described herein, and determining if disease progression has been reduced or renal morphology is improved. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 : Human glomerular endothelial microvessels in the 3D matrix, (a)

ULEX staining of human GEC microvessel networks in the 3D matrix at x40 power. Inset: lumenisation of GEC cords revealed by GFP lentivirus transfected GECs in triculture (MCs and podocytes not labelled) showing coalescence of vacuoles to form lumen, (b) Scanning electron microscopy of GEC microvessel showing the lumen (arrow) and (c) abluminal surface with fenestrae (arrow), (d) Quantification of GEC network arborisation with TGFfi (10ng/ml) or BMP7 (100ng/ml) treatment and co-treatment as assessed by cord number, total cord length, average cord length and branching points (n=3, standard deviation bars, 2- tailed Student's t test).

Figure 2: Mesangial nodule formation in 3D. (a) MC only 3D culture without the addition of TGFp shown by H and E staining of paraffin embedded sections, (b) MC nodule formation in the 3D culture in the presence of TGFp shown by H and E staining of paraffin embedded sections. Human collagen type IV (left inset) & human collagen type l/lll (right inset) in nodules, (c) High power view of MC nodule by scanning electron microscopy in cross section, (d) MC nodule count and COL1a, COL4a RNA quantification (n=3, standard deviation bars, 2-tailed Student's t test), (e) Effect of ALK5 inhibition on MC nodule formation (n=3, standard deviation bars, 2-tailed Student's t test), (f) Effect of SMAD2 or SMAD3 knockdown in MCs on nodule formation (n=3, standard deviation bars, 2-tailed Student's t test). Figure 3: Western blot of canonical responses after co-stimulation, (a)

GECs and (b) MCs with TGFp (10ng/ml) and BMP7 (100ng/ml). (c) The only difference in co-stimulation was seen in pSMAD2 in GECs (Black bars) and pSMAD1/5 in MCs (White bars). The graph shows quantification of this with densitomerty analysis of pSMAD2 and pSMAD 1/5 response in MCs and GECs in response to co-stimulation (n=3, standard deviation bars, 2-tailed Student's t test).

Figure 4: TGFp receptor changes in 3D culture of MCs in response to TGFp and B P7 & Phospho-SMAD expression in human chronic kidney disease tissue, (a)

TGFp/BMP receptor changes in MC mono-3D culture in response to TGFp (10ng/ml) and BMP7 (100ng/ml) stimulation and co-stimulation by quantitative reverse transcriptase PCR analysis (n=3, standard deviation bars, 2-tailed Student's t test), (b) Phospho-SMAD (1 , 2 and 3) activity in glomeruli from patients with diabetic nephropathy and normal human kidney tissue. Representative immunohistochemistry images and quantification by percentage of positive nuclei per glomerulus (diabetic nephropathy n=3 and FSGS n=3, control tissue n=6, 2-tailed Student's t test).

Figure 5: 3D culture of human podocytes. (a) Scanning electron microscopy of podocytes in the 3D matrix, (b) High powered scanning electron microscopy view of podocyte foot process in the 3D matrix, (c) CTGF release by podocytes in 2D (black bars) and 3D (white bars) by quantitative reverse transcriptase PCR analysis in response TGFp (10ng/ml) and BMP7 (100ng/ml) stimulation and co-stimulation (fold change compared to control, n=3, standard deviation bars, 2-tailed Student's f test). Figure 6: 3D triculture of human GECs, MCs and Podocytes. (a) Culture of GECs (Labeled with ULEX-FITC), Podocytes (Labeled with PKH-Red) and MCs (Labelled CAMC-Blue) at 24 hours, (b) Association of GEC (ULEX-FITC) and Podocyte (PKH-Red) in tricultures at 24 hours, (c) Scanning electron microscopy of GEC-Podocyte interaction at 24 hours. Insert shows high powered view of foot process with cord, (d) TGFfi induced nodules by phase microscopy (e) Immunofluorescence of nodule showing GEC network loss, podocyte detachment and increased MC number, (f) Effect of BMP7 on TGF induced nodule formation in triculture (n=3, standard deviation bars, 2-tailed Student's t test), (g) Effect of ALK5 inhibition & CTGF neutralising antibody (nAb) on TGF induced nodule formation and and COL1a, COL4a RNA quantification in triculture (n=3, standard deviation bars, 2-tailed Student's t test).

Figure 7: 3D culture system analysis of idiopathic focal segmental glomerulosclerosis (FSGS). Actin changes within glomerular endothelial cells within the 3D system in response to urokinase-type-plasminogen-activator receptor treatment (b) versus control (a).

DETAILED DESCRIPTION OF THE INVENTION

Cell Culture

According to one particular aspect of the invention which may be mentioned, there is provided a cell culture comprising glomerular endothelial cells, mesangial cells and podocytes. The advantage of this triculture of glomerular cells is the formation of a glomerular vascular network. Although signalling pathways can be studied in cell cultures of individual cell types, the current invention provides a cell culture comprising all three glomerular cells where the interactions between all three cell types can be studied. The cell culture described herein has significant advantages, in particular in respect to modelling glomerular disease for use in identifying therapeutic targets.

References to the term "cell culture" as used herein, refer to maintaining cells in an artificial environment comprising the use of items selected from: a suitable vessel; a medium; growth factors; hormones; gasses; and a matrix, and includes 2-dimensiona! and 3~dimensionai ceil culture. Furthermore, it would be known that techniques employed in cell culture include media changes, passaging cells, and fransfecting cells. In one embodiment, the glomerular endothelial cells and/or mesangial cells and/or podocytes are mammalian derived. In a further embodiment, the glomerular endothelial cells and/or mesangial cells and/or podocytes are human derived. One suitable example of glomerular endothelial cells includes those which may be obtained from ScienCell Research Laboratories, San Diego, CA, USA as catalogue #4000. One suitable example of mesangial cells includes those which may be obtained from ScienCell Research Laboratories, San Diego, CA as catalogue #4200. One suitable example of podocytes includes those which may be obtained from Celprogen, Torrance, CA, USA as catalogue #36036-08-T25. In one embodiment, the cell culture comprises at least 100,000 mesangial cells, at least 20,000 glomerular endothelial cells and at least 5,000 podocytes.

In a further embodiment, the cell culture comprises at least 200,000 mesangial cells, at least 40,000 glomerular endothelial cells and at least 10,000 podocytes.

In a further embodiment, the cell culture comprises at least 300,000 mesangial cells, at least 50,000 glomerular endothelial cells and at least 20,000 podocytes.

In a further embodiment, the cell culture comprises 330,000-340,000 mesangial cells, 50,000-70,000 glomerular endothelial cells and 20,000-24,000 podocytes.

