SP39
Mechanism dependant markers of human tubular damage in vitro.
K-Cadherin, NGAL and KIM1 detect different types of tubular injury
S Yates, R Davies, MEC Dockrell
SWT Institute for Renal Research, St Helier Hospital, Wrythe Lane, Carshalton, London,
SM5 1AA.
Introduction: K-cadherin is a proximal tubule cell specific junctional protein found in man but
not murine species. In previous studies we have shown a significant loss of k-cadherin in
proximal tubule cells with treatment of TGFβ and subsequently reported its loss in diabetic
nephropathy. It is not clear whether the loss of k-cadherin is specific or simply relates to
nephrotoxicity. Some renal biomarkers have been used in the assessment of different
nephrotoxic agents. In this study we aimed to compare k-cadherin loss with other markers and
investigate the value of a panel of renal biomarkers which could be used in vitro to assess the
nephrotoxic effects of medical agents, environmental pollutants and profibrotic agents such as
aristolochic acid.
Methods: Primary human proximal tubule epithelial cells were cultured at 37°C in
supplemented media with 0.25% fcs. At 90-95% confluence cells were treated with: Cisplatin
(10, 30, 100 µM), Cadmium ( 1, 3, 10 µM), LPS (30, 100, 300 ng/ml) and aristolochic acid (10,
30, 100uM) or vehicle for 24 hours. Supernatants and lysate were collected and used to measure
the levels of KIM1& NGAL expression, N-acetyl-β-D-glucosaminidase (NAG) & Lactate
Dehydrogenase (LDH) activity using plate based assays and full length K-cadherin expression
using western blot analysis.
Results: LDH, which is an indicator of cell death, was only raised in cells treated with Cadmium
at a concentration of 10uM. None of the other treatments showed any indication of overt cell
death. Cells treated with aristolochic acid showed a marked dose dependant loss of K-cadherin
after 24 hours. Cisplatin showed a less marked dose dependant effect on K-cadherin and also
show an increase in KIM1 at all doses. Cadmium and LPS had no significant effect on Kcadherin expression. Aristolochic acid and Cadmium did not have a dose dependant increase in
the other markers tested, but LPS did increase NGAL levels compared to vehicle.
Conclusion: The work here along with our previous work with TGF β supports the hypothesis
that the loss of K-Cadherin from primary human tubule epithelial cells appears to be associated
with fibrotic insults. The fact that cadmium did not induce loss of K-Cadherin was unexpected
as it has been demonstrated extensively that Cadmium can induce cadherin loss acutely. To our
knowledge there has been nor prior investigation on K-Cadherin, and in this study we did not
investigate N-Cadherin expression, another member of the cadherin family known to be
expressed in the proximal tubule. Furthermore, as we saw no effect of cadmium in this study it
may be that cytotoxicity in human renal cells may require higher concentrations than murine
cells. The fact that LPS selectively regulated NGAL expression and cisplatin induced Kim-1
demonstrate that single biomarkers are inadequate for broad screens of nephrotoxic agents and a
panel taking into consideration multiple mechanism of cell damage is needed