Activation of Tubular Epithelial Cells in Diabetic Nephropathy

Activation of Tubular Epithelial Cells in DiabeticNephropathyMichael Morcos,

1Ahmed A.R. Sayed,

1Angelika Bierhaus,

1Benito Yard,

2Rudiger Waldherr,

3

Wolfgang Merz,4

Ingrid Kloeting,5

Erwin Schleicher,6

Stefani Mentz,1

Randa F. Abd el Baki,1

Hans Tritschler,7

Michael Kasper,8

Vedat Schwenger,1

Andreas Hamann,1

Klaus A. Dugi,1

Anne-Marie Schmidt,9

David Stern,9

Reinhard Ziegler,1

Hans U. Haering,6

Martin Andrassy,1

Fokko van der Woude,2

and Peter P. Nawroth1

Previous studies have shown that renal function in type2 diabetes correlates better with tubular changes thanwith glomerular pathology. Since advanced glycationend products (AGEs; AGE-albumin) and in particularcarboxymethyllysine (CML) are known to play a centralrole in diabetic nephropathy, we studied the activationof nuclear factor �B (NF-�B) in tubular epithelial cellsin vivo and in vitro by AGE-albumin and CML. Urinesamples from healthy control subjects (n � 50) and type2 diabetic patients (n � 100) were collected and testedfor excretion of CML and the presence of proximaltubular epithelial cells (pTECs). CML excretion wassignificantly higher in diabetic patients than in healthycontrol subjects (P < 0.0001) and correlated with thedegree of albuminuria (r � 0.7, P < 0.0001), while therewas no correlation between CML excretion and HbA1c

(r � 0.03, P � 0.76). Urine sediments from 20 of 100patients contained pTECs, evidenced by cytokeratin 18positivity, while healthy control subjects (n � 50)showed none (P < 0.0001). Activated NF-�B could bedetected in the nuclear region of excreted pTECs in 8 of20 patients with pTECs in the urine sediment (40%).Five of eight NF-�Bp65 antigen–positive cells stainedpositive for interleukin-6 (IL-6) antigen (62%), whileonly one of the NF-�B–negative cells showed IL-6 posi-tivity. pTECs in the urine sediment correlated posi-tively with albuminuria (r � 0.57, P < 0.0001) and CMLexcretion (r � 0.55, P < 0.0001). Immunohistochemis-try in diabetic rat kidneys and a human diabetic kidneyconfirmed strong expression of NF-�B in tubular cells.

To further prove an AGE/CML-induced NF-�B activa-

tion in pTECs, NF-�B activation was studied in cultured

human pTECs by electrophoretic mobility shift assays

(EMSAs) and Western blot. Stimulation of NF-�B bind-

ing activity was dose dependent and was one-half max-

imal 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-�B activation

was demonstrated in pTECs transfected with a NF-�B–

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-�Bp65

and NF-�B–dependent IL-6 expression could be inhib-

ited using the soluble form of the receptor for AGEs

(RAGE) (soluble RAGE [sRAGE]), RAGE-specific anti-

body, or the antioxidant thioctic acid. In addition tran-

scriptional activity and IL-6 release from transfected

cells could be inhibited by overexpression of the NF-�B–

specific inhibitor �B�. The findings that excreted

pTECs demonstrate activated NF-�B and IL-6 antigen

and that AGE-albumin and CML lead to a perpetuated

activation of NF-�B in vitro infer that a perpetuated

increase in proinflammtory gene products, such as IL-6,

plays a role in damaging the renal tubule. Diabetes 51:

3532–3544, 2002

There is growing evidence that tubular injury is amajor feature in the development of renal dys-function in type 2 diabetes (1–9). Tubular cellsare are not only affected secondary to glomeru-

lar injury but are also primary targets for pathologicalinfluences in diabetes (1–4,8–15). Typical glomerulopathyis present in only one-third of type 2 diabetic patients withmicroalbuminuria, while another third demonstrates nor-mal renal structure. The last one-third has no or absentglomerular changes but disproportional severe tubuloin-terstitial lesions (2,16–18). In addition, histological studiesof chronic renal diseases confirmed that renal functioncorrelates better with tubular and interstitial changes thanwith glomerular changes (2,19,20). This indicates thatrenal pathology in diabetes is only in part explained byglomerulopathy (1–4,20). It has been shown that renaltubular damage can even precede microalbuminuria in theabsence of glomerular proteinuria (11,12,21–24). This fa-vors the hypothesis that pathologic reactions leading todiabetic nephropathy may first occur in the peritubular

From the 1Department of Internal Medicine 1 and Department of Nephrology,University of Heidelberg, Heidelberg, Germany; the 2Department of Nephrolo-gie, University Hospital of Mannheim, Mannheim, Germany; the 3Gemein-schaftspraxis fur Pathologie, Heidelberg, Germany; 4Biochemiezentrum,University of Heidelberg, Germany; the 5Department of Laboratory AnimalScience, Institute of Pathophysiology, Faculty of Medicine, University Greifs-wald, Greifswald, Germany; the 6Department of Medicine, University ofTuebingen, Tuebingen, Germany; 7Asta Medica, Frankfurt, Germany; the8Department of Anatomy, Department of Pathology and Institute of FoodChemistry, Technical University Dresden, Dresden, Germany; and the 9Col-lege of Surgeons, Columbia University, New York, New York.