In a further embodiment, there is provided a cell culture comprising glomerular endothelial cells, mesangial cells and podocytes in a ratio of at least 1 : 1 : 1. In a further embodiment, there is provided a cell culture comprising glomerular endothelial cells, mesangial cells and podocytes in a ratio of at least 1 :2: 1 or at least 1 :3:1.

In a further embodiment, there is provided a cell culture comprising glomerular endothelial cells, mesangial cells and podocytes in a ratio of at least 5: 1 : 1 or at least 10: 1 :1 or at least 5:2:1 or at least 10:2:1.

In a further embodiment, there is provided a cell culture comprising glomerular endothelial cells, mesangial cells and podocytes in a ratio of at least 16:3:1. In a further embodiment, there is provided a cell culture comprising glomerular endothelial cells, mesangial cells and podocytes in a ratio of 16:3:1. Optimisation of the media and cell ratios allowed triculture of the three glomerular cell types in 3-dimensions to create a structure that reflects the glomerulus. In the cell culture described herein glomerular endothelial cells surprisingly form vessels with lumens and fenestrae which associate intimately with podocytes, as presented in Figures 1 and 6. According to a further particular aspect of the invention which may be mentioned, there is provided a method of producing the cell culture as described herein which comprises combining the cells (i.e. the glomerular endothelial cells, mesangial cells and podocytes) in a medium. In one embodiment, the medium comprises components selected from: basal medium; serum; cell growth supplement; and antibiotics. Examples of suitable serum include fetal bovine serum. Examples of suitable antibiotics include penicillin and streptomycin. In one particular embodiment, the medium comprises RPMI 1640 (Gibco by Thermo Fisher, UK), fetal bovine serum, penicillin, streptomycin, insulin, apo-transferrin, sodium selenite, and EGCS (ScienCell Research Laboritories, San Diego, CA, USA). Surprisingly the combination provided in this embodiment promoted optimal growth of all 3 cell types.

It will be apparent to the skilled person that the cell culture will require maintenance through application of cell culture techniques. In a further embodiment, the cell culture is maintained by periodic application of new medium. In still a further embodiment, the cell culture is maintained by application of new medium at least every 24 hours, such as every 48 hours.

In one embodiment, the cells exhibit normal renal morphology. References to the term "normal renal morphology" as used herein, may refer to inclusion of morphological features selected from any or all of the following features:

lumenised cords formed via vacuolisation;

lumenised cords formed by glomerular endothelial cells via vacuolisation;

fenestrae identifiable by electron microscopy;

fenestrae identifiable by electron microscopy formed by glomerular endothelial cells;

mesangial cells randomly dispersed;

mesangial cells associated with collagen fibres;

cord-like structures that lack lumens;

cord-like structures that lack lumens formed by mesangial cells;

podocytes randomly dispersed;

podocytes with characteristic foot processes;

lumenised cords formed by glomerular endothelial cells via vacuolisation to which podocytes associate; and lumenised cords formed by glomerular endothelial cells via vacuolisation with which podocytes associate and mesangial cells surround but which are not intimately associated.

In one embodiment, the cells exhibit normal renal morphology within at least 12 hours, such as within at least 24 hours.

Matrix

According to a further particular aspect of the invention which may be mentioned, there is provided a matrix comprising glomerular endothelial cells, mesangial cells and podocytes. Advantages provided by the use of the matrix as described herein, include the application of a 3-dimensional system where the signalling pathways between the three glomerular cells can be studied, such as the association of glomerular endothelial cells with podocytes presented in Figure 6. References to the term "matrix" as used herein, refer to a platform used to facilitate the growth of 3-dimensional cellular structures including extracellular matrix and scaffold systems such as hydrogel matrices and solid scaffolds.

In one embodiment, the matrix is an extracellular protein scaffold. In a further embodiment, the matrix is an extracellular protein scaffold wherein said extracellular protein scaffold comprises collagen and fibronectin. In still a further embodiment, the matrix is an extracellular protein scaffold wherein said extracellular protein scaffold comprises type 1 collagen and plasma fibronectin. An example of suitable type 1 collagen is rat tail type 1 collagen. An example of suitable plasma fibronectin is human plasma fibronectin. In still a further embodiment, the matrix comprises type 1 collagen, plasma fibronectin, HEPES, NaHCOs buffered M199 medium and HCI.

In one embodiment, the matrix has a pH greater than 7, such as pH 7-8, in particular pH 7.4. As presented in the Examples described herein, favourable results were obtained at a pH 7.4.

According to a further particular aspect of the invention which may be mentioned there is provided a method of producing the matrix as described herein which comprises:

(a) combining the cells (i.e. the glomerular endothelial cells, mesangial cells and podocytes) and components of the matrix either collectively, individually or in any combination thereof; and

(b) polymerisation of said matrix. References to the term "polymerisation" as used herein, refer to the formation of polymer chains or 3-dimensional networks. In one embodiment, said polymerisation typically takes at least 10 minutes, such as 15-25 minutes, in particular 20 minutes.

In one embodiment, said method is conducted at greater than 3°C, such as 3-10°C, in particular 4°C. As presented in the Examples described herein, favourable results were obtained at a temperature of 4°C.

In an alternative embodiment, said polymerisation of step (b) comprises incubating the matrix at at least 25°C, such as 30-45°C, in particular 37°C. It will be apparent to the skilled person that the matrix comprising glomerular endothelial cells, mesangial cells and podocytes will require maintenance through application of cell culture techniques. In a further embodiment, the matrix comprising glomerular endothelial cells, mesangial cells and podocytes is maintained by periodic application of new medium. In still a further embodiment, the matrix comprising glomerular endothelial cells, mesangial cells and podocytes is maintained by application of new medium at least every 24 hours, such as every 48 hours.

Renal Disease Model

According to a first aspect of the invention, there is provided a renal disease model comprising:

a matrix which comprises or includes glomerular endothelial cells, mesangial cells and podocytes; and

an agent which induces renal disease morphology. The renal disease model described herein has significant advantages over the current animal and monolayer cell culture models by forming glomerular vascular networks which provide an in vitro platform for the study of renal disease aetiology.

In one embodiment, the renal disease model is a glomerular disease model. In a further embodiment, the renal disease model is a glomerulosclerosis model. The renal disease model described herein has significant advantages as a glomerulosclerosis model because histological changes characteristic of glomerulosclerosis are seen (Figure 6). References to the term "renal disease" as used herein, refer to any disease state where the kidneys fail to adequately filter waste products from the blood. Examples of such diseases include but are not limited to: nephropathy; nephritis; nephrosis; kidney failure; chronic kidney disease; primary renal chronic kidney disease; systemic chronic kidney diseases; renal insufficiency; end stage renal failure; nondiabetic renal failure; acute kidney injury; acute-on-chronic kidney failure; and associated pathophysiology.

