Regulation of renal proximal tubular epithelial cell hyaluronan generation: implications for diabetic nephropathy.
BACKGROUND
Proximal tubular cells (PTCs) contribute to pathological changes in the renal interstitium by the generation of cytokines and alterations in the composition of the extracellular matrix. Hyaluronan (HA) is a ubiquitous connective tissue polysaccharide that regulates cell function and tissue remodeling. In the current study, we investigated the regulation of HA generation by PTCs.


METHODS
Primary cultures of human PTCs were grown to confluence and stimulated under serum-free conditions with either interleukin-1 (IL-1) or 25 mmol/L D-glucose. Alterations in HA generation were detected by enzyme-linked immunosorbent assay, and alterations in HA synthase gene expression were examined by reverse transcription-polymerase chain reaction. Subsequently, the mechanisms of IL-1 beta and glucose-induced alterations in HA were examined utilizing HK-2 cells.


RESULTS
Stimulation of human PTCs (HPTCs) with either IL-1 beta or 25 mmol/L D-glucose led to a significant increase in the HA concentration in the culture supernatant. In contrast, stimulation of HPTCs with transforming growth factor-beta1, basic fibroblast growth factor, or platelet-derived growth factor-AB did not stimulate HA production. The addition of IL-1 beta or 25 mmol/L D-glucose also increased HA generation in HK-2 cells and was associated with the induction of HAS2 mRNA. HAS3 mRNA was constitutively expressed and was not influenced by the addition of either stimulus. HAS1 mRNA expression was not detected in either unstimulated or stimulated cells. Inhibition of gene transcription or protein synthesis abolished HA production in response to either IL-1 beta or glucose. Inhibition of nuclear factor-kappa B (NF-kappa B) activation either by sulindac or by the proteosome inhibitor (PSI) abrogated both IL-1 beta and glucose-mediated alteration in HA synthesis.


CONCLUSIONS
This study demonstrates, to our knowledge for the first time, that increased HA synthesis in response to either IL-1 beta or elevated 25 mmol/L D-glucose is associated with NF-kappa B-activated transcription of HAS2.
Jones
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Institute of Nephrology, University of Wales College of Medicine, Heath Park, Cardiff, Wales, United Kingdom.
Jones
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Phillips
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D001483:Base Sequence; D002478:Cells, Cultured; D017931:DNA Primers / Q000235:genetics; D003928:Diabetic Nephropathies / Q000378:metabolism*; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D015870:Gene Expression; D005947:Glucose / Q000494:pharmacology; D014453:Glucuronosyltransferase / Q000235:genetics / Q000378:metabolism; D016695:Glycosyltransferases; D006801:Humans; D000076002:Hyaluronan Synthases; D006820:Hyaluronic Acid / Q000096:biosynthesis*; D007375:Interleukin-1 / Q000494:pharmacology; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008565:Membrane Proteins; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D013467:Sulindac / Q000494:pharmacology; D014166:Transferases; D029867:Xenopus Proteins
D017931:DNA Primers; D007375:Interleukin-1; D008565:Membrane Proteins; D016328:NF-kappa B; D012333:RNA, Messenger; D029867:Xenopus Proteins; D013467:Sulindac; D006820:Hyaluronic Acid; D014166:Transferases; D016695:Glycosyltransferases; D014453:Glucuronosyltransferase; C056944:HAS1 protein, Xenopus; C101754:HAS2 protein, human; C105458:HAS3 protein, human; C491096:Has2 protein, Xenopus; D000076002:Hyaluronan Synthases; D005947:Glucose