Address correspondence and reprint requests to Michael Morcos, MD,Department of Internal Medicine 1, University of Heidelberg, Bergheimerstr58, 69115 Heidelberg, Germany. E-mail: [email protected].

Received for publication 25 February 2002 and accepted in revised form 13September 2002.

M.M. and A.A.R.S. contributed equally to this study.AGE, advanced glycation end product; CC, contingency coefficient; CML,

carboxymethyllysine; ELISA, enzyme-linked immunosorbent assay; EMSA,electrophoretic mobility shift assay; I�B, inhibitory �B; LPS, lipopolysaccha-ride; NF-�B, nuclear factor �B; pTEC, proximal tubular epithelial cell; RAGE,receptor for AGEs; sRAGE, soluble RAGE; TA, thioctic acid.

3532 DIABETES, VOL. 51, DECEMBER 2002

microcirculation, where they induce oxidative injury(2,11,12) and subsequent tubular damage.

Tubular cells are direct targets for enhanced glucoselevels present in diabetes. Glucose uptake of tubular cellsis independent of insulin, resulting in a direct relation ofthe plasma glucose concentration to the intracellularglucose level of tubular cells (7,14,25–27). In addition,excess glucose in the glomerular filtrate leads to enhancedproximal tubular glucose reabsorption, further augment-ing the effects of hyperglycemia on intracellular glucoseefflux within the proximal tubule (28,29). On exposure toglucose, tubular cells secrete vasoactive hormones likeangiotensin II (due to the activation of the local intrarenalrenin-angiotensin system), transforming growth factor �and matrix proteins (14,30–40). Glucose-dependent meta-bolic pathways and vasoactive hormones may directlyinfluence tubular and interstitial cells, leading to renaldysfunction caused by nonglomerular mechanisms (7,9,10,14,30,31). It has recently been demonstrated that highintracellular glucose levels lead to the enhanced formationof advanced glycation end products (AGEs), in particularcarboxymethyllysine (CML)-modified proteins and thesubsequent activation of the redox-sensitive transcriptionfactor NF-�B (41).

AGEs such as CML have the potential to directly targetthe renal tubular system. The renal tubule, particularly itsproximal segment, is exposed to the glomerular effluent,containing large quantities of AGEs, in particular in diabe-tes. Furthermore, in diabetes, tubular cells are exposed toenhanced levels of circulating AGEs by the peritubularcapillary network (2,26,39,42–57). The proximal tubule is asite of reabsorption and catabolism of circulating AGEsfound in diabetes. AGEs are taken up by pTECs in thelysosomal apparatus and lead to cellular hypertrophy dueto decreased protein breakdown resulting from reducedlysosomal proteinase activities, with a concomitant stim-ulation of protein synthesis (43,49). The accumulation ofAGEs in renal tubules is reduced by treatment with aninhibitor of advanced glycation, aminoguanidine (43,57–60). Thus, formation of AGEs might play a central role inthe development of tubular dysfunction in diabetic ne-phropathy (48,57,60–63). AGEs activate intracellular sig-nal transduction systems with the consecutive generationof free oxygen radicals, leading to activation of the redox-sensitive transcription factor NF-�B and induction ofNF-�B–controlled genes such as interleukin-6 (IL-6) (54,

56,59,63,64–76). AGEs activate various intracellular sec-ond messengers, including mitogen-activated proteinkinase (33,34,54,57,59,63,67–77). Furthermore, the nitricoxide synthase activity is inhibited by early glycation endproducts as well as AGEs in rabbit tubular epithelial cellsin vitro (57,78).

The effects of AGE proteins such as CML are mediatedby binding of AGEs to various distinct cellular receptors,which can be found on different cell types. One of thesereceptors is the receptor for AGEs (RAGE) (59,63,68–76,79–94). RAGE is a 35-kDa receptor of the IgG super-family, which is expressed by a variety of cells, includingendothelial cells, tubular epithelial cells, and other celltypes (57,59,68–76,79–84,89–97). Increased RAGE expres-sion could be demonstrated in tubular cells in diabeticnephropathy (59,79,95,97).

This raises the question of whether binding of AGEs toRAGE might induce pTEC activation and tubular dysfunc-tion. This study investigates the hypothesis that in diabe-tes, AGE-albumin and/or CML-modified albumin interactwith tubular cells in a RAGE-dependent manner, thusinducing oxidative stress and subsequent activation ofNF-�B and NF-�B–controlled genes. This may lead todamage of the renal tubulus system and appearance ofNF-�B and IL-6–positive pTECs in the urine of diabeticpatients.

