Protein uptake disturbs collagen homeostasis in proximal tubule-derived cells.
BACKGROUND
Interstitial fibrosis is of major importance for the deterioration of renal function, leading to uremia. Interaction of filtered proteins with proximal tubular cells is important for the onset and development of tubulointerstitial damage.


METHODS
We investigated the effects of protein endocytosis on collagen homeostasis and signaling pathways of proximal tubule-derived cells (OK cells, LLC-PK1 cells), which express the endocytic machinery typical for the proximal tubule (megalin and cubilin), and compared it to renal epithelial cells with low endocytic activity (MDCK, IHKE1, NHE3-deficient OK cells). Collagen homeostasis was assessed by proline incorporation, ELISA, and Western blot. Matrix metalloproteinase (MMP) activity was assessed by gelatinase assay. Signaling pathways were monitored by reporter gene assay.


RESULTS
Albumin, glycated albumin, fatty acid-free albumin, or globulins led to an increase of secreted collagen (types I, III, and IV) in OK and LLC-PK1 cells. In cells with low protein uptake activity, albumin exposure inhibited collagen secretion. Western blot analysis showed an increase of cellular collagen. MMP activity was significantly decreased by albumin exposure. Furthermore, albumin exposure led to activation of the NF-kappa B-, AP1-, NFAT-, SRE-, and CRE-pathways. Inhibition of NF-kappa B, PKC, or PKA partially reversed the effects of albumin. In addition, inhibition of albumin endocytosis reduced collagen secretion and activation of the signaling pathways. Discussion. The data show that endocytic uptake of proteins disturbs collagen homeostasis in proximal tubular cells. This disturbed matrix homeostasis probably supports the progression of interstitial fibrosis, which is of importance for the development of renal insufficiency.
Wohlfarth
Verena
V
Physiologisches Institut, Universität Würzburg, Würzburg, Germany.
Drumm
Karina
K
Mildenberger
Sigrid
S
Freudinger
Ruth
R
Gekle
Michael
M
D000418:Albumins / Q000493:pharmacokinetics*; D000818:Animals; D015153:Blotting, Western; D003094:Collagen / Q000032:analysis / Q000378:metabolism*; D017364:Collagenases; D004797:Enzyme-Linked Immunosorbent Assay; D018093:Gelatinases / Q000378:metabolism; D017930:Genes, Reporter; D006706:Homeostasis / Q000502:physiology; D007687:Kidney Tubules, Proximal / Q000166:cytology* / Q000378:metabolism*; D018374:LLC-PK1 Cells; D009893:Opossums; D011392:Proline / Q000493:pharmacokinetics; D015398:Signal Transduction / Q000502:physiology; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics; D013552:Swine
D000418:Albumins; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers; D003094:Collagen; D011392:Proline; D017364:Collagenases; D018093:Gelatinases