Impaired transcription factor interplay in addition to advanced glycation end products suppress podocalyxin expression in high glucose-treated human podocytes

Abstract

Podocalyxin represents a Wilms' tumor suppressor protein (WT1)-regulated differentiation marker for glomerular epithelium. We provide evidence concerning mechanisms involved in the regulation of podocalyxin expression following long-term exposure to increased (25 mM) glucose levels. Prolonged culture of conditionally immortalized human podocytes in 25 mM glucose induced suppression of podocalyxin expression both at the protein and mRNA levels, whereas WT1 protein levels remained unaltered. WT1 interacted with another transcription factor, CRE-binding protein (CBP). This association was decreased by 40% in the presence of 25 mM glucose. Chromatin immunoprecipitation assays on chromatin from podocytes cultured in 25 mM glucose revealed reduced WT1 binding to podocalyxin promoter sequences, probably resulting from impaired WT1-CBP interactions. We explored the possible role of glucose-induced adducts (advanced glycation end products; AGEs) in impairing interactions between WT1 and CBP, with the use of aminoguanindine, an inhibitor of AGE formation. Podocytes were cultured in the simultaneous presence of 20 mM aminoguanidine and 25 mM glucose, and podocalyxin protein levels were examined. Aminoguanidine effectively prevented downregulation of podocalyxin protein levels but could not restore podocalyxin levels once expression was suppressed. Thus increased glucose apparently impaired the ability of WT1 to initiate transcription in part by decreased association of WT1 with CBP. Administration of aminoguanidine concomitant with increasing glucose levels in our in vitro model system protected from glucose-induced "silencing" of the podocalyxin gene, suggesting that AGEs play an important role in suppressing its expression in diabetic conditions.

Vitamin D Receptor Activators Upregulate and Rescue Podocalyxin Expression in High Glucose-Treated Human Podocytes

“…Nuclear protein extracts were obtained as described [7]. Cells were scraped off and solubilized in buffer A (20 m M HEPES, pH 7.9, 10 m M KCl, 1 m M EDTA, 1 m M DTT, 1 m M PMSF, 0.1 m M NaO 3 VO 4 , 0.2% Nonidet P-40, 10% glycerol, and protease inhibitors).…”

“…Luminal podocytic membrane possesses negative surface charge maintained mainly by podocalyxin (PC), the latter regulating podocyte morphology, as well as foot process formation and maintenance [4,5]. Glucose induces PC suppression in glomeruli of streptozotocin- diabetic rats in vivo [6], and in human glomerular epithelial cells (HGEC) in vitro [5,7]. Suppressed PC expression, in HGEC, could not be restored by reverting glucose concentration to normal levels for either short or longer time intervals [7].…”

“…Glucose induces PC suppression in glomeruli of streptozotocin- diabetic rats in vivo [6], and in human glomerular epithelial cells (HGEC) in vitro [5,7]. Suppressed PC expression, in HGEC, could not be restored by reverting glucose concentration to normal levels for either short or longer time intervals [7]. …”

“…Nephrin reduction in HGEC is glucose-induced [7] and may be related to side effects of glycated albumin/AGEs [9]. …”

“…The phenotype of HGEC fully agrees with parental podocytes, since they express visceral glomerular epithelial cell markers such as PC, CALLA, cytokeratin, α3β1 integrin, α1 and α5 chains of collagen IV and WT1. Moreover, HGEC have already been used for studies of expression and function [5,7]. …”