RESEARCH DESIGN AND METHODS

Reagents. Reagents were obtained as follows: HEPES buffer solution,L-glutamine, penicillin-streptomycin mixture, and PBS, pH 7.4, were obtainedfrom Biowhittaker (Walkerville, MD). pTEC medium and IL-6 ELISA wereobtained from Promocell (Heidelberg, Germany). FCS was from Gibco/BRL(Dreieich, Germany). [�-32P]ATP� (3,000 Ci/mmol at 10 Ci/ml), Hybond-N-Nylonfilter, ECL-nitrocellulose membranes, ECL detection reagents, and Hy-perfilm X-ray films were obtained from Amersham (Braunschweig, Germany).PMSF, thioctic acid (TA), and the Limulus assay were purchased from Sigma(Deisenhofen, Germany). Poly dI/dC was from Pharmacia (Freiburg, Germa-ny). Polyclonal anti-RAGE antibodies, generated in goat with recombinantRAGE prepared in E. coli as antigen, were a gift from Dr. M.A. Shearman(Merck, Sharpe & Dome, Essex, U.K.). Vectastain ABC kit was purchased fromVector Laboratories (Burlingame, CA). Anti-p65, -p50, -p53, -cREL, -relB, and-�� and the respective second antibodies were obtained from Santa Cruz(Heidelberg, Germany). Monoclonal anti-p65 antibodies specific for activatedNF-�Bp65 and Fugen 6 transfection reagent were obtained from Roche(Mannheim, Germany). Soluble RAGE (sRAGE) preparations used throughoutthis study have previously been described in detail (70,71,83,92,96) and weregenerously provided by Drs. Schmidt and Stern (Columbia University, NewYork). The kit for the determination of CML antigen was kindly provided byRosemarie Kientsch-Engels (Roche AG, Penzberg, Germany).

TABLE 1Patient characteristics of 50 healthy control subjects, 50 type 2 diabetic patients without nephropathy (defined as normal albuminexcretion �20 mg/l), 50 type 2 diabetic patients with overt nephropathy (defined as macroalbuminuria �200 mg/l), and 50 patientswithout diabetes but with macroalbuminuria �200 mg/l.

Healthy controlsubjects

Type 2 diabetes,no nephropathy

Type 2 diabetes,overt nephropathy

Nondiabetickidney disease

n 50 50 50 50HbA1c (%) 5.1 � 0.5 7.4 � 1.1 9 � 1.9 5.7 � 0.3Diabetes duration (years) — 8.3 � 4.3 14.2 � 8.2 —Creatinine (mg/dl) 0.9 � 0.2 1 � 0.2 1.25 � 0.6 2 � 2.1Osmolality (mOsmol/kg) 922 � 1,050 964 � 656 940 � 533 340 � 279Albuminuria (mg/l) 10.2 � 4.7 15 � 3.3 439 � 431 1,000 � 640CML (�g/ml) 0.1 � 0.3 0.6 � 0.5 2.3 � 1.99 0.6 � 1.1Cytokeratin 18 positive (n) 0 0 20 0

Data are means � SD. CML, CML excretion in the urine.

M. MORCOS AND ASSOCIATES

DIABETES, VOL. 51, DECEMBER 2002 3533

Animal experiments. Kidneys obtained from diabetic BB/O(ttawa)K(arls-burg) rats were used as diabetes model. This animal model is described indetail by Kloting et al. (98). Diabetes was present for 57 � 9 days at an age of104 � 16 days. Rats were treated with a continuous infusion of insulin at 2units/24 h, and the blood glucose level was kept at �20 mmol/l. Kidneys fromnormal Sprague-Dawley rats served as a control. Paraffin-embedded kryostat

sections from rat kidneys were prepared from normal and diabetic animalsaccording to a standard protocol.Immunohistochemistry. Immunohistochemistry was performed on paraffin-embedded tissues of a human kidney (obtained from kidney biopsy) and ratkidneys by indirect immunoperoxidase technique. Detection of signals wasperformed with the Vectastain ABC kit (Vector Laboratories) according to the

FIG. 1. NF-�B in rat and human kidney. Paraffin-embedded tissue specimens of rat kidneys and a human diabetic kidney were stained with apolyclonal antibody against NF-�Bp50 and -p65 antigen (indicated as brown color). Control animals showed no NF-�Bp50 (A) and no NF-�Bp65antigen (C), while in diabetic rats, NF-�Bp50 (B) and NF-�Bp65 antigen (D) were detectable in tubular cells and mainly present in theperinuclear region. Human diabetic kidney expressed NF-�Bp50 (E) and NF-�Bp65 antigen (F).

NF-�B ACTIVATION IN RENAL TUBULAR CELLS


manufacturer’s instructions as described (68–70,75,99–101). Peroxidase ac-tivity was visualized with 0.05% 3,3-diaminobenzidine-tetrahydrochloride(Serva, Heidelberg, Germany) before the sections were counterstained withMayer’s hematoxylin. Controls for immunstaining were included in all stain-ings by omission of the primary antibody and its replacement by PBS andmatching concentrations of normal rabbit serum (data not shown). Inaddition, blocking peptides were included in some of the reactions to confirmspecificity (data not shown).Patients. For the investigations, each patient gave informed consent and thestudy was approved by the ethical committee of the Department of Medicine,University of Heidelberg, and performed in accordance with the Declarationof Helsinki.

Urine samples from healthy control subjects (n 50) and type 2 diabeticpatients (n 100) were collected from morning spot urine and immediatelystored at 20°C. Information about the clinical and laboratory data from thepatients is given in Table 1.

