Advanced Glycation End Products Suppress Neuropilin-1 Expression in Podocytes by a Reduction in Sp1-Dependent Transcriptional Activity

Abstract

Background: Neuropilin-1 (NRP1) is a transmembrane glycoprotein, initially defined as a receptor for members of the semaphorin family. We observed that NRP1 expression was downregulated by the addition of advanced glycation end products-modified bovine serum albumin (AGE-BSA). The present study was undertaken to unravel the molecular mechanisms underlying AGE-BSA-mediated NRP1 suppression. Methods: Expression of NRP1 was analyzed in podocytes. The transcriptional activity of the NRP1 promoter was investigated using wild-type and mutant NRP1 promoter reporter constructs. Electrophoretic mobility assays were performed. Results: NRP1 expression was downregulated in podocytes by the addition of AGE-BSA. In contrast, phorbolester induced NRP1 mRNA and protein expression. The wild-type promoter transcriptional activity was significantly reduced when podocytes were treated with AGE-BSA compared with control, unmodified BSA. Point mutations of proximal and distal Sp1-like sites inhibited basal NRP1 promoter activity. AGE-BSA failed to further suppress transcriptional activity of these constructs. Double mutation of the Sp1A and Sp1B binding sites completely abolished NRP1 transcriptional activity. Gel shift analysis showed a specific binding of the Sp1 transcription factor. Treatment of podocytes with AGE-BSA revealed a decrease in Sp1 binding to consensus sequences, but no effect on AP1 binding. Conclusions: AGE-BSA inhibits NRP1 promoter transcriptional activity in podocytes by reducing the binding ability of the Sp1 transcription factor to attach to the NRP1 promoter.

Erythropoietin Protects Podocytes from Damage by Advanced Glycation End-Products

“…As previously reported [9,10], AGE-BSA treatment of podocytes leads to a significant decrease in NRP1 mRNA and protein expression (fig. 8a–c).…”

“…In our study in differentiated cultured mouse podocytes, erythropoietin facilitated a partial protection against AGE-BSA-mediated podocyte damage. AGE-induced upregulation of p27 Kip1 expression with concomitant cell cycle arrest and podocyte hypertrophy as well as AGE-mediated downregulation of NRP1 resulting in reduced podocyte migration have previously been identified as key events in podocyte damage associated with diabetic nephropathy [9,10]. Since AGE-BSA-mediated effects on p27 Kip1 (induction) and NRP1 expression (suppression) are different and are both counteracted by epoetin-β or CERA, we believe that these protective effects are specific and do not merely represent a general cytoprotection.…”

“…In cultured podocytes, AGE-mediated hypertrophy and cell cycle arrest are linked to a mechanism involving the cell-cycle-regulatory protein p27 Kip1 [8]. Furthermore, AGEs suppress neuropilin-1 expression (NRP1) of podocytes [9,10]. Suppression of NRP1 in podocytes has also been noted in diabetic db/db mice and biopsies from patients with diabetic nephropathy [9].…”

Type VIII Collagen Modulates TGF-β1-induced Proliferation of Mesangial Cells

“…58 1 ϫ 10 3 cells were seeded in 96-well plates; after incubation in RPMI supplemented with 0.1% FCS for 24 hours, the cells were treated with 2.5 ng/ml human recombinant TGF-␤1 (Peprotech, Hamburg, Germany) for 24 hours. Mesangial cells were then labeled with BrdU for 4 hours at 37°C, and the incorporation was determined with the assay kit following the manufacturer's instructions.…”

“…Western Lightning Chemiluminescence Reagent Plus (ECL; PerkinElmer LAS, Boston, MA) was used for detection of signals, and the images were captured using a Fuji LAS-3000 imaging system (Fujifilm Life Science, Düs-seldorf, Germany) as described previously. 58,59 Immunohistochemistry for FGF21 Expression Paraffin-embedded kidneys were sectioned at 4 m. For the detection of FGF21, a rabbit polyclonal to FGF21 (Abcam, Cambridge, UK) with a concentration of 0.30 mg/ml was used in a 1.100 dilution (this antibody reacts with human and murine FGF21). Detection was performed with the Vectastain ABC kit (Vector Laboratories, Burlingame, CA) using an anti-rabbit secondary antibody as described previously.…”