S4(W)
DOWN REGULATION OF THIAMINE TRANSPORTER EXPRESSION IN HUMAN
TUBULAR EPITHELIAL CELLS BY HIGH GLUCOSE CONCENTRATION IN VITRO
AND INCREASED THIAMINE CLEARANCE IN DIABETES
Larkin, J, Rabbani, N, Zehnder D, Thornalley, P
Warwick Medical School, Clinical Sciences Research Institute, University of Warwick,
University Hospital, Coventry
BACKGROUND: Increased renal clearance of thiamine (vitamin B1) is common in diabetic
patients linked to decreased renal reuptake of thiamine and is a risk predictor of decline in renal
function. Two recent clinical studies evaluating thiamine supplements to correct thiamine loss in
patients with type 2 diabetes and microalbuminuria showed decreased urinary albumin excretion
and reversal of microalbuminuria. The aim of our investigation is to elucidate the mechanism of
decreased renal reuptake of thiamine in diabetes. Thiamine is actively scavenged from
glomerular filtrate by two thiamine transporter proteins, THTR-1 and THTR-2, encoded by
genes SLC19A2 and SLC19A3. The location of these transporters in human kidneys and the
effect of high glucose concentration on transporter expression in the human tubular epithelial
HK-2 cell line in vitro and human tubular epithelial cells in primary culture were investigated.
METHODS: Renal location of THTR-1 and THTR-2 was investigated by
immunohistochemical staining of paraffin-embedded human kidneys. HK-2 cells and freshly
extracted human proximal tubular epithelial cells were grown in primary culture in medium
containing low and high glucose concentrations (5 or 26 mmol/L, respectively) with 4 nmol/L
thiamine and the expression of SLC19A2 and SLC19A3 investigated.
RESULTS: Immunohistochemical staining of human kidneys showed particularly intense
staining of THTR-1 and THTR-2 in the proximal tubule.
When human proximal tubular epithelial cells were incubated in high glucose concentration,
SLC19A2 and SLC19A3 mRNA was decreased (−76% and −56% respectively; p<0.001).
Thiamine transporter protein levels were also decreased in high glucose concentration (THTR-1
−77%; THTR-2 −83%; both p<0.05). Concomitantly, forward: reverse apparent permeability of
monolayers of proximal tubule epithelial cells to [3H]thiamine was decreased 23% in high
glucose concentration (p<0.001).
A comparison of the primary cultures of cells to the HK-2 proximal tubule cell line revealed
marked differences between the two models. THTR-2 protein was undetectable by western blot
or immunohistochemistry in HK-2 cells and SLC19A3 mRNA was 120-fold less abundant in
HK-2 cells (p<0.001). HK-2 cells also had a lower content of thiamine metabolites (31.3 ± 4.1
versus 57.9 ± 8.9 pmol/mg protein respectively; p< 0.01). This thiamine metabolite pool was all
held in the form of thiamine pyrophosphate in the primary cultures but the HK-2 cells held
significant percentages as thiamine monophosphate (18%) and free thiamine (6%).
CONCUSIONS: We conclude that the proximal tubule is the likely major site in the kidney of
reuptake of thiamine from glomerular filtrate. The decreased renal reuptake of thiamine in
diabetes is likely due to hyperglycaemia-induced decreased expression of thiamine transporters
in the renal tubular epithelium. The validity of the HK-2 cell line as a model for the proximal
tubule when studying thiamine metabolism is in doubt because of marked differences in
thiamine transporter expression and thiamine metabolites compared to human tubular epithelial
cells in primary culture.
This research was funded by Diabetes UK.