Patients with nondiabetic kidney disease were suffering from glomerulo-nephritis, systemic lupus erythematodes, or vasculitis.Quantification of CML formation by enzyme-linked immunosorbent

assay. Quantification of urinary CML was performed using a commerciallyavailable kit as previously described (102).Immunocytochemistry. Immunocytochemistry staining was performed asdescribed in detail elsewhere (70,79,87). In brief, fresh urine samples wereobtained from healthy control subjects (n 50) and type 2 diabetic patients(n 100). After direct centrifugation, the supernatant was decanted and theconcentrated cellular material was deposited on glass slides by cytocentrifu-gation (Shandon Cytospin). Tubular epithelial cells were identified on May-Grunwald/Giemsa (Pappenheim) staining and by positive reactions withantibodies against cytokeratin 18 and neutral endopeptidase. In a second step,cells were stained with a monoclonal mouse antibody against NF-�Bp65antigen (Roche) and a FITC-labeled monoclonal mouse antibody against IL-6antigen using a standard protocol. The NF-�B antibody recognizes activatedNF-�Bp65 (103). Staining was performed using anti–NF-�Bp65 and anti–IL-6 ata concentration of 0.01 �g/ml for 60 min at room temperature.Cell culture. Human renal pTECs were obtained from adult kidneys aftersurgery from the unaffected parts of kidneys obtained from tumornephrec-

tomy or kidney biopsies, as described (104–106). Proximal tubular epithelialcells (pTECs) were characterized by staining with FITC-labeled cytokeratin 18antibodies and an antibody against neutral endpeptidase as described inimmunocytochemistry. For electrophoretic mobility shift assay (EMSA) anal-ysis, Western blot, and enzyme-linked immunosorbent assay (ELISA), pTECsfrom the same passage were used (fourth to fifth passage). Before stimulatingpTECs with AGE-albumin or CML, cells were cultivated without growthfactors for 5 days. Where indicated, cells were preincubated with sRAGE (asoluble and truncated form of the receptor RAGE) (70,71,75,83,92,93,96) in athreefold molar excess (1.5 �mol/l) compared with AGE-albumin or CML, aRAGE-specific antibody (20 ng/�l), or TA (200 �mol/l).Preparation and characterization of AGE-albumin and CML. AGE-albumin was prepared as previously described (70,96,107). The extent oflysine modifications in the AGE preparations varied up to 36%. In vitrosynthesis of CML-albumin was performed as previously described by Schlei-cher and colleagues (52,96). Assays for endotoxin showed AGE-albumin andCML preparations to contain virtually undetectable levels of lipopolysaccha-ride (LPS) (�10 pg/ml at a protein concentration of 5 mg/ml according to theLimulus assay [Sigma]).EMSAs. After stimulation of pTECs with AGE-albumin and CML with theconcentrations and time points indicated in the figure legends (Figs. 3, 4, and6), nuclear proteins from pTECs were harvested as described elsewhere(67,70,96,99,100) and assayed for transcription factor binding activity usingthe NF-�Bp65 consensus sequence: 5�-AGTTGAGGGGACTTTCCCAGGC-3�.Specificity of binding was ascertained by competition with a 160-fold molarexcess of unlabeled consensus oligonucleotides and supershift experiments.For supershift experiments, nuclear extracts were preincubated with antibod-ies against NF-�Bp65, -p50, -p52, -cRel, and -relB prior to stimulation asdescribed elsewhere (50,51,56,67,69,70,96,99,100,108–111). All experimentswere performed at least three times.Immunoblot (Western blot) analysis. Cytoplasmic and nuclear fractionswere prepared as previously described in detail (69,70,99,100). Western blotwas performed as described. Membranes were incubated with primaryantibodies directed against NF-�B65 and -��. After washing, the secondaryantibody (horse radish peroxidase–coupled rabbit IgG) was added andincubation was continued for 30 min. Immunoreactive proteins were detectedwith the ECL-Western blot System (Amersham Pharmacia, Braunschweig,Germany) and subsequent autoradiography for 2 min. All experiments wereperformed three times.Plasmids. The simian virus 40–driven luciferase control plasmid pGL2-control, the promoterless plasmid “pGL2-basic,” and the �-galactosidasecontrol plasmid “pSV-Gal” were obtained from Promega (Heidelberg, Germa-ny). The plasmid NF-�B-Luc, which contains four tandem copies of the NF-�Bconsensus sequence fused to a TATA-like promoter region from the Herpessimplex virus thymidine kinase promoter, was purchased from Clontech(Heidelberg, Germany). The I�� expression plasmid was kindly provided byDr. Baeuerle (Tularic Inc.).Transient transfection experiments. For transfection experiments, pTECsgrowing in the logarithmic phase were transfected as described (67,70,93,96,99,100,112) using Fugen 6 transfection reagent (Roche) according to themanufacturer’s instructions. Before cells were stimulated with AGE-albuminor CML (concentrations and time points are indicated in the figure legends),medium was changed and cells were kept without growth factors and FCS.Cotransfection was performed as previously described (69,70,96,100) with a�B�-overexpressing plasmid. After 42 h, cells were washed with 37°C warmNaCl 0.9% for two times and harvested as described elsewhere (69,70,96). Forinhibition experiments, TA (200 �mol/l), sRAGE (1,500 nmol/l), or anti-RAGE(20 ng/�l) was combined with AGE-albumin or CML (500 nmol/l). The ratio ofluciferase activity to �-galactosidase activity served to normalize luciferaseactivity (112). Each experiment was performed in triplicates, and experimentswere repeated at least three times.Determination of IL-6 antigen. The supernatant from NF-�Bp65 or inhibi-tory �B (I�B)�-transfected and AGE- or CML-stimulated cells was harvestedand IL-6 antigen determined by ELISA. The ELISA for determination of IL-6was performed using a commercially available kit (Promocell) according tothe instructions of the manufacturer. The experiment was performed intriplicates and repeated at least three times.Statistical analysis. All values are given as mean, with the bars showing SDs.For statistical analysis, Student’s t test, Fisher’s test, Mann-Whitney U test,Pearson correlation, �2 median test, and determination of contingency co-efficient (CC) were performed. P � 0.05 was considered statistically significant.

