Influence of high glucose concentrations on the expression of glycosaminoglycans and N-deacetylase/N-sulphotransferase mRNA in cultured skin fibroblasts from diabetic patients with or without nephropathy.
BACKGROUND
The Steno hypothesis postulates that a genetic defect in the regulation of the production of heparan sulphate by renal and non-renal cells determines susceptibility for the development of proteinuria and macro-angiopathy in patients with diabetic nephropathy (DN).


METHODS
To test this hypothesis, skin fibroblasts isolated from type II diabetic patients with overt DN, micro-albuminuria, or without DN and from non-diabetic patients (n=8 for each group) were cultured in the presence of 5 or 25 mM D-glucose or in 25 mM L-glucose, and tested for the expression of N-deacetylase/N-sulphotransferase (NDST) 1 and 2 by semi-quantitative RT--PCR. Proteoglycan production was measured by means of metabolic labelling.


RESULTS
In each group of patients, 25 mM D-glucose significantly reduced the incorporation of [3H]glucosamine (P<0.01), but not [35S]sulphate. The quantity of NDST 1 mRNA expression did not differ between the four groups. In the non-diabetic group only, 25 mM D-glucose significantly increased NDST 1 mRNA expression (P<0.01). In contrast, NDST 2 mRNA expression was reduced by 25 mM D-glucose in all groups (P<0.01). In the diabetic patients, NDST 2 mRNA was significantly reduced compared with the non-diabetic patients. No differences were found between patients with or without nephropathy. In mesangial cells (MC), NDST expression was not influenced by glucose.


CONCLUSIONS
Since NDST 1 and 2 are not differentially expressed in patients with or without nephropathy and, in MC, the mRNA expression hereof is not influenced by glucose as in skin fibroblasts, our data do not support the Steno hypothesis.
Yard
Benito
B
Vth Medical Clinic, Klinikum Mannheim, Faculty for Clinical Medicine, Heidelberg University, Mannheim, Germany. benito.yard@med5.ma.uni-heidelberg.de
Feng
Yuxi
Y
Keller
Hanno
H
Mall
Christa
C
van Der Woude
Fokko
F
D000368:Aged; D000581:Amidohydrolases / Q000235:genetics*; D002478:Cells, Cultured; D003924:Diabetes Mellitus, Type 2 / Q000235:genetics* / Q000378:metabolism*; D003928:Diabetic Nephropathies / Q000235:genetics* / Q000378:metabolism*; D005260:Female; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D005944:Glucosamine / Q000378:metabolism; D005947:Glucose / Q000494:pharmacology*; D006025:Glycosaminoglycans / Q000096:biosynthesis*; D019812:Heparan Sulfate Proteoglycans / Q000096:biosynthesis; D006801:Humans; D008297:Male; D008875:Middle Aged; D008954:Models, Biological; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012867:Skin / Q000187:drug effects / Q000378:metabolism; D015238:Sulfotransferases / Q000235:genetics*
D006025:Glycosaminoglycans; D019812:Heparan Sulfate Proteoglycans; D012333:RNA, Messenger; C113066:NDST2 protein, human; D015238:Sulfotransferases; C021350:heparitin sulfotransferase; D000581:Amidohydrolases; D005947:Glucose; D005944:Glucosamine