References to the term "glomerular disease" as used herein, refer to any disease state where the glomerular is affected. Examples of such diseases include but are not limited to: glomerulonephritis; and glomerulosclerosis.

The glomerular filtration unit is a multicellular structure consisting of a complex basement membrane partitioning glomerular endothelial cell (GEC)-lined capillaries and mesangial cells (MCs) on the side facing the blood from podocytes that face into Bowman's space (Suh, J.H., and Miner, J.H (2013) Nat Rev Nephrol 9, 470-477).

In order to capture some of this complexity, the inventors have developed a 3-dimensional in vitro human glomerular cell culture model of glomerulosclerosis. The inventors first used the 3D system to culture each of the three main human glomerular cells in isolation, and then refined the system to form an in vitro human glomerular vascular structure through 3- dimensional triculture of human glomerular endothelial cells, mesangial cells and podocytes. The inventors demonstrate that TGFp induces a glomerulosclerotic phenotype in this model, which can be used to screen therapeutic targets. Although the signalling pathways can be studied in culture of individual cell types, the mechanism involves an interaction between all three types and identification of therapeutic targets depends on the 3D culture of all three glomerular cells.

Regardless of the aetiology, glomerulosclerosis is characterised by expansion of matrix within the mesangio-capillary compartment, initially "thickening" the basement membrane and eventually replacing the glomerulus entirely (Schlondorff, D., and Banas, B. (2009) J Am Soc Nephrol 20, 1179-1187) and may refer to nodular glomerulosclerosis, focal segmental glomerulosclerosis, idiopathic focal segmental glomerulosclerosis (FSGS) and associated renal fibrosis. Causes of glomerulosclerosis include but are not limited: glomerulonephritis (including but not limited to focal segmental glomerulosclerosis); diabetes; drug use;

infection; chronic renal disease; inherited genetic problems; obesity; reflux neuropathy; sickle cell disease; and AIDS. Therefore, references to the term "glomerulosclerosis" as used herein, refer to fibrotic glomerular scarring, the pathophysiology of which includes features selected from but not limited to: a loss of the glomerular endothelial cell vascular structures; podocyte detachment; nodule formation; and matrix deposition. References to the term "model" as used herein, refer to an in vitro platform which emulates in vivo cells, such as animal models and ceil culture models, !n one embodiment, the model is a 3-dimens!onal cell culture which may be grown in a bioreactor, a small capsule in which the cells can grow into spheroids, or a 3-dimensional cell colony. According to a further aspect of the invention, there is provided a method of producing the renal disease model as described herein which comprises:

(a) combining the cells (i.e. the glomerular endothelial cells, mesangial cells and podocytes) and components of the matrix either collectively, individually or in any combination thereof;

(b) polymerisation of said matrix; and

(c) incubating the product obtained in step (b) with an agent that induces renal disease.

In one embodiment, step (b) is conducted for at least 12 hours, such as at least 24 hours, in particular 24 hours.

In one embodiment, step (c) is conducted for at least 12 hours, such as at least 24 hours, in particular 24 hours. It will be apparent to the skilled person that the renal disease model will require maintenance through application of cell culture techniques. In a further embodiment, the renal disease model is maintained by periodic application of new medium. In still a further embodiment, the renal disease model is maintained by application of new medium at least every 24 hours, such as every 48 hours.

According to a further aspect of the invention, there is provided a method of identifying a compound for the treatment of renal disease comprising contacting a test compound with the model as described herein, and determining if disease progression has been reduced or renal morphology is improved.

It will be appreciated that disease progression in the context of renal disease is deviation from normal renal morphology, such as the appearance of renal disease morphology. References to the term "renal disease morphology" as used herein, refer to morphological features which may be selected from any or all of the following features:

a loss of arborisation of vascular networks;

a loss of arborisation of vascular networks formed by glomerular endothelial cells;

formation of large nodules;

formation of large nodules containing cells and matrix proteins;

formation of large nodules by mesangial cells containing cells and matrix proteins;

an increase in collagen type I and IV mRNA;

formation of large nodules and an increase in collagen type I and IV mRNA within and around the nodules;

formation of large nodules by mesangial cells and an increase in collagen type I and IV mRNA within and around the nodules;

an increase in connective tissue growth factor mRNA; and

an increase in connective tissue growth factor mRNA by podocytes and reduced interaction between glomerular endothelial cells and podocytes.

In one embodiment, there is provided a method of identifying a compound for the treatment of renal disease, such as glomerular disease, in particular glomerulosclerosis, comprising contacting a test compound with the model as described herein, and determining if disease progression is reduced or renal morphology is improved.

According to a further aspect of the invention, there is provided a compound identified by the methods as described herein.

According to a further aspect of the invention, there is provided a compound as described herein for use in the treatment of renal disease, such as glomerular disease, in particular glomerulosclerosis. Agent

References to the term "agent" as used herein, refer to a compound or substance capable of producing an effect. Therefore, the term "an agent which induces renal disease" as used herein, refers to a compound or substance, the presence of which results in renal disease or a more advanced form of renal disease.

In one embodiment, the agent induces deviation from normal renal morphology. In a further embodiment, the agent induces renal disease morphology. In one embodiment, the agent is a member of the TGFp signalling pathway.

TGF-β has been identified as an important downstream mediator of glomerulosclerosis, and a potential therapeutic target for preventing the fibrotic changes that accompany many glomerular diseases. BMP7 may act as an anti-fibrotic agent, counteracting the effects of TGF . Border et al. (Shihab, F.S. et al. (1995) J Am Soc Nephrol 6, 286-294; Yamamoto, T. et al. (1996) Kidney Int 49:461-469) showed a significant increase in all three TGF isoforms in the glomeruli of patients with glomerulosclerosis associated with a range of chronic kidney diseases and chronic allograft nephropathy compared to controls.

Quantification of glomerular TGF i mRNA in diabetic patients by glomerular dissection revealed much higher levels of TGF 1 compared to controls (Iwano, M. et al. (1996) Kidney Int 49, 1120-1126). This correlated with the intensity of collagen IV staining within the extracellular matrix, and also with serum HbA1c. Aortic and renal vein sampling of TGF in diabetic patients undergoing elective cardiac catheterisation revealed a net renal production of TGF$, compared to controls which demonstrated net renal extraction of TGF& (Sharma, K. et al. (1997) Diabetes 46, 854-859).