RESULTS

NF-�B activation in the kidney. Paraffin-embedded tis-sues from diabetic BB/O(ttawa)K(arlsburg) (98) rat kid-

FIG. 2. NF-�Bp65 and IL-6 antigen in urinary excreted tubular epithe-lial cells. A and B: Cells were obtained from the urine of a type 2diabetic patient. Detection of pTECs was performed using cytokeratin18 antigen (A) and neutral endopeptidase (B) as markers. The figuresshow a marked positivity in scattered cells for cytokeratin 18 antigen(A) or neutral endopeptidase (B). C: Detection of NF-�Bp65 antigen inpTECs using an antibody recognizing activated NF-Bp65. D: Singlecells were stained with an antibody recognizing IL-6-antigen. A– D:Magnification 550�.



neys were prepared to demonstrate NF-�Bp65 and -p50antigen. Diabetes was present for 57 � 9 days and mani-fested at an age of 104 � 16 days. Normal Sprague-Dawleyrats served as a control group. In diabetic animals, NF-�Bp50 and -p65 antigen was present in the nuclear regionof tubular cells (Fig. 1B and D), while in control animals,no NF-�Bp50 and -p65 antigen could be detected in tubularcells (Fig. 1A and C). Interestingly, in diabetic kidneys,NF-�Bp50 and -p65 was mainly present in tubular cells andnot in glomerular cells. To confirm these data, kidneyspecimens derived from a kidney biopsy of a patient withdiabetic nephropathy were stained with the same antibod-ies. Tubular epithelial cells showed a marked staining forNF-�Bp65 and -p50 antigen (Fig. 1E and F). Again, NF-�Bp65 and -p50 were mainly present in the renal tubularsystem.Excretion and activation of tubular epithelial cells in

diabetic patients. Urine was collected from 50 healthycontrol subjects and 100 patients with type 2 diabetes (50patients with normal albumin excretion [�20 mg/l] and 50with macroalbuminuria [�200 mg/l]). Mean serum creati-nine and urea had been normal. In addition, urine from 50nondiabetic patients with macroalbuminuria [�200 mg/l]was collected. Patient characteristics are shown in Table1. Urinary excretion of CML-modified proteins was deter-mined by ELISA. Diabetic patients showed significantlyhigher urinary CML levels than healthy control subjects(P � 0.0001) or nondiabetic patients with macroalbumin-

uria �200 mg/l (P � 0.0001). These results are in contrastto a recently published study that showed a decreasedurinary excretion of CML in diabetic patients with im-paired renal function (102). This is most probably due toan increase in CML excretion, while renal function is notseverely impaired. With decreasing renal function, CMLcan be excreted only to a lesser extent. When the diabeticpatients were further analyzed, a positive correlationbetween albuminuria and excretion of CML antigen (r 0.7, P � 0.0001) was found, while there was no correlationbetween CML excretion and HbA1c (r 0.03, P 0.76).

The presence of pTECs in the urine was evidenced usingantibodies against cytokeratin 18 and neutral endopepti-dase. Single or scattered pTECs (Fig. 2A and B) werefound in the urine of 20 of 100 type 2 diabetic patients butin none of the healthy control subjects (P � 0.0001).Positive staining for activated NF-�Bp65 antigen wasrecognized in 8 of 20 (40%) patients, in some but not allexcreted pTECs (Fig. 2C). Furthermore, to indirectly as-sess the transcriptional consequences of NF-�Bp65 activa-tion, slides were incubated with an antibody against IL-6.In five of eight patients positive for NF-kBp65, IL-6 antigencould be detected (62%) in single cells (Fig. 2D), whereasin the preparations negative for NF-�Bp65, only one ex-pressed the IL-6 antigen (1/12).

pTEC positivity in the urine sediment showed a strongcorrelation to the degree of albuminuria (�2 median test 25, CC 0.63) and CML excretion (�2 median test 12.5,

FIG. 3. NF-�B binding activity in pTECs in vitro: EMSA. pTECs wereeither left untreated (Cont) or stimulated with AGE-albumin or CMLas described in RESEARCH DESIGN AND METHODs. Nuclear extracts wereprepared (see RESEARCH DESIGN AND METHODs) and studied for the pres-ence of NF-�Bp65. The position of NF-�B is indicated by an arrow (65kDa). The results shown are representative of at least three indepen-dent experiments. One representative experiment is shown. Doseresponse: cells were incubated for 6 h with the indicated concentra-tions of AGE-albumin (A) or CML (B). Time course: cells wereincubated for the indicated periods with 500 nmol/l AGE-albumin (C)or CML (D). Nonglycated human albumin did not induce NF-�Bp65 (E).



FIG. 4. Characterization of NF-�B bindingactivity by “supershift” analysis. To furthercharacterize the NF-�B subunits, contribut-ing to the observed “shift,” cells were stim-ulated with 500 nmol/l AGE-albumin for 3 h,nuclear extracts were prepared, and 2.5 �gof antibodies against NF-�Bp50, -p52, -p65,-cRel, or -RelB antigen were included in bind-ing reaction. The position of NF-�B and the“supershift” is indicated by arrows. �, anti-body; Cons, 160-fold excess of unlabelledconsensus oligonucleotide; Control, un-stimulated pTECs; H, heat-inactivated AGE-albumin.

