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Isolation of Modified Ubiquitin as a Neutrophil Chemotaxis
Inhibitor from Uremic Patients
GERALD COHEN, MICHAEL RUDNICKI, and WALTER H. HORLDivision of Nephrologv. Departnieizt of Medicine, Univei�s-itv of Vienna, Vienna, Austria.
Abstract. Uremic toxins are factors that accumulate in the
serum and penitoneal cavity of unemic patients. They are re-
sponsible for a variety of functional disturbances and also
contribute to the increased risk of infection by interfering with
essential functions of the unspecific immune response. From
the peritoneal effluent of penitoneal dialysis (PD) patients, a
pcptide was isolated by applying three different chromato-
graphic methods. This peptide inhibits the chemotactic move-
ruent of pobyruorphonuclear leukocytes (PMNL) in an in vitro
assay in a concentration-dependent, nonreversible manner, and
therefore belongs to the group of urcruic toxins. Amino acid
sequencing showed that the isolated peptide has the same
amino terminal sequence as ubiquitin. The peptide also reacted
with anti-ubiquitin antibodies in a Western blot experiment,
but had a more acidic isoebectnic point than ubiquitin. By using
affinity chromatography, anti-ubiquitin antibody binding frac-
tions were isolated from all PD and hemodialysis (HD) patients
investigated. These fractions contained the same acidic band
and also significantly inhibited PMNL chemotaxis. Ubiquitin
per se had no effect on PMNL cheruotaxis. Therefore, it is
concluded that from PD and HD patients a modified form of
ubiquitin was isolated, and this modification was responsible
for its inhibitory effect. (J Am Soc Nephrol 9: 451-456, 1998)
One of the major factors causing the high incidence of mor-
bidity and mortality among uremic patients undergoing hemo-
dialysis (HD) and penitoneal dialysis (PD) treatment is the
increased risk of bacterial infections (1-3). Between 15 and
20% of all cases of mortality among hemodialysis patients are
due to infection (summarized in reference 4). An impairment
of the unspecific immune defense system, especially of poly-
ruorphonuclear leukocytes (PMNL), is primarily responsible
for this situation (5). PMNL are cells of the first-line immune
defense and are also the main immune cells in the penitoneal
cavity during bacterial infections (6).
A variety of factors contribute to the diminished functions of
PMNL found in uremia. Toxic substances that directly inter-
fere with PMNL activities accumulate in the serum of uremic
patients and are partly responsible for their susceptibility to
bacterial infections. A number of urcmic toxins have already
been purified and characterized. In our laboratory, a gnanubo-
cyte inhibiting protein with homology to Ig bight chains has
been isolated (7). We could show that free Ig light chains per
se are able to interfere with essential PMNL functions (8). A
granubocyte inhibiting protein with homology to p2-micro-
globulin was also isolated from hemodialysis patients (9) and
from patients undergoing continuous ambulatory penitoneab
dialysis (CAPD) treatment ( 10). Angiogenin was purified from
uremic patients as a PMNL degranulation-inhibiting protein
( 1 1 ), and complement factor D was shown to adversely affect
Received February 10, 1997. Accepted August 1 1, 1997.Correspondence to Dr. Gerald Cohen. Medizinische Universit#{228}tsklinik III.
Wahringer GUrtel 18-20. A-l090 Vienna, Austria.
l()46-6673/0903-045 I $03.()0/0
Journal of the American Society of Nephrology
Copyright 0 1998 by the American Society of Nephrology
PMNL functions (12,13). Furthermore, p-cresol was identified
as a uremic solute that impairs the respiratory burst activity of
PMNL (14).
In the present study, we describe the isolation of a modified
version of ubiquitin from the penitoneal effluent of PD patients
and from the ultrafiltrate of high-flux HD patients. We show
that the purified protein significantly inhibits PMNL chemo-
taxis, whereas unmodified ubiquitin has no effect on this
essential PMNL function.