Two animal studies have shown a causative effect for TGF& in glomerulosclerosis. A transgenic mouse model with increased plasma levels of JGF& exhibit renal lesions highly representative of the glomerular scarring seen in chronic kidney disease (Sanderson, N. et al. (1995) Proc Natl Acad Sci U S A 92, 2572-2576; Kopp, J.B. et al. (1996) Lab Invest 74, 991-1003). In vivo transfection of TGF6 into the rat kidney by injecting a plasmid containing the TGFp gene through the renal artery induced glomerulosclerosis with extracellular matrix expansion and proteinuria (Isaka, Y. et al. (1993) J Clin Invest 92, 2597-2601). Increased expression of TGFp mRNA and protein in glomerular cells is seen in rats after induction of diabetes with streptozotocin (Park, I.S. et al. (1997) Diabetes 46, 473-480). Heterozygosity for TGF-β type II receptor attenuates the degree of mesangial expansion after streptozotocin treatment (Kim, H.W. et al. (2004) Kidney Int 66, 1859-1865). References to the term "TGFp signalling pathway" as used herein, refer to the gene regulatory pathway comprising TGFp superfamily ligands, type II receptors, type I receptors, receptor-regulated SMADs, coSMADs, ALK receptors and their regulators.

It is less clear whether BMP7 expression is altered in glomerulosclerosis. A study (De Petris, L, et al. (2007) Nephrol Dial Transplant 22, 3442-3450) assessed BMP7 expression in biopsies from diabetic patients with glomerulosclerosis, and found a significant decrease in BMP7 expression compared to normal biopsies. A larger study found no difference in BMP7 levels within the glomeruli of patients with CKD compared to controls, but did find a significant decrease within the interstitium (Bramlage, CP. et al. (2010) BMC Nephrol 11 , 31). Tail vein injection of BMP7 in streptozotocin treated mice reversed proteinuria, restored glomerular filtration rate (GFR), and decreased the percentage of sclerotic glomeruli (Wang, S. et al. (2003) Kidney Int 63, 2037-2049). Studying BMP7 as an anti-fibrotic agent has been hampered due to technical challenges in producing large enough quantities of bioactive BMP7 (Nematollahi, L. et al. (2012) Avicenna J Med Biotechnol 4, 178-185). Sugimoto et al. administered a novel ALK3 agonist (a BMP7 receptor) systemically in five mouse models of renal fibrosis, and showed that it reversed fibrosis in each case (Sugimoto, H. et al. (2012) Nat Med 18, 396-404). However, this was predominantly in the tubule-interstitial

compartment, with only minor effects on glomerulosclerosis. In one embodiment, the agent increases mRNA for ALK5 and/or decreases mRNA for ALK1.

In a further embodiment, the agent decreases TGFpRII/ALK1/ALK5 trimerisation and/or increases TGFpRII/ALK5 dimerisation. In a further embodiment, the agent increases SMAD2/3 phosphorylation and/or decreases SMAD 1/5 phosphorylation.

As described in the Examples herein, these effects are associated with the addition of TGFp, which induces a phenotype characteristic of glomerulosclerosis.

In a further embodiment, the agent is TGFp. TGFp induces a phenotype characteristic of glomerulosclerosis, therefore the renal disease model presented herein advantageously provides the possibility for in vitro study of glomerulosclerosis and related renal diseases. In one embodiment, the agent is present at a concentration of at least 1 ng/ml, such as at least 5 ng/ml, in particular at least 10 ng/ml, such as 10 ng/ml.

Kits

According to a further aspect of the invention, there is provided the use of a kit, comprising the matrix as defined herein and an agent which induces renal disease, as a renal disease model. According to a further particular aspect of the invention which may be mentioned, there is provided a kit comprising the matrix as described herein and an agent which induces renal disease. According to a further particular aspect of the invention which may be mentioned, there is provided the use of the kit as described herein as a renal disease model.

Therapy

According to a further particular aspect of the invention which may be mentioned, there is provided a matrix as described herein or a component thereof for use in the treatment of renal disease. In one embodiment, the glomerular endothelial cells and/or mesangial cells and/or podocytes cells are used as explants for the treatment of renal disease. In a further embodiment, a portion of the matrix comprising glomerular endothelial cells and/or mesangial cells and/or podocytes cells are used as explants for the treatment of renal disease.

The following studies and protocols illustrate embodiments of the methods described herein:

MATERIALS AND METHODS

Cell 2D monocultures

Human GECs were cultured in medium supplied by the vendor consisting of 500 ml of basal medium, 25 ml of FBS, 5 ml of endothelial cell growth supplement (ECGS) and 5 ml of penicillin/streptomycin solution. Human mesangial cells (MCs) were cultured in supplied medium consisting of 500 ml of basal medium, 10 ml of fetal bovine serum (FBS), 5 ml of mesangial cell growth supplement, and 5 ml of penicillin/streptomycin solution (glomerular endothelial cells (GECs), MCs, media and supplements were all from ScienCell Research Laboratories, San Diego, CA. All plastic ware was from Corning Incorporated, Corning, NY. The cells were used between passages 2 and 6. Both cell types were grown on tissue culture media treated plates. In addition, for GECs the surface was coated with gelatin (Sigma-Aldrich, MO, USA). Flasks were incubated at 37°C with 3 ml of 0.1 % gelatin solution for 10 minutes, after which time the solution was removed by aspiration. Human podocytes were cultured in the supplier's complete medium with serum (Celprogen, Torrance, CA). Cells were used between passages 2 and 6. Formation of 3D culture

GECs, MCs and podocytes in monoculture or co-culture were suspended in a solution of rat tail type 1 collagen (1.5 mg/ml; BD, NJ, USA) and human plasma fibronectin (90 pg/ml; Millipore, MA, USA) in 25 mM HEPES and 1.5 mg/ml NaHC03 buffered M199 medium at

4°C, and the pH was adjusted to 7.4 using 0.1 M HCI. The suspension was then pipetted into 48 well plates in a volume of 320μΙ with a GEC number of 0.5 x 10 ⁶ cells per well

(monoculture), podocyte number of 250,000 per well (monoculture) and a MC number of 500,000 (monoculture). For co-cultures MC: 330,000-340,000 GEC: 50,000-70,000 podocytes: 20,000-24,000 were added per well (a ratio of 16:3:1). After the matrix had polymerised, 0.5ml of triculture media (consisting of 100mls RPMI 1640 (Gibco™ by Thermo Fisher, UK), 2mls FBS, 1 ml penicillin/streptomycin, 1 ml insulin (1mg/ml), Apo-transferrin (1 mg/ml), sodium selenite (3.4 uM) (in ITS mix) and 1ml ECGS (supplements all from ScienCell Research Laboratories, San Diego, CA) were pipetted onto the top of the gel, and this media was changed every second day for cultures maintained longer than 24 hours. For confocal microscopy 10μΙ of the matrix with the same density and ratio of cells was pipetted into Ibidi μ-slides (Ibidi®, Germany) and 40μΙ of the triculture media was placed on top of the matrix after it had polymerised.