FIG. 5. AGE- and CML-dependent NF-�B activation in vitro: Western blot. pTECs were stimulated with AGE-albumin and CML, and cytoplasmicand nuclear extracts were obtained as described in RESEARCH DESIGN AND METHODS. For analysis, a monoclonal antibody against NF-�Bp65 and I�B�antigen was used. The results shown are representative of at least three independent experiments. One representative experiment is shown. Thelocalization of NF-�Bp65 antigen in the nucleus and cytoplasm (Cytopl.) and I�B in the cytoplasm is indicated by arrows. Dose dependence:stimulation for 6 h with various concentrations of AGE-albumin (A) or CML (B). Time dependence: stimulation with 500 nmol/l AGE-albumin (C)or CML (D) over a period of 30 min to 5 days. Cont, unstimulated cells.



CC 0.46 (113), leading to the hypothesis that CML-modified proteins may induce NF-�Bp65 positivity inpTECs.NF-�B activation in cultured pTECs. In vitro experi-ments were performed using cultivated human pTECs toprove that tubular cells indeed have the ability to activateNF-�B in response to increased AGEs. pTECs were stim-ulated with either AGE-albumin or CML as described inRESEARCH DESIGN AND METHODS. When cultured pTECs wereincubated over 6 h with either AGE-albumin (Fig. 3A) orCML (Fig. 3B), a dose-dependent activation of NF-�B wasobserved in EMSA (Fig. 3A and B). NF-�B binding activitywas half maximal at 250 nmol/l AGE-albumin and 500nmol/l CML. Normal nonglycated human albumin did notinduce NF-�Bp65 (Fig. 3E). Heat inactivation of AGE-albumin over 12 h at 100°C abolished inducible NF-�Bbinding activity (data not shown).

AGE-albumin and CML induced NF-�B binding activityin a time-dependent manner (Fig. 3C and D). An early startof NF-�B activation was observed already at 30 min,reaching a first maximum after 6 h (Fig. 3C, lane 4). Aftera decrease between 12 and 24 h (Fig. 3C, lanes 5 and 6), asecond peak could be observed after 4 days (Fig. 3C, lane

7). This time course is similar to the data obtained in aprevious study (70). Stimulation of pTECs with CMLrevealed similar data compared with stimulation withAGE-albumin (Fig. 3D).

Supershift analysis (Fig. 4) revealed that NF-�Bp65 and-p50 (lanes 4 and 6) constituted the major protein contrib-uting to the shift observed, while NF-�Bp52, -cRel, and-RelB did not participate in the binding reaction. NF-�Bbinding activity was suppressed using a sixfold excess ofunlabeled oligonucleotides (lane 9). Heat-inactivated AGEdid not activate NF-�B in cultured pTECs (lane 1).

Western blot analysis corresponded well to the bindingactivity demonstrated in EMSA analysis. After AGE-albu-min or CML stimulation, NF-�Bp65 antigen translocationwas dose dependent (Fig. 5A and B). The decrease in

cytoplasmic p65 antigen occurred after 60 min, simulta-neously with the increase in nuclear p65 antigen (Fig. 5C

and D). Correspondingly, I�B� degradation was also doseand time dependent (Fig. 5A–D). A reconstitution ofcytoplasmic NF-�Bp65 antigen was observed at 3–4 days(lanes 8 and 9), a time point of maximal NF-�Bp65 binding

FIG. 6. Inhibition of NF-�Bp65 binding activity TA and inhibition of ligand-RAGE interaction: EMSA analysis. pTECs were either left untreatedor stimulated for 6 h with 500 nmol/l AGE-albumin (AGE) (A and C) or CML (B and D) in the presence of 200 �mol/l TA (A and B), sRAGE (1,500nmol/l), or RAGE antibody (20 ng/�l) (C and D). The data shown are representative of at least three independent experiments. Onerepresentative experiment is shown. The position of NF-�Bp65 is indicated by arrows. NF-�Bp65 activation induced by AGE-albumin (A and C,lane 2) or CML (B and D, lane 2) is inhibited by TA (A and B, lane 3), sRAGE (C and D, lane 3), or RAGE-specific antibody (C and D, lane 4).

FIG. 7. Inhibition of NF-�Bp65 nuclear translocation by TA andinhibition of ligand-RAGE interaction. Western blot. Before stimula-tion over 6 h with 500 nmol/l AGE-albumin, pTECs were incubated withTA (200 �mol/l), sRAGE (1,500 nmol/l), or RAGE-antibody (20 ng/�l).After 6 h, nuclear and cytoplasmic extracts were obtained for immuno-blot as described in RESEARCH DESIGN AND METHODS. The data shown arerepresentative of at least three independent experiments. The positionof NF-�Bp65 and I�B� is indicated by arrows. NF-�B activation byAGE-albumin (A and B, lane 2) could be inhibited by TA (A, lane 3),sRAGE (B, lane 3), or RAGE antibody (B, lane 4).



activity (Fig. 3) and nuclear translocation (Fig. 5C and D).A previous report demonstrated that long-lasting NF-�Bp65 activation is associated with increased NF-kB syn-thesis, overriding I�B�, explaining simultaneous nuclearand cytoplasmic NF-�Bp65 antigen (70). Consistently, astrong loss of �� antigen was observed after a 12-hstimulation, but not after 4 days (Fig. 5C and D), presum-ably because NF-�Bp65 drives the de novo synthesis ofI�B� (70).