Materials and Methodsisolation of the Chemotaxis-inhibiting Protein
Patients. The chemotaxis-inhibiting protein (CIP) was originally
isolated from the effluent of a patient (patient I ) undergoing CAPD,
using three different chromatographic methods. The patient was
treated with 2 L of PD solution four times daily. CIP was also isolated
by affinity chromatography from the same patient undergoing nightly
intermittent PD (NIPD) treatment and from two other CAPD patients
(patients 2 and 3) treated in the same way as patient I . From theultrafiltrate of three HD patients (patients 4 to 6), CIP was isolated as
well. Informed consent was obtained from all patients. Care was taken
that the patients did not have any clinical signs or symptoms of
infection interfering with our study. The serum C-reactive protein
concentration was lower than the detection limit (0.5 rug/dI). The PD
effluents were without any laboratory findings responsible for perito-
neal infections.
isolation of CIP by Three Chromatographic Methods
High molecular weight proteins were removed from the CAPD
effluent by using a polysulfone membrane with a cutoff of approxi-
mately 60 kD (F60; Fresenius, Oberursel, Germany). Then, the CAPD
effluent was concentrated about 30-fold, and the NIPD effluent was
concentrated about 100-fold with Cuprophan membranes, with a
cutoff of approximately 5 kD (Hemoflow E3 or E4, Fresenius).
Afterward, the concentrate was chromatographed on a fast-protein
452 Journal of the American Society of Nephrology
liquid chromatographic (FPLC) system (Pharmacia LKB Biotechnol-
ogy, Uppsala, Sweden), and the protein was detected with a variable
wavelength monitor (VWM 2141, Pharmacia) at wavelengths of 225
and 214 nm.Ion Exchange Chromatography. In a first step, the concentrate
was applied to FPLC, using an ion exchange Mono Q HR 10/10
column equilibrated with 20 mM Tnis-HC1, pH 8.0. The flow rate was4 ml/min. The biological activity (see below) was found in the
unbound material and was concentrated by lyophilization. resus-pended in water, and dialyzed against 200 ruM sodium phosphate
buffer, pH 7.2, using a Spectra/Por molecular-porous membrane(Spectrum, Houston, TX) with a molecular cutoff of approximately 1
kD. The bound material that was inactive in our in vitro assay was
eluted with 1 M NaCI in 20 mM Tris-HC1, pH 8.0. to regenerate the
column for next use.
Size Exclusion Chromatography. The sample was then chro-
matographed on FPLC with a Superdex 75 prep grad column using200 mM sodium phosphate buffer, pH 7.2, and a flow rate of 1ml/min. The protein peak containing the biological activity was in the
molecular weight range of 8 to 10 kD, was concentrated by lyophi-
lization, dissolved in water, and dialyzed against water.
Reversed-Phase Chromatography. The material was furtherchromatographed on FPLC with a Resource 1 -ml reversed-phase
chromatography column (Pharmacia), using a gradient from 0 to
100% acetonitnile with 0.05% (vol/vol) trifluoroacetic acid (Aldrich
Chemical Co., Milwaukee, WI) with a corresponding gradient of
water from 100 to 0%. The flow rate was 4 mI/mm. Peak fractions
have been extensively dialyzed against phosphate-buffered saline
(PBS; pH 7.2). The fraction that had been eluted at 30% acetonitrile
showed the highest biological activity and was used for further in-vestigations.
Isolation of C/P by Immunoaffinitv Chromatography
Concentrates of the PD effluents were prepared as described above.
After identification by amino acid sequencing of CIP as being homol-ogous to ubiquitin, concentrates of the PD effluents that were prepared
as described above and ultrafiltrates obtained from HD patients were
passed through an immunoaffinity column specific for ubiquitin. For
the preparation of this column, anti-ubiquitin antiserum (U-5379,
Sigma) was coupled onto a N-hydroxysuccinimide-activated HiTrap
affinity column (Pharmacia), according to the manufacturer’s instruc-
tions. After washing the column with PBS, the bound material was
eluted by lowering the pH (0.2 M Glycine-HCI, pH 2.8). The fractionswere neutralized immediately after elution by a 1/5 vol of I M
Tris-HC1, pH 8.0. The protein-containing fractions were identified by
the bicinchoninic acid protein assay (Pierce, Rockford, IL). They werepooled, dialyzed against PBS diluted with water 1 to 40, lyophilized,
and then taken up in water in a 40th of the original volume so that the
sample was finally dissolved in I X PBS.
