Functional expression of TRPV4 in human collecting duct (HCD) cells: a role in diabetic nephropathy

Squires, Paul E. and Hills, Claire E. and Ronco, Pierre M. and Bland, Rosemary (2008) Functional expression of TRPV4 in human collecting duct (HCD) cells: a role in diabetic nephropathy. In: 43rd Annual Meeting of the European-Association-for-the-Study-of-Diabetes, Sep 18-21, 2007, Amsterdam, Netherlands.

Full content URL: http://dx.doi.org/10.1007/s00125-008-1117-6

Full text not available from this repository.

Item Type:Conference or Workshop contribution (Paper)
Item Status:Live Archive

Abstract

Background and aims: According to the World Health Organisation (WHO
Fact sheet 312, Sept 2006) diabetes is among the leading causes of kidney failure
with a10-20% mortality. When hyperglycaemia spills over into the urine
(glucosuria), loss of glucose causes osmotic drag and diuresis and exposes
the kidney to various physical and nutrient stresses. In collecting duct epithelium,
sodium absorption is linked to cell volume regulation and depends
upon detection of these mechanical stimuli. Prolonged exposure of renal
epithelial to osmotic changes and increased fluid flow has led to a number
of suggestions concerning the mechanisms involved in stimulus-response
coupling. One mechanism involves the calcium permeable transient receptor
potential-vanilloid-4 (TRPV4) channel. Gated by changes in osmolarity or
membrane stretch, these mechano-sensitive channels reputedly regulate cell
volume via a Ca2+ dependent mechanism. In the present study, mechanical
stimulation was employed as a surrogate form of osmotic stress to elucidate
a role for TRPV4 in cell volume regulation in a novel model system of the
human collecting duct.
Materials and methods: RT-PCR and western blot analysis were used to
determine mRNA and protein expression for TRPV4 in a human collecting
duct (HCD) cell line. Fura-2-microfluorimetry was used to assess generation
of touch evoked calcium transients. Knockdown of TRPV4 expression was
achieved using a TRPV4 specific siRNA and the siLentGene U6 Casette RNA
Interferance System different siRNA protocols.
Results: Bands corresponding to TRPV4 were identified from HCD mRNA
and protein. Mechanical stimulation of a single HCD cell evoked a transient
increase in cytosolic calcium ([Ca2+]i) that propagated between neighbouring
cells within a cluster (5 separate experiments). Touch evoked changes in
[Ca2+]i were still observed under Ca2+-free conditions, however the basal to
peak amplitude of the response was only 35% of that obtained in the presence
of extracellular calcium (P<0.05 n=6). Pre-incubation with the Ca2+-ATPase
inhibitor thapsigargin (1μM) was used to assess contribution of intracellular
Ca2+-stores on touch-evoked changes in [Ca2+]i. Whilst removal of extracellular
calcium alone, failed to negate the generation of touch evoked Ca2+
transients, the chelation of extracellular calcium in conjunction with the depletion
of Ca2+-stores completely prevented touch-evoked changes in
[Ca2+]i(P<0.05; n=6). Confirmation of a role for TRPV4 in the generation of touch
evoked Ca2+-transients was assessed by siRNA knockdown. Transient transfection
of HCD cells with TRPV4 siRNA reduced TRPV4 protein expression
by approximately 40% (n=4; P<0.01 compared to control), whilst mechanical
stimulation of individual RFP-tagged anti-TRPV4 cells failed to significantly
elevate [Ca2+]i, compared to control cells observed in the same field of view
under identical culture conditions.
Conclusion: These data confirm a functional role for TRPV4 in generation
of touch evoked Ca2+-signals between coupled cells of the human collecting
duct and suggest that knockdown of TRPV4 expression inhibit the cells ability
to both detect and respond to osmotic signals via a Ca2+-dependent mechanism.
These data have implications for altered fluid and electrolyte handling
in the diabetic kidney.

Additional Information:Diabetologia (2008) 51:[Suppl1]S1–S588
Keywords:Diabetes, bmjdoi
Subjects:Library of Congress Subject Areas > Q Science > QP Physiology
Library of Congress Subject Areas > R Medicine > RC Internal medicine
Divisions:College of Science > School of Life Sciences
ID Code:14073
Deposited On:20 Apr 2011 15:17

Repository Staff Only: item control page