Stimulation assays: 3D stimulation assays to TGF3 and BMP7

For stimulation 10 ng/ml TGF (R&D Systems, MN, USA) or 100 ng/ml BMP7 (R&D

Systems, MN, USA) were added to the 0.5ml (for 48 well) or 40μΙ (for Ibidi μ-slides) of media placed on top of each matrix.

Imaging of 3-dimensional cultures

The cultures were followed in real time by phase contrast microscopy and at relevant timepoints by epifluorescence microscopy [using a Leica DM I 3000B manually inverted microscope with ImagePro software (MediaCybernetics, MD, USA)] or confocal microscopy [using a Leica TCS SP5 microscope with Leica Application Suite software, Wetzlar, Germany]. For labelling GECs were either transfected with GFP or whole mount immunostained for Ulex europeus Agglutinin I (ULEX, Vector Laboratories, CA, USA). GECs were transduced with green fluorescent protein (GFP) using lentiviral vector containing the human polypeptide chain elongation factor-1 (EF-1) promoter by using 10μΙ virus in 4000μΙ media per approximately 300,000 GECs. Cells were counted and plated into 6-well TC treated plates (Corning Incorporated, Corning, NY), left in culture 24 hours and then culture medium was replaced and cells were treated with lentivirus for 48 hours. After treatment GECs were ready to be counted and incorporated into 3D culture. Whole mount

immunostaining was carried out using fluorescein-labelled Ulex europaeus agglutinin I (FL1061 , Vector Laboratories). Matrices were washed three times for one hour each wash with phosphate buffered solution, and then fixed for one hour in 4% paraformaldehyde. They were then washed another three times for 30 minutes each, and then incubated with Uiex europaeus agglutinin i overnight at 4°C on a rocker. Podocytes were labelled with PKH26 dye before implantation into the matrix using the manufacturer's protocol (Sigma-Aldrich, MO, USA). MCs were labelled with Celltracker™ Blue CMAC dye before implantation into the matrix using the manufacturer's protocol (Life Technologies, Thermo Fisher Scientific, MA, USA).

For standard histology of 3D cultures of MCs matrices were embedded in histogel (Thermo Fisher Scientific, MA, USA) according to the manufacturer's instructions, fixed in 4% paraformaldehyde overnight, paraffin wax embedded for sectioning and the slides were stained with hematoxylin and eosin (H&E).

For scanning electron microscopy the 3D matrices were washed in normal saline, fixed in 2% glutaraldehyde in 0.1 M PIPES buffer pH 7.4 in 0.1 M sodium cacodylate buffer for 14 hours, dehydrated with increasing concentrations of ethanol to 100%, frozen in liquid nitrogen, and fractured with a razor blade and hammer. They were then critical point dried, mounted on Cambridge SEM stubs with Silver DAG, sputter coated with 10nm of gold and viewed in an FEI XL30 FEG scanning electron microscope operated at 5keV (FEI, Oregon, USA).

Staining of human collagen in MC 3D cultures

After 3D culture of MCs matrices were embedded in histogel (Thermo Fisher Scientific, MA, USA), fixed in 4% paraformaldehyde overnight and paraffin wax embedded for sectioning and staining for human collagen type IV (mouse anti-human collagen IV monoclonal antibody 2150-0121 , Bio-Rad, CA, USA) and human collagen type l/lll (rabbit anti-human collagen type l/lll polyclonal antibody 2150-2210, Bio-Rad, CA, USA). After automated antigen retrieval processing (PT Link Machine, DAKO, Denmark), the sections were washed three times for five minutes in wash buffer (EnVision Flex Wash Buffer, DAKO, Denmark), blocked for ten minutes in blocking reagent (EnVision FLEX Peroxidase-blocking reagent, DAKO, Denmark). After washing a further three times for five minutes in wash buffer, they were incubated with primary antibody diluted in substrate buffer (EnVision Flex Substrate Buffer, DAKO, Denmark) for one hour at room temperature, and then washed three times for five minutes in wash buffer. After incubation with secondary antibody

(EnVision FLEX/HRP, DAKO, Denmark) for 30 minutes at room temperature, three further five minute washes were performed, and 3,3-diaminobenzidine tetrahydrochloride (DAB) solution (EnVision FLEX DAB, DAKO, Denmark) was added to visualise the antibody, and H&E counterstain was performed. Acellular matrix (consisting of rat type I collagen) was used as a negative control, and normal human kidney tissue was used as a positive control.

SMAD2 and SMAD3 siRNA knockdown of mesangial cells

For siRNA knockdown of MCs a 20μΜ solution of lyophilised siRNA in 1x siRNA buffer (5x, Dharmacon CO, USA) was used. MCs plated on day one at 150,000 per well (in supplied medium consisting of 500 ml of basal medium, 10 ml of fetal bovine serum (FBS), 5 ml of mesangial cell growth supplement) were transfected on day three. Wells were first washed with 2ml / well of Optimem 1 (Invitrogen, CA, USA) for two hours and then changed to fresh Optimem 1.6ml / well. Four tubes containing DharmaFECT 1 , a non-targeting control pool (siCP), ON-TARGETP/i/s™ siGENOME™ Smartpool s SMAD2 and s\SMAD3 were made up as per manufacturer's instructions (Invitrogen, CA, USA). Lipoplexes were left on the cells for 4 hours, and then removed and normal growth medium added. They were then left overnight before use in stimulation assays. For SMAD3 knockdown the same method was also carried out using DharmaFECT 2 and 4. Knockdown was confirmed by immunoblotting for total SMAD2 and total SMAD 3.

ALK5 inhibition and CTGF neutralising antibody in 3D matrices

For inhibition of ALK5 in 3D, 616456 compound (Millipore, MA, USA) was added to the 0.5 ml of media on top of the 3D matrices at a concentration of 2 μΜ in the presence of at 10 ng/ml. For neutralisation of CTGF, CTGF neutralising antibody (ab109606, Abeam, Cambridge, UK) was used at a concentration of 8pg/ml.