Characterization of C/P
Molecular Characterization. Sodium dodecyl sulfate-polyac-
rylamide gel clectrophoresis (SDS-PAGE). 8 to 25% gels, or high-density gels and isoelectric focusing (p1 range, 3 to 9) was performed
using the Phast System (Pharmacia). For the reducing SDS-PAGE,
10% (vol/vol) beta-mercaptoethanol was added to the sample buffer,
and the sample was boiled for 3 ruin before loading. The reactivity ofCIP with an anti-ubiquitin antibody was tested by Western blotting.
After electrotransfer of the proteins from the SDS polyacrybamide gelsto a nitrocellulose membrane, the transferred proteins were detectedwith an anti-ubiquitin antiserum (U-5379, Sigma), horseradish perox-
idase-labeled goat-anti-rabbit antibodies, and the enhanced chemilu-
minescence detection system (Amersharu International, Little Chab-
font, Buckinghamsire, United Kingdom). The amino acid sequence
was determined by automatic Edruan degradation (model 476A; Ap-
plied Biosystems, Foster City, CA) in the protein chemistry laboratory
of the Institute for Biochemistry of the University of Vienna by Dr. R.
Prohaska.
PMNL Isolation
PMNL were isolated as described previously ( 15). Briefly, 10 ml of
the venous whole blood of healthy donors was anticoagulated with 10 jd
of heparin sodium (Liquemine Roche iv; Hoffmann-Roche, Bascl, Swit-
zerland) and put on top of 10 ml of Ficoll-Paque (Pharmacia). After
approximately 45 mm, most of the erythrocytes sedimented into the
Ficoll layer. A Percoll (Pharmacia) step gradient (63% vol/vol and 72%
vob/vol) was used to separate granulocytes, lymphocytes, monocytes, and
thrombocytes that stayed in the plasma supernatant. The PMNL band was
isolated, the PMNL were washed twice in PBS, and the cells counted in
a cell counter (Cell Dyn 610, Abbott, Wiesbaden, Germany). The via-
bility of the PMNL obtained by this protocol was greater than 95% as
determined by the trypan blue (Sigma) exclusion assay.
Chemotaxis Assay
We used the under-agarose method ( 16) to test the inhibitory effect
of our fractions or of the purified factor on PMNL chemotaxis. PMNL
were resuspended in PBS at a concentration of I X I0� cells/lO �l in
PBS or in PBS containing the isolated peptide at a final concentrationof 20 j.�g/ml and incubated for 15 ruin at 37#{176}Cbefore starting the
chemotaxis assay. N-formyl-ruethionyl-leucyl-phenylalanine (Sigma),
dissolved in Hank’s buffer with Ca2� and Mg2� (Seromed, Bio-
chrom, Berlin, Germany), was used as a chemoattractant at a concen-
tration of2.5 X l0� M. Afterward, the agarose plates were incubated
for 100 ruin at 37#{176}C.Then the cells were fixed with methanol andparaformaldehyde and stained with Gierusa (Merck, Darmstadt, Ger-
many). The distance the cells migrated under the agarose was mea-
sured under the microscope. For each isolate, between eight and 12
independent assays were performed. The results were statistically
evaluated by paired analyses.
ResultsInitial isolation and Characterization of C/P
We concentrated the penitoncab effluent obtained from pa-
tient 1 undergoing CAPD treatment, as described in Material
and Methods. To obtain a pure substance that shows chemo-
taxis inhibiting activity, we used three different chromato-
graphic methods (see Material and Methods). After each pun-
fication step, i.e. , ion exchange chromatography, gel filtration,
and reversed-phase chromatography, we controlled the biolog-
icab activity of the individual fractions in our in vitro chemo-
taxis assay. Furthermore, we checked the purity of the most
active fractions by nonreducing SDS-PAGE. Figure 1A shows
the progress of purification using the concentrated CAPD
effluent of patient 1 , resulting in the pure protein with chemo-
taxis inhibiting activity. Reducing (Figure 1 B, lane a) and
nonreducing (Figure 1B, lane b) SDS-PAGE of CIP show a
single band, indicating that we obtained a pure pcptidc that
does not consist of subunits connected by disulfide bridges.