As shown by EMSA (Fig. 3), NF-�Bp65 activation startedafter 30 min, reaching a first maximum after 6 h and asecond maximum at 4 days after CML stimulation and thusresembled the activation pattern observed for AGE-albu-min. In Western blot analysis, however, translocation ofNF-�Bp65 from the cytoplasm into the nucleus was al-ready observed 30 min after AGE stimulation, but not 6 hafter CML stimulation, which might be due to a lesserstimulatory effect of CML compared with AGE-albumin.Since EMSAs are much more sensitive than Western blots,it is reasonable to assume that a weaker CML-dependentNF-�B induction can be monitored in EMSA but is notevident in Western blot analysis.

All effects could be reduced using the antioxidant TA.The reduction could be observed in EMSA analysis andWestern blot (Fig. 6A and B and Fig. 7A). To investigate

whether the AGE-albumin– and CML-induced NF-�Bp65activation is RAGE dependent, coincubation with sRAGEand a specific RAGE-antibody was performed. sRAGE andRAGE antibody decreased NF-�Bp65 activation in EMSA(Fig. 6C and D) and Western blot (Fig. 7B).Transcriptional activity of NF-�B. Transient transfec-tion of cultured pTECs, using a NF-�B consensus-drivenluciferase reporter plasmid, was performed to demon-strate that increased NF-�B binding activity (Fig. 3) andnuclear translocation (Fig. 5) is functionally significantand results in increased NF-�B–dependent gene expres-sion. A dose-dependent activation could be demonstratedwhen pTECs were stimulated with AGE-albumin or CML(Fig. 8A and B), corresponding well to the data concerningNF-�B activation in EMSA analysis (Fig. 3) and Westernblot (Fig. 5). When cells were stimulated for 6 h with 500nmol/l AGE-albumin or CML, respectively, an 8- (CML) to10-fold increase (AGE) in luciferase activity was observed(Fig. 8A–D). This was not the case when cells werestimulated with control albumin. Overexpression of ��and treatment with the antioxidant TA reduced luciferaseactivity (Fig. 8C and D). Furthermore, NF-�B activation isRAGE dependent, since AGE-albumin–induced bindingactivity was markedly inhibited by addition of excesssRAGE and RAGE antibody, as described in RESEARCH

FIG. 8. Transcriptional activity of NF-�Bp65. pTECs were transiently transfected with the reporter plasmid NF-�B-Luc (see RESEARCH DESIGN AND

METHODS). After transfection, cells were left unstimulated, stimulated with control albumin, or stimulated with various concentrations ofAGE-albumin (A) or CML (B) for 6 h. Where indicated, cells were cotransfected with a �B�-overexpressing plasmid (C and D, lane 4) or treatedwith 200 �mol/l TA (C and D, lane 5), 1,500 nmol/l sRAGE (C and D, lane 6), or 20 ng/�l RAGE antibody (C and D, lane 7). After harvest, luciferaseactivity was determined in the cell lysates. Values are expressed as relative luc units. Three independent experiments were performed withidentical results. One representative experiment is shown.



DESIGN AND METHODS (Fig. 8C and D). Due to experimentallimitations of transient transfection experiments, the timeof stimulation could not be exceeded for �42 h.

AGE-albumin- and CML-mediated pTEC activation re-sulted not only in increased expression of luciferase butalso in de novo synthesis of IL-6 antigen (studied as amodel of a NF-�B–driven gene). When the supernatants ofthe transfected cells (transfection data are shown in Fig. 8)were analyzed for IL-6 antigen, a dose-dependent increasewas observed (Fig. 9A and B). IL-6 antigen release wasreduced by overexpression of I�B or addition of theantioxidant TA. Furthermore, the IL-6 antigen induction isRAGE dependent, since sRAGE and RAGE antibody re-duced IL-6 release (Fig. 9C and D).

DISCUSSION

Human pTECs are not only passive bystanders in thedevelopment of diabetic nephropathy, but they also re-spond actively to hyperglycemia and AGEs by inducingNF-�B activation and NF-�B–dependent gene expressionin vitro and in vivo. One defined AGE generated bylipoxidation and glycoxidation in diabetic nephropathy isCML (44,52,61,70,114). The presence of CML-modifiedproteins in the urine of type 2 diabetic patients and the in

vitro observation that CML is a potent inducer of sustainedNF-�B activation in pTECs suggest that CML might play arole in the development of diabetes renal complications. Inaddition, the observation that type 2 diabetic patientsdemonstrated excretion of tubular cells that was positivefor activated NF-�Bp65 and IL-6 antigen implies that theAGE/CML-RAGE–mediated NF-�B activation is function-ally significant.

Indirect evidence for the role of NF-�B activation indiabetic nephropathy has already been given from clinicalstudies in which an increase in oxidative stress correlatedwith renal function and NF-�B activation in patients withtype 2 diabetes (99,100,115–118). We found that increasedCML excretion in type 2 diabetes correlates to the excre-tion of NF-�Bp65 and IL-6 antigen–positive pTECs.