Immunoblotting

GECs or MCs were grown in 6 cm dishes to ~80% confluence and treated with 10 ng/ml TGF6 (R&D Systems, MN, USA) and / or 100 ng/ml BMP7 (R&D Systems, MN, USA) in mesangial cell or endothelial cell medium as described above. At 6 hours the media was aspirated and the cells washed with ice cold PBS, which was then aspirated. The dishes were then placed on ice and the cells scraped in 350μΙ RIPA buffer (ThermoFisher Scientific, MA, USA) with protease inhibitors (Roche, Switzerland), transferred to an Eppendorf tube and agitated on ice three times for ten seconds every two minutes, and centrifuged at 1200 rpm for 20 minutes at 4°C. The supernatant was aspirated to another Eppendorf tube for storage at -80°C. Protein concentrations were determined using a BCA protein assay kit (Thermo Fisher Scientific, MA, USA). Proteins (15 g) in Laemmli sample buffer were separated by SDS-polyacrylamide gel (10%) electrophoresis and then transferred to polyvinylidene fluoride membrane. Blots were blocked in 5% dried milk powder / 0.05% Tween 20 in PBS for one hour at room temperature or overnight at 4°C. After blocking they were immunoblotted with rabbit anti-pSMAD2 (3108, Cell Signalling, MA, USA), rabbit anti- pSMAD1/5 (9516, Cell Signalling, MA, USA), rabbit anti-pSMAD3 (1880-1 , Epitomics, Abeam, Cambridge, UK), rabbit anti-tSMAD2 (3122, Cell Signalling, MA, USA), rabbit anti- tSMAD3 (9513, Cell Signalling, MA, USA), or mouse anti-β actin (A5441 , Sigma-Aldrich, MO, USA) for two hours (40 minutes for actin) at room temperature or overnight at 4°C. This was followed by incubation for two hours at room temperature with anti-mouse (1 :5000, DAKO, Denmark) or anti-rabbit IgG horseradish peroxidise conjugate (1 :4000, DAKO, Denmark) and detection by enhanced chemiluminescence according to the manufacturer's instructions (West Pico for β actin and West Dura for SMADs, Thermo Fisher Scientific, MA, USA).

Quantitative reverse transcriptase PCR analysis (qPCR)

RNA was isolated from 3D matrices by trizol extraction. 1 ml of trizol was added per 300μΙ of matrix and vortexed for 15 seconds, left at room temperature for 15 minutes, and vortexed for a further 15 seconds. Chloroform (0.2 mis per 1 ml of trizol used) was added, shaken vigorously for 5 seconds, left to stand for 3 minutes at room temperature, and then centrifuged at 12000g at 4°C for 15 minutes. The RNA (aqueous phase) was removed and a half volume of 100% ethanol added. This was then added to the spin columns of the RNA isolation kit (Mo Bio RNA Isolation kit, MoBio, CA, USA), centrifuged at 8000 rpm for 1 minute at 4°C, washed with 500μΙ of aqueous ethanol solution of guanidine thiocyanate, and centrifuged at 8000 rpm for 1 minute at 4°C. 45μΙ of tris-HCI (pH 7.5), sodium chloride and magnesium chloride solution and 5μΙ of DNase was added to the filter and incubated at room temperature for 15 minutes. 400 μΙ of aqueous solution of guanidine thiocyanate was then added and centrifuged at 8000 rpm for 1 minute at 4°C. This was repeated twice with 500 μΙ of ethanol solution and then centrifuged at 10,000 rpm for 2 minutes at 4°C. This washed twice with aqueous solution of ethanol and centrifuged at 8000 rpm for 1 minute at 4°C each time. It was then centrifuged at 10,000 rpm for 2 minutes at 4°C. Finally, it was eluted with 30 μΙ of RNase-free water, incubated for 2 minutes and centrifuged at 8000 rpm for 1 minute at 4°C. DNase-digested total RNA (300-700 ng) was reverse transcribed using a high capacity cDNA reverse transcription kit (Mo Bio, CA, USA) as described in the

manufacturer's instructions. qPCR reactions were prepared with 45 ng of cDNA using the SYBR® Green Jumpstart™ Taq Readymix™ (Sigma-Aldrich, MO, USA) containing 200 nM of the relevant sense and antisense primers and 10 nM fluorescein (Invitrogen, NY, USA). Reactions were amplified on an iCycler (Bio-Rad, CA, USA) using Quantitect Primers for COL1A 1, COL4A1, CTGF, MMP2, TGFBR2, ALK1, ALK5, ALK3, ALK6 and ALK2 (Qiagen, Germany). The relative expression of target mRNAs was normalized to the housekeeping genes B2M or GAPDH using the ΔΔΟΤ method and expressed as the fold-change relative to the control.

Assessment of TGF3 MCs on 2D coated surfaces

MCs were plated onto non-treated plastic, tissue culture treated plastic, or plastic coated with collagen by incubation overnight with 1.5 mg / ml type I rat tail collagen (BD, Franklin Lakes, N.J., USA) in PBS at 37°C before removal by aspiration, or fibronectin [0.1 mg/ml human fibronectin Millipore, Billerica, Mass., USA in PBS], which was left on the dish overnight at 37°C before aspiration. MCs were cultured in 6 cm culture dishes (Corning, NY, USA) at low density. 10 ng/ml of TGF$ in 3 ml of MC media (described above) was used to culture the cells, and this media was changed every 24 hours. The cells were then followed by phase contrast microscopy using a Leica DM I 3000B manually inverted microscope until they were 3 days post confluent. Staining of human collagen in MC 3D cultures

After 3D culture of MCs matrices were embedded in histogel (Thermo Fisher Scientific, MA, USA) according to manufacturer's instructions. They were then fixed in 4%

paraformaldehyde overnight and paraffin wax embedded for sectioning and staining for human collagen type IV (mouse anti-human collagen IV monoclonal antibody 2150-0121 , Bio-Rad, CA, USA) and human collagen type l/lll (rabbit anti-human collagen l/lll polyclonal antibody 2150-2210, Bio-Rad, CA, USA). After automated antigen retrieval processing (PT Link Machine, DAKO, Denmark), the sections were washed in wash buffer (EnVision Flex Wash Buffer, DAKO, Denmark) for five minutes three times, blocked for ten minutes in blocking reagent (EnVision FLEX Peroxidase-blocking reagent, DAKO, Denmark), then washed in wash buffer again for five minutes three times. They were incubated in the relevant collagen primary antibody diluted in substrate buffer (EnVision Flex Substrate Buffer, DAKO, Denmark) for one hour at room temperature and then washed for five minutes three times in wash buffer. Sections were incubated with secondary antibody (EnVision FLEX/HRP, DAKO, Denmark) for 30 minutes at room temperature, after which time three five minute washes were performed with wash buffer. 3,3-diaminobenzidine

tetrahydrochloride (DAB) solution (EnVision FLEX DAB, DAKO, Denmark) was added to visualise the antibody, and sections were counterstained with H&E. Acellular matrix

(consisting of rat type I collagen) was used as a negative control, and normal human kidney tissue was used as a positive control.

