Connective tissue growth factor (CTGF) is a member of an emerging family of immediate-early gene products that coordinate complex biological processes during differentiation and tissue repair. Here we describe the role of CTGF in integrin-mediated adhesive signaling and the production of extracellular matrix components in human mesangial cells. The addition of CTGF to primary mesangial cells induced fibronectin production, cell migration, and cytoskeletal rearrangement. These functional responses were associated with recruitment of Src and phosphorylation of p42/44 MAPK and protein kinase B. The inhibition of CTGF-induced p42/44 MAPK or phosphatidylinositol 3-kinase (PI3K)/protein kinase B pathway activities abrogated the induction of fibronectin expression. In addition, anti-β₃integrin antibodies attenuated the activation of both the p42/44 MAPK and protein kinase B and the increase in fibronectin levels. CTGF also induced mesangial cell migration via a β₃integrin-dependent mechanism that was similarly sensitive to the inhibition of the p42/44 MAPK and PI3K pathways, and it promoted the adhesion of the mesangial cells to type I collagen via up-regulation of α₁ integrin. Transient actin cytoskeletal disassembly was observed following treatment with the ligand over the course of a 24-h period. CTGF induced the loss of focal adhesions from the mesangial cell as evidenced by the loss of punctate vinculin. However, these processes are p42/44 MAPK and PI3K pathway-independent. Our data support the hypothesis that CTGF mediates a number of its biological effects by the induction of signaling processes via β₃ integrin. However, others such as actin cytoskeleton disassembly are modulated in a β₃integrin/MAPK/PI3K-independent manner, indicating that CTGF is a complex pleiotropic factor with the potential to amplify primary pathophysiological responses.

Connective tissue growth factor [CTGF]/CCN2 is a prototypic member of the CCN family of regulatory proteins. CTGF expression is up‐regulated in a number of fibrotic diseases, including diabetic nephropathy, where it is believed to act as a downstream mediator of TGF‐ β function; however, the exact mechanisms whereby CTGF mediates its effects remain unclear. Here, we describe the role of CTGF in cell migration and actin disassembly in human mesangial cells, a primary target in the development of renal glomerulosclerosis. The addition of CTGF to primary mesangial cells induced cell migration and cytoskeletal rearrangement but had no effect on cell proliferation. Cytoskeletal rearrangement was associated with a loss of focal adhesions, involving tyrosine dephosphorylation of focal adhesion kinase and paxillin, increased activity of the protein tyrosine phosphatase SHP‐2, with a concomitant decrease in RhoA and Rac1 activity. Conversely, Cdc42 activity was increased by CTGF. These functional responses were associated with the phosphorylation and translocation of protein kinase C‐ ζ to the leading edge of migrating cells. Inhibition of CTGF‐induced protein kinase C‐ ζ activity with a myristolated PKC‐ ζ inhibitor prevented cell migration. Moreover, transient transfection of human mesangial cells with a PKC‐ ζ kinase inactive mutant (dominant negative) expression vector also led to a decrease in CTGF‐induced migration compared with wild‐type. Furthermore, CTGF stimulated phosphorylation and activation of GSK‐3 β . These data highlight for the first time an integrated mechanism whereby CTGF regulates cell migration through facilitative actin cytoskeleton disassembly, which is mediated by dephosphorylation of focal adhesion kinase and paxillin, loss of RhoA activity, activation of Cdc42, and phosphorylation of PKC‐ ζ and GSK‐3 β . These changes indicate that the initial stages of CTGF mediated mesangial cell migration are similar to those involved in the process of cell polarization. These findings begin to shed mechanistic light on the renal diabetic milieu, where increased CTGF expression in the glomerulus contributes to cellular dysfunction.