As demonstrated here, pTECs respond to exogenouslyadded AGE-albumin and CML with an NF-�B activationthat meets the requirements of RAGE-dependent NF-�Bactivation, as evidenced by NF-�B binding activity thatlasted �4 days and NF-�B–dependent gene expression(69,70). In vivo, pTECs are exposed not only to AGEspresent in the urine but also to glucose leading directly tointracellular AGE formation (41). An additional source ofintracellular CML formation is the inflammatory reaction,

FIG. 9. Transactivation capacity of NF-�B. Determination of IL-6 antigen in the cell supernatant after stimulation of transfected pTECs for 6 hwith various concentrations of AGE-albumin (A) or CML (B). AGE-albumin–induced (C, lane 3) or CML-induced increase (D, lane 3) in IL-6antigen could be reversed by overexpression of I�B� (C and D, lane 4), TA (C and D, lane 5), sRAGE (C and D, lane 6), and anti-RAGE (C andD, lane 7). Three independent experiments were performed with identical results. One representative experiment is shown.



as we have shown that pTECs in diabetic patients are inpart positive for IL-6. Cytokines and inflammatory agents,as they occur in time remodeling, are associated withintracellular CML formation and activation of NF-�Bp65. Itremains unknown whether pTECs are subject to extra-cellular- and/or intracellular-mediated CML responses indiabetes. Until now, only cell responses to extracellularAGEs, for example via RAGE, have been reported. Theinvolvement of RAGE in AGE-dependent pTEC activationwas confirmed by competition of NF-�B activation bysRAGE and an RAGE blocking antibody. This in agree-ment with recent data showing reduction of vascularhyperpermeability by scavenging RAGE ligands (75,91,92,119,120) and increased diabetic nephropathy in diabeticanimals overexpressing RAGE (93). Furthermore, there isevidence that RAGE-ligand interaction contributes to sus-tained NF-�Bp65 activation (41,68–70,83,121,122). Thelong-lasting nature of RAGE-dependent NF-�Bp65 activa-tion corresponds well to the p65 and p50 positivity oftubular cells in the tissue sections shown in Fig. 1. Onewould not expect all cells to be positive, even in serialsections, if NF-�B activation in humans would be as shortlasting as in tissue culture after tumor necrosis factorstimulation. Thus, in diabetes, autoregulatory negativefeedback loops are shut down. As shown previously, onemechanism is excessive de novo synthesis of p65, over-riding I�B� inhibition (70,123–125). Since many stimuliresult in NF-�B activation, one has to assume that mech-anisms for cell- and disease-specific activation must exist.

It has been demonstrated that different stimuli in differ-ent renal diseases lead to disease-specific activation ofcertain NF-�B subunits. Using LPS as stimulus, or otherrenal models like rats with ureteric obstruction or immunecomplex nephritis, different activation patterns could beobserved (50,51,55,56,108–111,126). This suggests that dif-ferences in the NF-�B activation pattern in response to agiven stimulus might determine the selection of the genesactivated. Therefore, we investigated which activationpattern is present in human pTECs due to AGE-albuminstimulation. We and others show two specific DNA-proteincomplexes (50,51,56,111). Supershift analysis revealed thatthe slower-migrating complex is composed of NF-�Bp65and -p50. In accordance with other studies, the faster-migrating band could not be depleted could by NF-�Bp65,-p50, -p52, -cREL, or -relB antibodies but could by unla-beled oligonucleotide. To define whether the complex is dueto the binding of a coactivator protein such as CBP/p300(111), however, is beyond the scope of this study.

The observation that excreted pTECs demonstrate bothNF-�B and IL-6 antigen activation led us to speculate thata perpetuated increase in proinflammtory gene productssuch as IL-6, depending on perpetuated NF-�B activation(as demonstrated in vitro), might be central in damagingthe renal tubule. This view is emphasized by the fact thatnot only cytokines, but also metalloproteinases such asMMP9, are controlled by NF-�B. MMP-9 has been impli-cated to contribute to proteinuria in Heymann nephritisand therefore might be a good candidate for the destruc-tion of pTECs in the course of diabetic nephropathy (127).

Further studies are needed to define which mechanismsare activated by perpetuated NF-�B activation and finally

result in the destruction of the tubulus, as evidenced bythe excretion of pTECs in overt diabetic nephropathy.

ACKNOWLEDGMENTS

This work was supported by grants from the University ofHeidelberg (to M.M. and A.B.) and the University ofTubingen (to A.B. and P.P.N.), the IZKF program of theUniversity of Tubingen (to P.P.N.), and Asta Medica (toA.B. and P.P.N.). P.P.N. performed part of this work duringthe tenure of a Schilling professorship. A.B., B.Y., E.S.,H.U.H., F.v.W., and P.P.N. were supported by the DeutscheForschungsgemeinschaft. A.-M.S. and D.S. were supportedby grants from the USPHS, the American Heart Associa-tion (New York affiliate), and the Juvenile Diabetes Foun-dation. A.A.R.S. is supported by a grant from the ArabRepublic of Egypt.

We thank Dr. M.A. Shearman (Merck, Sharpe & Dome)for providing anti-RAGE antibodies, Dr. Baeuerle (TularicInc.) for providing I�� expression plasmid, and Dr.Kientsch-Engels (Roche AG) for providing the CML-ELISAkit. Part of this report was presented at the meeting of theGerman Diabetes Society 2001 (Aachen, Germany).

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Activation of Tubular Epithelial Cells in Diabetic Nephropathy

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