Human kidney tissue staining Kidney tissue from patients with diabetic nephropathy (n=3) or focal segmental

glomerulosclerosis (n=3) and histologically normal tissue from the normal poles of kidneys removed for tumours (n=6) was obtained from the Cambridge University Hospitals Tissue Bank with ethical approval (07/Q0108/49). The tissue was fixed in 4% paraformaldehyde, paraffin wax embedded for sectioning, and stained with rabbit anti-pSMAD2 (3108, Cell

Signalling, MA, USA), rabbit anti-pSMAD3 (1880-1 , Epitomics, Abeam, Cambridge, UK), and rabbit anti-pSMAD1 (9553, Cell Signalling, MA, USA). After automated antigen retrieval processing (PT Link Machine, DAKO, Denmark), the sections were washed in wash buffer (EnVision Flex Wash Buffer, DAKO, Denmark) for five minutes three times, blocked for ten minutes in blocking reagent (EnVision FLEX Peroxidase-blocking reagent, DAKO,

Denmark), then washed in wash buffer again for five minutes three times. Sections were incubated in the relevant collagen primary antibody diluted in substrate buffer (EnVision Flex Substrate Buffer, DAKO, Denmark) for one hour at room temperature and then washed for five minutes three times in wash buffer. Sections were incubated with secondary antibody (EnVision FLEX/HRP, DAKO, Denmark) for 30 minutes at room temperature, after which time three five minute washes were performed with wash buffer. 3,3-diaminobenzidine tetrahydrochloride (DAB) solution (EnVision FLEX DAB, DAKO, Denmark) was added to visualise the antibody, and sections were counterstained with H&E. Human lung tissue was used as a positive control.

Statistics

Quantification of cord networks and nodule counts within the 3-D matrix was performed by taking random images through the matrix with phase contrast or fluorescence microscopy at low power using an Axiovert 200 M Carl Zeiss microscope or Leica DM I 3000B manually inverted microscope. Images were analysed using Image J software (NIH, USA) to quantify cord length (total and average length), branching points (nodes), tube width and cord number as well as MC nodule number. Differences between treatments were analysed using an unpaired two-tailed Student t test. Experiments were repeated three times (biological replicates).

Study Approval

Tissue used was obtained from Cambridge University Hospitals Tissue Bank with ethical approval by NHS Health Research Authority, UK (REC Reference 07/QO108/49). EXAMPLE 1 : TGFp causes glomerular endothelial cell vascular network rarefication in 3D culture which is prevented by BMP7 Human GECs cultured on tissue culture treated plastic form a confluent monolayer with a cobblestone appearance (data not shown). GECs cultured in a 3D collagen and fibronectin matrix form networks of cords surviving up to 6 days (Fig. 1a). Narrow lumens appear to form by coalescence of intracellular vacuoles by about 24 h (Fig. 1a insert and 1 b) and fenestrae can be identified by electron microscopy (Fig. 1c). TGFp induces a loss of arborisation of vascular networks consistent with capillary rarification seen in

glomerulosclerosis, but co-treatment with BMP7 prevents this TGF -induced decrease in arborisation (Fig. 1d). EXAMPLE 2: TGFp causes mesangial cell nodule formation with matrix deposition in 3D culture, which is not prevented by BMP7

MCs cultured on plastic coated with or without rat tail collagen type I- or fibronectin grow to confluence and do not alter their morphology in response to TGFβ (data not shown). In 3D culture individual MCs randomly disperse throughout the matrix (Fig. 2a). Some MCs intimately associate with collagen fibres, forming cord-like structures that lack lumens (data not shown). TGFp treatment of 3D MC cultures promotes, within 24 h, formation of large nodules (Fig. 2b, c), containing both cells and matrix proteins (Fig. 2b, c). Human collagen type I and IV mRNA was also increased in the TGFp-treated group compared to controls (Fig. 2d) and was located within and around the nodules; human collagen was not detected in non-nodular areas or within the rat tail type I collagen matrix scaffold (data not shown). BMP7 did not modulate TGFp-mediated collagen I and IV mRNA expression or nodule formation (Fig. 2d).

EXAMPLE 3: BMP7 modulates TGFp SMAD responses in glomerular endothelial cells but not mesangial cells

In view of the different effects of BMP7 on TGFp-induced GEC network loss and TGFp- induced MC nodule formation, we assessed the SMAD responses in the two cell types to treatment with TGF with or without BMP7. In GECs, TGFp led to phosphorylation of both SMAD2 and SMAD3; BMP7 prevented TGFp induced SMAD2 phosphorylation, but not SMAD3 phosphorylation (Fig. 3a, c). BMP7 induced phosphorylation of SMAD1/5, which was not prevented by TGFp. In MCs BMP7 did not prevent TGFp-induced SMAD2 or SMAD3 phosphorylation (Fig. 3b, c). TGFp also decreased the BMP7 SMAD1/5 phosphorylation. In other words, BMP7 modulates TGFp SMAD responses in GECs but not MCs. EXAMPLE 4: TGFp induced mesangial cell nodule formation 3D culture, which is prevented by ALK5 inhibition TGFp increased mRNA for ALK5 and decreased mRNA for ALK1 in 3D MC cultures (Fig. 4a). This is likely to decrease TGFpRII/ALK1/ALK5 trimerisation and increase

TGFpRII/ALK5 dimerisation (Upton, P.D., and Morrell, N.W. (2009; Curr Opin Pharmacol 9, 274-280), thereby increasing SMAD2/3 phosphorylation, and decreasing SMAD1/5 phosphorylation. These changes in receptor mRNA expression were not prevented by co- treatment with BMP7 (Fig. 4a). An ALK5 inhibitor decreased TGF -induced nodule formation (Figure 2e). SMAD2 siRNA did not alter the number of nodules formed (Fig. 2f), whereas SMAD3 siRNA prevented TGFp-induced nodule formation (Fig. 2f). This is consistent with an increase in phospho-SMAD3 but not phospho-SMAD2 as seen in human

glomerulosclerosis (Fig. 4b).

EXAMPLE 5: Podocytes exhibit characteristic morphology in 3D culture and upregulate CTGF in response to TGFp

Podocytes cultured within the 3D matrix were randomly dispersed and maintained characteristic foot processes (Fig. 5a, b). Treatment with TGFp did not alter morphology, but increased connective tissue growth factor (CTGF) mRNA, particularly in 3D culture (Fig. 5c).