The sequence of the 16 N-terminal amino acids of CIP is
Mct-Gln-Ilc-Phc-Val-Lys-Thr-Leu-Thr-Gly-Lys-Thr-Ibe-Thr-
67.0
43.0
30.0
20.1
1 4.4
1 7.2014.60
8.246.38
2.56
abmabcdM ab
Modified Ubiquitin as PMNL Chemotaxis Inhibitor 453
A B C
!�Figure 1. (A) Progress of chemotaxis-inhibiting protein (CIP) purification. Nonreducing sodium dodecyl sulfate-polyacrylamide gel electro-
phoresis (SDS-PAGE) on an 8 to 25% gradient gel. Lane a, concentrated peritoneal dialysis (PD) effluent from patient I ; lanes b through d,
active fractions after ion exchange (b), size exclusion (c), and reversed-phase chromatography (d). M, high molecular weight marker. Molecular
weight is indicated in kilodabtons. (B) SDS-PAGE ofCIP on a high-density gel. Reducing (a) and nonreducing (b) conditions. m, low molecular
weight marker. Molecular weight is indicated in kibodaltons. (C) Western blotting experiment. Lane a, commercially available ubiquitin; lane
b, purified CIP.
Lcu-Glu. This is exactly the same primary structure as found at
the N terminus of ubiquitin (17).
Nonreducing SDS-PAGE of commercially available ubiq-
uitin (Figure 2. lane a) and of CIP (Figure 2, lane b) shows that
both peptides comigrate, and therefore have about the same
size. The molecular mass of ubiquitin predicted from its amino
acid sequence is 8.5 kD ( 17). The anomalous migration of
ubiquitin as a 5.5-kD band on the SDS polyacrybamide gel
indicates the incomplete unfolding of the polypcptide chain.
This finding is consistent with the highly globular compact
conformation of ubiquitin (1 8). In a Western blotting cxpcni-
ment (Figure bC), we found that CIP is able to react with
anti-ubiquitin antibodies. Isoclectric focusing shows that CIP
has an isoclectric point of 5.2 (Figure 3, lane a). This is much
more acidic than the isoelectnic point of unmodified ubiquitin
of approximately 7 (Figure 3, lane b) (19).
inhibitory Effect of C/P on PMNL Chemotaxis
Figure 4 shows that CIP significantly inhibited the cheruo-
tactic movement of PMNL from healthy donors. On the other
30.0
20.114.4 I
--- _
Mabcdefgh I
Figure 2. Nonreducing SDS-PAGE on an 8 to 25% gradient gel. M,
high molecular weight marker. Lane a, commercially available ubiq-
uitin; lane b, CIP isolated from the continuous ambulatory PD
(CAPD) effluent of patient I by three chromatographic methods: lanesc and d, CIP isolated from the nightly intermittent PD effluent of
patient I by affinity chromatography: lanes e and f, CIP isolated by
affinity chromatography from the CAPD effluent of patients 2 and 3,
respectively: lanes g through i, CIP isolated by affinity chromatogra-
phy from hemodialysis (HD) patients 4, 5, and 6, respectively.
hand, commercially available unmodified ubiquitin had no
effect. Furthermore, we showed that the inhibitory effect of
CIP was not reversible (Figure 4). Nevertheless, the viability of
PMNL was not influenced in the presence of CIP, as deter-
mined by the trypan blue exclusion assay (data not shown). We
also found that CIP inhibits PMNL chemotaxis in a concen-
tration-dcpendent manner (Table 1 ). We tested the ability of
CIP to attract normal PMNL. This was not the case within the
concentration range tested (2.5 X lO_6 M to 2.5 X b0” M)
(data not shown).
