title: Activation of tubular epithelial cells in diabetic nephropathy.
abstract: Previous studies have shown that renal function in type 2 diabetes correlates better with tubular changes than with glomerular pathology. Since advanced glycation end products (AGEs; AGE-albumin) and in particular carboxymethyllysine (CML) are known to play a central role in diabetic nephropathy, we studied the activation of nuclear factor kappaB (NF-kappaB) in tubular epithelial cells in vivo and in vitro by AGE-albumin and CML. Urine samples from healthy control subjects (n = 50) and type 2 diabetic patients (n = 100) were collected and tested for excretion of CML and the presence of proximal tubular epithelial cells (pTECs). CML excretion was significantly higher in diabetic patients than in healthy control subjects (P < 0.0001) and correlated with the degree of albuminuria (r = 0.7, P < 0.0001), while there was no correlation between CML excretion and HbA(1c) (r = 0.03, P = 0.76). Urine sediments from 20 of 100 patients contained pTECs, evidenced by cytokeratin 18 positivity, while healthy control subjects (n = 50) showed none (P < 0.0001). Activated NF-kappaB could be detected in the nuclear region of excreted pTECs in 8 of 20 patients with pTECs in the urine sediment (40%). Five of eight NF-kappaBp65 antigen-positive cells stained positive for interleukin-6 (IL-6) antigen (62%), while only one of the NF-kappaB-negative cells showed IL-6 positivity. pTECs in the urine sediment correlated positively with albuminuria (r = 0.57, P < 0.0001) and CML excretion (r = 0.55, P < 0.0001). Immunohistochemistry in diabetic rat kidneys and a human diabetic kidney confirmed strong expression of NF-kappaB in tubular cells. To further prove an AGE/CML-induced NF-kappaB activation in pTECs, NF-kappaB activation was studied in cultured human pTECs by electrophoretic mobility shift assays (EMSAs) and Western blot. Stimulation of NF-kappaB binding activity was dose dependent and was one-half maximal at 250 nmol/l AGE-albumin or CML and time dependent at a maximum of activation after 4 days. Functional relevance of the observed NF-kappaB activation was demonstrated in pTECs transfected with a NF-kappaB-driven luciferase reporter plasmid and was associated with an increased release of IL-6 into the supernatant. The AGE- and CML-dependent activation of NF-kappaBp65 and NF-kappaB-dependent IL-6 expression could be inhibited using the soluble form of the receptor for AGEs (RAGE) (soluble RAGE [sRAGE]), RAGE-specific antibody, or the antioxidant thioctic acid. In addition transcriptional activity and IL-6 release from transfected cells could be inhibited by overexpression of the NF-kappaB-specific inhibitor kappaBalpha. The findings that excreted pTECs demonstrate activated NF-kappaB and IL-6 antigen and that AGE-albumin and CML lead to a perpetuated activation of NF-kappaB in vitro infer that a perpetuated increase in proinflammtory gene products, such as IL-6, plays a role in damaging the renal tubule.
authors.affiliation: Department of Internal Medicine 1, University of Heidelberg, Bergheimerstrasse 58, 69115 Heidelberg, Germany. michael_morcos@med.uni-heidelberg.de
authors.forename: Michael
authors.initials: M
authors.lastname: Morcos
authors.forename: Ahmed A R
authors.initials: AA
authors.lastname: Sayed
authors.forename: Angelika
authors.initials: A
authors.lastname: Bierhaus
authors.forename: Benito
authors.initials: B
authors.lastname: Yard
authors.forename: Rüdiger
authors.initials: R
authors.lastname: Waldherr
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Merz
authors.forename: Ingrid
authors.initials: I
authors.lastname: Kloeting
authors.forename: Erwin
authors.initials: E
authors.lastname: Schleicher
authors.forename: Stefani
authors.initials: S
authors.lastname: Mentz
authors.forename: Randa F
authors.initials: RF
authors.lastname: Abd el Baki
authors.forename: Hans
authors.initials: H
authors.lastname: Tritschler
authors.forename: Michael
authors.initials: M
authors.lastname: Kasper
authors.forename: Vedat
authors.initials: V
authors.lastname: Schwenger
authors.forename: Andreas
authors.initials: A
authors.lastname: Hamann
authors.forename: Klaus A
authors.initials: KA
authors.lastname: Dugi
authors.forename: Anne-Marie
authors.initials: AM
authors.lastname: Schmidt
authors.forename: David
authors.initials: D
authors.lastname: Stern
authors.forename: Reinhard
authors.initials: R
authors.lastname: Ziegler
authors.forename: Hans U
authors.initials: HU
authors.lastname: Haering
authors.forename: Martin
authors.initials: M
authors.lastname: Andrassy
authors.forename: Fokko
authors.initials: F
authors.lastname: van der Woude
authors.forename: Peter P
authors.initials: PP
authors.lastname: Nawroth
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D003924:Diabetes Mellitus, Type 2 / Q000652:urine; D003928:Diabetic Nephropathies / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology* / Q000652:urine; D004848:Epithelium / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology; D017127:Glycation End Products, Advanced / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000378:metabolism; D007684:Kidney Tubules / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology*; D008239:Lysine / Q000031:analogs & derivatives* / Q000378:metabolism; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011910:Rats, Brattleboro; D017207:Rats, Sprague-Dawley; D000067759:Receptor for Advanced Glycation End Products; D011971:Receptors, Immunologic / Q000378:metabolism; D012016:Reference Values; D012709:Serum Albumin / Q000378:metabolism; D014158:Transcription, Genetic; D014556:Urine / Q000166:cytology
chemical_list: D017127:Glycation End Products, Advanced; D016328:NF-kappa B; D000067759:Receptor for Advanced Glycation End Products; D011971:Receptors, Immunologic; D012709:Serum Albumin; C048496:N(6)-carboxymethyllysine; D008239:Lysine