Down regulation of thiamine transporter expression in human tubular epithelial cells by high glucose concentration in vitro and increased thiamine clearance in diabetes
James R Larkin, Naila Rabbani, Daniel Zehnder and Paul J Thornalley
Poster at British Renal Society Conference, Birmingham, UK (2011)
Background and Aims: 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%).
Conclusions: We conclude that the proximal tubule is the likely major site in the kidney of reuptake of thiamine from the 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.