Affinity Chromatography and Characterization of Anti-
Ubiquitin Antibody Binding Fractions
Our data suggested that we isolated a modified form of
ubiquitin from the penitoneab effluent and that this modification
is responsible for the inhibiting effect on PMNL cheruotaxis. In
a second set of experiments, we prepared an affinity column
specific for ubiquitin and applied the penitoneal effluent ob-
tamed from the same patient (at this time undergoing NIPD
5.1
6.0
6.5
M abcdefgFigure 3. Isoelectnic focusing of CIP isolated from the CAPD effluent
of patient I by three chromatographic methods (a), commercially
available ubiquitin (b), and CIP isolated from patients 2 through 6 by
affinity chromatography (c through g). M, p1 marker.
120
1 00
80
% 60distancemigrated
40
20
0
120
1 00
80
% 60distance
migrated
40
20
0
a CIP, chemotaxis-inhibiting protein; PMNL, polymorphonuclear
beukocytes: fMLP, N-formyl-methionyl-leucyl-phenylalanine.
454 Journal of the American Society of Nephrology
- 1 ir U
Figure 4. Chemotactic movement toward N-formyl-ruethionyl-leucyl-
phenylalanine (fMLP) of polymorphonuclear leukocytes (PMNL)
from healthy subjects in the presence of CIP purified from patient 1
(1 ), commercially available ubiquitin (U), or after preincubation with
and subsequent removal of CIP purified from patient 1 ( 1 r). Mean ±
SEM. *P < 0.05: � < 0.01.
Table 1. Effect of different concentrations of CIP on the
chemotactic movement of PMNL from healthy
subjects toward fMLP”
�g/ml % Migration (mean ± SEM)
0 100
4 101±3
9 96±5
15 75±2
20 46±7
treatment) onto the column. Figure 2 (lanes c and d) shows the
result of an SDS-PAGE of the material bound to the anti-
ubiquitin affinity column from two different purification cx-
periments. We obtained in both cases a single band migrating
at the same distance as ubiquitin.
We further applied the concentrated CAPD effluent from two
other PD patients (patients 2 and 3) and from three HD patients
(patients 4, 5, and 6) onto the anti-ubiquitin affinity column.
SDS-PAGE of the bound material (Figure 2, lanes e through i)
showed a single band comigrating with that of ubiquitin.
Isoclectric focusing of the material ebuted from the anti-
ubiquitin column showed that the fractions isolated from the
other patients contained a component with an acidic isoclectnic
point of approximately 5.2 (Figure 3, lanes c through g) that
most likely corresponds to a modified form of ubiquitin.
Chemotaxis-/nhibiting Activity of Anti- Ubiquitin
Antibody Binding Fractions
We tested all fractions obtained by immunopurification in
our in vitro chemotaxis assay. Figure 5 shows that the samples
- U 1’ 1’’ 2 3 4 5 6
Figure 5. Chemotactic movement toward fMLP of PMNL from
healthy subjects in the presence of commercially available ubiquitin
(U) and of immunopunified CIP from patient I (1 ‘, I”), the PD patients2 and 3 (2 and 3, respectively), and from HD patients 4, 5, and 6 (4,
5, and 6, respectively). *P < 0.05; **P < 0.01.
obtained from all PD patients and from all HD patients signif-
icantly inhibited the cheruotactic movement of PMNL.
DiscussionIn the present study, we isolated from PD and HD patients a
pcptidc with homology to ubiquitin and showed that it has the
ability to inhibit the chemotactic movement of PMNL in a
concentration-dependent manner.
Uremic patients have an increased risk of infection, and as a
result they have a high incidence of morbidity and mortality
(20). In fact, despite intensive research and improved clinical
conditions within the past 20 years, bacterial infections are still
one of the leading causes of death in patients with chronic renal
failure. Because PMNL play a key role in the unspecific
immune defense against bacterial infections, the function of
PMNL isolated from uremic patients has been studied inten-
sively.
These investigations of the PMNL-ruodubating activities of
uremic toxins arc an important contribution to a better under-
standing of the immune status of patients with chronic renal
failure and to future applications in diagnosis and therapy. The
PD effluent represents an important starting material for the
isolation of uremic toxins: The fact that many disturbed func-
tions of PMNL can be corrected by PD treatment is an mdi-
cation for the presence of uremic toxins in the peritoncal cavity
of PD patients. Furthermore, it was shown that addition of
CAPD effluents to PMNL of healthy donors markedly de-
pressed phagocytosis (2 1 ) and also interfered with their ability
to kill ingested Staphylococcus epidertnidis (22).