EXAMPLE 6: TGFp leads to mesangial nodule formation with loss of glomerular networks and podocyte detachment in 3D triculture of glomerular cells and is prevented with combined ALK5 inhibition and CTGF neutralisation

Optimisation of the media and cell ratios allowed triculture of the three glomerular cell types in 3D (Fig. 6a). GECs formed lumenised cords via vacuolisation (Fig. 1 a insert) to which podocytes associated within 24 h (Fig. 6b, c). MCs surrounded these networks but were not intimately associated with either the GECs or podocytes, which maintained their foot processes (Fig. 6c insert). Within 24 h, treatment with TGFp induced nodule formation (Fig. 6d), reduced interaction between GECs and podocytes, caused loss of GEC network arborisation, (Fig. 6e) and increased collagen I and IV expression (Fig. 6f), changes characteristic of glomerulosclerosis. BMP7 co-treatment did not alter this phenotype (Fig. 6f). ALK5 inhibition was less effective at reducing TGFp-induced nodule formation in triculture of the three different glomerular cells compared to its effects in MC monoculture, whereas treatment of the tricultures with a CTGF neutralising antibody in conjunction with ALK5 inhibition prevented TGFp-induced nodule formation (Fig. 6g). ALK5 inhibition reduced the increased collagen I and IV expression (Fig. 6g). EXAMPLE 7: 3D culture system analysis of idiopathic focal segmental glomerulosclerosis (FSGS) FSGS is an irreversible scarring disease of the kidneys with unknown cause. The fact that FSGS can occur in a normal kidney transplanted into a person who has FSGS as the cause of kidney failure in their own kidneys points towards a circulating factor as the cause of FSGS. Many factors have been identified from serum of FSGS patients, including urokinase-type- plasminogen-activator receptor (Konigshausen and Sellin (2016) Biomed Res int. Savin et al

(2012) Kidney Res and Clin Practice 31(4), 205-213). However, study of how these factors may affect cells within a human kidney, and hence potential therapies, is limited by lack of representative human model systems. The 3D system described herein provides an excellent tool for interrogating this by adding such factors to the culture medium. These factors then diffuse through into the matrix containing the cells. This provides a rapid throughput method for assessing their effects, in turn allowing study of the pathways leading to these responses, and to the effects of potential therapies. This potential for disease modelling has been confirmed for urokinase-type-plasminogen-activator receptor (Jefferson and Shankland

(2013) Kidney Int 84(2), 235-238).

METHOD AND RESULTS

Glomerular endothelial cells and podocytes were cultured within the 3D system as described herein. They were cultured for 24 hrs. In the treatment group urokinase-type-plasminogen- activator receptor (UPAR, R & D systems) was added to the culture medium at a concentration of 20ng/ml. At the 24 hour time point the matrix was fixed for 30 minutes with 4% paraformaldehyde, washed with phosphate buffered solution-tween (PBST) three times for 5 mins each time. It was then blocked with 5% bovine serum albumin in PBST for 1 hour. They were then incubated overnight at 4 degrees centigrade with phalloidin antibody conjugated with 488 fluor (ThermoFisher) and then washed three times in PBST before DAPI mounting solution was applied (Vector Laboratories). They were then viewed by epifluorescence microscopy (Leica systems). The UPAR treated group exhibited less continuous GEC networks and striated actin (Figure 7b) compared to controls (Figure 7a).

Discussion

The 3D culture of human glomerular cells described here reveals their ability to assemble into a glomerular vascular structure when cultured in a 3D matrix at optimised ratios and media. In this structure GECs form vessels with lumens and fenestrae, which associate intimately with podocytes. TGFp induces a phenotype characteristic of glomerulosclerosis in this model. This includes loss of GEC vascular structures, podocyte detachment, nodule formation and matrix deposition. These aspects provide quantifiable measures that can be used to assess signalling targets involving cross talk between cells, which cannot be assessed in traditional 2D monolayer cultures. BMP7 prevented the GEC component of the glomerulosclerotic phenotype but not the MC component. This may explain the less impressive effects of treatment with BMP7 (or BMP7 ligand) on glomerulosclerosis compared to tubulointerstitial fibrosis in animal models. In murine models renal fibrosis has been shown to be ameliorated by BMP7 (Wang, S., et al. (2003). Kidney Int 63, 2037-2049). The results presented herein suggest that in the glomerulus different targets need to be modulated in different cell types, and BMP7 treatment alone will not be sufficient to ameliorate glomerulosclerosis. The results also suggest that downstream the TGFp and BMP7 pathways interact in different ways in different glomerular cell types. This was supported by the observation that in GECs, but not MCs, BMP7 is able to influence TGFp induced SMAD phosphorylation.

The ability of TGFp to modulate its own receptors has been previously described. For example, high concentrations of TGFp downregulate TGFpRII and III receptors in human osteoblasts (Gebken, J., et al. (1999) J Endocrinol 161 , 503-510). Such changes are believed to form a feedback loop to dampen down responses in the presence of high concentrations of TGF (Gebken, J., et al. (1999) supra). In the MCs assayed here, TGFp increased the expression of ALK1 and decreased the expression of ALK5 in the 3D system. This was not preventable by BMP7. The response of a cell to TGFp is influenced by the heterodimeric or trimeric receptor formed to accept the ligand. TGFp associated with a heterodimer of TGFbRII and ALK5 signals through phosphorylation of SMAD2 and SMAD3, whereas TGFp associated with a heterotrimer of TGFbRII, ALK1 and ALK5 signals thorough SMAD1 , SMAD5 and SMAD8 (Upton, P.D., and Morrell, N.W. (2009) Curr Opin Pharmacol 9, 274-280). In the homeostatic state this may provide a balanced response to TGFp, but an increase in ALK5 and decrease in ALK1 will tip this balance towards an increase in the formation of the heterodimeric receptor and phosphorylation of SMAD2 and SMAD3. This may exacerbate nodule formation by MCs, and provides the rationale for inhibiting ALK5 as a means of preventing nodule formation. In 3D triculture neither BMP7 administration nor ALK5 inhibition prevented MC nodule formation and collagen deposition. This is likely to be due to the effects of CTGF released from podocytes, and is consistent with the exacerbation of glomerular damage in transgenic mice with podocytes overexpressing CTGF in diabetes induced by streptozotozin (Yokoi, H., et al. (2008) Kidney Int 73, 446-455). The combination of ALK5 inhibition and neutralising CTGF was needed to prevent the MC nodule formation in triculture. In summary the inventors have discovered the first 3D triculture of human glomerular cells with formation of a glomerular vascular network. TGFp induces a phenotype characteristic of glomerulosclerosis. The signalling pathways can be studied in culture of individual cell types, but the mechanism involves an interaction between all three types. Identification of therapeutic targets depends on the 3D model of all three glomerular cells described in this model.