Human induced pluripotent stem cell (hiPSC)-derived kidney organoids have prospective applications ranging from basic disease modelling to personalised medicine. However, there remains a necessity to refine the biophysical and biochemical parameters that govern kidney organoid formation. Differentiation within fully-controllable and physiologically relevant 3D growth environments will be critical to improving organoid reproducibility and maturation. Here, we matured hiPSC-derived kidney organoids within fully synthetic self-assembling peptide hydrogels (SAPHs) of variable stiffness (storage modulus, G′). The resulting organoids contained complex structures comparable to those differentiated within the animal-derived matrix, Matrigel. Single-cell RNA sequencing (scRNA-seq) was then used to compare organoids matured within SAPHs to those grown within Matrigel or at the air-liquid interface. A total of 13,179 cells were analysed, revealing 14 distinct clusters. Organoid compositional analysis revealed a larger proportion of nephron cell types within Transwell-derived organoids, while SAPH-derived organoids were enriched for stromal-associated cell populations. Notably, differentiation within a higher G’ SAPH generated podocytes with more mature gene expression profiles. Additionally, maturation within a 3D microenvironment significantly reduced the derivation of off-target cell types, which are a known limitation of current kidney organoid protocols. This work demonstrates the utility of synthetic peptide-based hydrogels with a defined stiffness, as a minimally complex microenvironment for the selected differentiation of kidney organoids.

Summary: Signalling interplay between transforming growth factor-β (TGFβ) and CCN2 [also called connective tissue growth factor (CTGF)] plays a crucial role in the progression of diabetic nephropathy and has been implicated in cellular differentiation. To investigate the potential role of microRNAs (miRNAs) in the mediation of this signalling network, we performed miRNA screening in mesangial cells treated with recombinant human CCN2. Analysis revealed a cohort of 22 miRNAs differentially expressed by twofold or more, including members of the miR-302 family. Target analysis of miRNA to 3′-untranslated regions (3′-UTRs) identified TGFβ receptor II (TβRII) as a potential miR-302 target. In mesangial cells, decreased TβRII expression was confirmed in response to CCN2 together with increased expression of miR-302d. TβRII was confirmed as an miR-302 target, and inhibition of miR-302d was sufficient to attenuate the effect of CCN2 on TβRII. Data from the European Renal cDNA Biopsy Bank revealed decreased TβRII in diabetic patients, suggesting pathophysiological significance. In a mouse model of fibrosis (UUO), miR-302d was increased, with decreased TβRII expression and aberrant signalling, suggesting relevance in chronic fibrosis. miR-302d decreased TGFβ-induced epithelial mesenchymal transition (EMT) in renal HKC8 epithelial cells and attenuated TGFβ-induced mesangial production of fibronectin and thrombospondin. In summary, we demonstrate a new mode of regulation of TGFβ by CCN2, and conclude that the miR-302 family has a role in regulating growth factor signalling pathways, with implications for nephropathic cell fate transitions.

Diabetic Kidney Disease (DKD) represents a significant global health burden and is recognised as the leading cause of end-stage renal disease. Kidney organoids derived from human induced Pluripotent Stem Cells (hiPSCs) have the potential to transform how we model renal disease and may provide personalised replacement tissues for patients with renal failure. However, kidney organoids remain poorly reproducible, and are structurally and functionally immature. Three-dimensional cultures that more appropriately mimic the complexity of the in vivo microenvironment are required to improve organoid maturation and structural authenticity. Here, we describe the application of semi-synthetic Gelatin Methacryloyl (GelMA) hydrogels as extracellular support matrices for the differentiation of hiPSC-derived kidney organoids. Hydrogels of defined mechanical strengths were generated by varying the concentration of GelMA solution in the presence of low concentration photo-initiator. After confirming a high level of mechanical stability of the hydrogels over extended culture periods, their effect on kidney organoid maturation was investigated. Organoids differentiated within GelMA hydrogels generated typical renal cell types including podocytes, tubular epithelia, renal interstitial cells, and some nascent vascularisation. Interestingly, kidney organoids derived within hydrogels that closely approximate the stiffness of the adult human kidney (∼5000-10,000 Pa) demonstrated improved podocyte maturation and were shown to upregulate renal vesicle-associated genes at an earlier stage following encapsulation when compared to organoids derived within softer hydrogels (∼400 Pa). A model of TGFβ-induced injury was also developed to investigate the influence of the mechanical environment in propagating early, fibrotic-like features of DKD within organoids. Growth within the softer matrix was shown to reduce pSMAD3 expression following TGFβ1 treatment, and accordingly ameliorate the expression of the myofibroblast marker α-Smooth Muscle Actin (α-SMA). This work demonstrates the suitability of GelMA hydrogels as mechanically-stable, highly-tuneable, batch-to-batch reproducible three-dimensional supports for hiPSC-derived kidney organoid growth and differentiation, and further substantiates the role of the biophysical environment in guiding processes of cell fate determination and organoid maturation.