R. Brunkhorst et al., ADVANCED GLYCOSYLATED ENDPRODUCTS (AGES) IN NONDIABETIC PATIENTS UNDERGOING DIALYSIS, Clinical nephrology, 46(4), 1996, pp. 265-266
Renal failure is associated with the accumulation of advanced glycosyl
ated endproducts as a consequence of an impaired renal clearance. We c
onfirmed the finding that in sera of ESRD patients the AGE-beta(2)-mic
roglobulin fraction is significantly elevated in contrast to healthy c
ontrols but also to diabetic patients without dialysis. Furthermore, w
e investigated the question, as to whether the continuously high gluco
se concentration in the abdominal cavity of CAPD patients leads to a l
ocal production of AGE. We found increased levels of AGE in the dialys
ate of CAPD patients after long dwell times as compared to the serum c
oncentrations. In immunohistochemical studies we proved the presence o
f high amounts of AGE in the mesothelial and submesothelial tissue of
patients on perennial CAPD. In in vitro experiments mesothelial cells,
cultured on glyosylated collagen, showed a disturbed, net-like growth
and an impaired matrix production. Further studies demonstrated that
the disturbed matrix production was paralleled by an impaired producti
on of the metalloproteinase gelatinase and its natural inhibitors TIMP
-1 and TIMP-2. These alterations may (at least in part) be responsible
for the long-term damage of the peritoneal membrane observed in CAPD
patients. The extent of the non-enzymatic and irreversible glycosylati
on of proteins (AGE synthesis) depends on the time and intensity of gl
ucose exposure. II is well established that AGE accumulate with age [C
erami et al. 1987] and in diabetic patients. AGE are supposed to cause
tissue damage, like e.g. arteriosclerosis by their stimulation of gro
wth factors and their influence on collagen synthesis [Vlassara et al.
1992]. In 1991 Makita described elevated serum AGE levels in patients
undergoing dialysis and found a significant correlation between serum
creatinine and AGE levels [Makita et al. 1991]. Hemodialysis lowered
the AGE levels only transiently (for approximately three hours) even w
hen high flux dialysis filters were applied. Normalization of renal fu
nction by successful kidney transplantation, however, leads to a rapid
fall of circulating AGE levels [Makita et al. 1994]. We measured seru
m AGE concentrations with a competitive ELISA (polyclonal antibody aga
inst AGE-bovine serum albumin) in four groups of patients: 1) Patients
undergoing dialysis (HD and PD, n=7) with diabetes mellitus, 2) Patie
nts undergoing dialysis (HD and PD, n=32) without diabetes mellitus, 3
) Non-uremic diabetic patients (n=24), 4) Healthy people serving as a
control (n=7). In order to determine glycosylated proteins of medium s
ize molecular weight, especially the fraction of the AGE beta(2)-micro
globulin, the fractions from 5,000 up to 20,000 kDa were purified by f
iltration and in addition, a beta(2)-microglobulin-affinity chromatogr
aphy was carried out. It became evident, that the AGE-beta(2)-microglo
bulin fraction was significantly elevated only in patients undergoing
dialysis (groups 1 and 2) in contrast to diabetic patients without dia
lysis and the healthy control group (groups 3 and 4). These results co
nfirm the findings of Miyata et al. and other investigators [Dolhofer-
Bliesener et al. 1995] and lead to the hypothesis that accumulation of
glycosylated beta(2)-microglobulin might be involved in the pathogene
sis of dialysis-associated amyloidosis by cytokine stimulation. This w
as further supported by Miyata et al. who found AGE beta(2)-microglobu
lin in the amyloid of patients on dialysis [1994].In a further clinica
l investigation we were interested in a special problem in patients tr
eated with peritoneal dialysis: During CAPD there is a continuously hi
gh glucose concentration in the abdominal cavity which might cause an
AGE formation in the peritoneal cavity or the peritoneal membrane, res
pectively AGE levels were measured in the serum and the dialysate (aft
er 2 and 12 hours of incubation of the dialysate) of 15 non-diabetic P
D patients. Tn two of these patients we prepared samples of the omentu
m for immunohistochemical staining, gained during abdominal surgery. T
he samples were screened for AGE products by incubating them with an A
GE antibody and application of the avidin-biotin complex method. The r
esults are means of two measurements in each of the 15 patients and ar
e given in U per mg of total protein (1U equals 50% reduction of the A
GE antibody binding in the ELISA). The serum showed a mean of 0.26+/-0
.12 U, the dialysate 0.04+/-0.01 U after 2 hours of incubation and 0.4
6+/-0.21 U after 10 hours. The difference between serum and dialysate
levels (after 10 hours of incubation) was significant (p <0.02, Studen
t's t-test). Immunhistochemical staining of the samples of the omentum
showed AGE accumulation in the mesothelial and submesothelial interst
itium of the peritoneal membrane. Our results prove an accumulation of
AGE in the dialysate and the peritoneal membrane of CAPD patients. Si
nce intraperitoneal AGE concentration (after long dwells) exceed serum
AGE concentration, we postulate an increased AGE synthesis in the mes
othelial and submesothelial tissue during CAPD and a ''washing out eff
ect'' after long incubation periods. These high concentrations of loca
l AGE could possibly cause damage to the peritoneal membrane, especial
ly in long-term treatment. Further experiments were carried out to inv
estigate possible influences of local AGE-accumulation on growth rate
and matrix synthesis of peritoneal mesothelial cells. Human mesothelia
l cells were grown on culture wells coated with glycosylated collagen
(AGE collagen). Mesothelial cells risen on wells coated only with coll
agen (without AGE) served as control. After 20 hours of culture signif
icant differences in cell growth rate and matrix synthesis were observ
ed under the microscope: Mesothelial cells grown on collagen showed th
eir typical polygonal structure (''cobblestone appearance''), whereas
cells grown on AGE collagen lost this structure and showed a longitudi
nal, net-like growth. Similar influences of AGE on cell growth were fo
und in cultures of synovial cells [Miyata 1994]. To get an idea of the
reasons for this pathological growth rate and matrix synthesis, we me
asured the gelatinase activity of the supernatants in the culture well
s with the help of a zymogram assay. In addition we determined gelatin
ase m-RNA with the PCR method. We found, that cells grown on AGE colla
gen produced gelatinase, whereas cells cultivated on collagen without
AGE did not. We got similar results for the gelatinase inhib