S8(T)
EPITHELIAL-MESENCHYMAL CROSSTALK IN THE REGULATION OF RENAL
PROXIMAL TUBULAR EPITHELIAL CELL FUNCTION
Luo, D, Steadman, R, Phillips, A, Meran, S
Institute of Nephrology, University Hospital of Wales, Cardiff
BACKGROUND: The function of epithelial/specialised cells in many tissues is regulated by
adjacent stroma. In cancer biology, during malignant transformation, epithelial cells undergo
changes similar to the process of EMT in renal fibrosis. Recent reports suggest the important
role of stromal fibroblasts in the initiation, as well as the progression of carcinomas. This
specialised “carcinoma-associated stroma” is reported to have an abundance of
myofibroblasts that express factors that support the survival, proliferation and malignant
transformation of epithelial cells in a paracrine fashion. Analogous to tumorigenesis,
fibrogenesis is associated with epithelial cell transdifferentiation and an abundance of
myofibroblasts. However, the importance of epithelial-stromal crosstalk within the kidney
and the potential role they play in progressive cortico-interstitial fibrosis has received little
attention.
AIM: To test the hypothesis that fibroblasts once transformed, or fibroblasts with the ability
to undergo myofibroblastic transformation, differentially regulate proximal tubular cell
phenotype when compared to fibroblasts that are resistant to this transformation.
METHODS: We utilised an established library of phenotypically heterogenous fibroblasts
which have been characterised with respect to their scarring (SF) (dermal) and non-scarring
(NSF) (oral mucosal fibroblasts) potentials. We have set up a 3D co-culture system using type
I collagen from rats tail to study the influence of these distinct fibroblast phenotypes on HK2
phenotype. The fibroblasts were grown within the gel, with the HK2 cells grown as
monolayers on the surface of the gel. Transwell inserts were used to separate the two cell
cultures for analysis.
RESULTS: Fibroblasts grown within gels generated increased levels of TGF-1 compared to
fibroblasts grown in 2D-culture. This increased TGF-1 resulted in increased alpha-smooth
muscle actin expression, suggesting that fibroblasts adopt a myofibroblast phenotype within
gels. SF phenotypes grown within gels also demonstrated more alpha-SMA expression
compared to NSF phenotypes. Work was then directed towards the effect of co-culture of SF
and NSF phenotypes on HK2 phenotype. Co-culture of HK2 cells with fibroblasts enhanced
their E-cadherin expression as compared to HK2 culture alone. Furthermore, co-culture of
HK2 with SF enhanced E-cadherin expression more than co-culture with NSF’s. Co-culture
of HK2 cells with fibroblasts also enhanced HK2 proliferation, and SF phenotypes appeared
to enhance this more than NSF’s. Differences in EDA-fibronectin and Hyaluronan Synthase-2
expression were also highlighted, with increased levels of both present in HK2/SF co-culture
as compared with HK2/NSF co-culture and HK2 monoculture. Differences in IL-1 and EGF
production were also identified, with HK2/fibroblast co-cultures (specifically HK2/SF)
demonstrating increased levels. Use of the ALK5 inhibitor did not abrogate the changes in Ecadherin seen in HK2/fibroblast co-cultures. However, increased levels of miR192 and
reduced ZEB-2 mRNA expression were identified in SF/HK2 cultures that may account for
the differences in E-Cadherin expression seen.
CONCLUSIONS: We have identified some interesting differences in HK2 phenotype,
proliferation, cytokine production and matrix generation when these cells are co-cultured with
fibroblasts, and when they are co-cultured with fibroblasts with scarring versus non-scarring
phenotypes. We have also identified changes in miR192, HAS2 and EGF Receptor expression
that may account for the changes in phenotype and proliferation seen.