To have a uremic toxin, the compound should be retained in
the uremic serum, and the concentration should be higher in the
ureruic than in the normal plasma. Although the majority of
proteins that appear in penitoncal effluent come from the vas-
cuban compartment, it is possible that some are generated from
Modified Ubiquitin as PMNL Chemotaxis Inhibitor
cells within the penitoneal cavity on within the peritoncab ruem-
brane. We therefore looked up this component in ureruic serum
ultrafiltrate to exclude that CIP is only extracted from perito-
neal effluent and produced in the peritoncuru. This study
provides evidence that the protein is present in the blood of
unemic patients. The adequate removal of ureruic solutes in the
higher molecular weight range during high-efficiency and
high-flux hemodiabysis has been studied (23).
An obvious consequence of the partial removal of PMNL-
inhibiting factors from the uremic serum by PD treatment and
consequently their presence in the peritoneal cavity is that these
toxins exert a negative influence on functions of peritoneab
PMNL. Whereas most of the cells within the peritoneal cavity of
a PD patient are macrophages in a stable situation, after the onset
peritonitis that is accompanied by a massive and rapid influx of
cells into the peritoncal cavity, more than 85% of the cells are
PMNL (6). Therefore, urcmic toxins affecting PMNL functions
seem to be at least partly responsible for the high susceptibility of
PD patients to peritonitis and, hence, for the increased risk of
morbidity and mortality in this group of patients.
Chemotaxis of PMNL is the first important step of the unspe-
cific immune response (24). Whereas the transendotheliab migra-
tion of PMNL is mainly regulated by interlcukin-8 (25), N-
formyb-methionyl peptides that arc generated by the invading
microorganisms are the main chcmoattractants in bacterial infec-
tions and induce PMNL finally to move to the site of infection
(26). Therefore, we used the peptide N-formyl-methionyl-leucyl-
phenylalaninc, one of the most potent within this group of che-
moattractants (27), in our in vitro chemotaxis assay.
We demonstrated that CIP significantly inhibits PMNL che-
motaxis (Figure 4). Because it has been described (28) that PD
treatment per se might induce the generation of a chcmoattrac-
tant, we tested this ability. However, CIP did not attract normal
PMNL.
Sequencing of the N terminus showed that the first 16 amino
acids of CIP arc identical to those of ubiquitin. Ubiquitin is a
polypcptide with a molecular weight of approximately 8.5 kD.
Its 76 amino acids are highly conserved in evolution. In our
control experiments, we used bovine ubiquitin that has the
same amino acid sequence as human ubiquitin (29). Ubiquitin
is present in all cells investigated so far, i.e. , in bacteria, yeast,
higher plants, and animals (30). It can be attached in an
ATP-nequining process to other proteins via an isopeptidc link-
age between its C-terminal carboxyl group and the epsilon-
amino groups of lysinc residues in the target protein. This
posttransbational protein modification has a variety of biobog-
ical effects. The most prominent function is the participation of
ubiquitin in the protcosomal protein degradation (3 1 ): Ubiq-
uitin covalently bound to proteins can target them to a complex
protcase called the 265 protcasome that degrades misfolded
proteins within the cell. Usually, the formation of ubiquitin
oligoruers is necessary for proteasome binding and subsequent
protein destruction (32). The fact that we isolated by affinity
chromatography only material that migrates as a single band in
SDS-PAGE (Figure 2) at the same distance as ubiquitin mdi-
cated that the anti-ubiquitin antibody-binding fractions con-
tamed only ubiquitin and/or a modified form of it, but no
ubiquitin oligoruers or other proteins with ubiquitin covabently
attached to them.
The increased protein degradation beading to muscle wasting
in uremia is caused by metabolic acidosis induced ATP- and
ubiquitin-dependent protcolysis (33,34). Furthermore, the
ubiquitination of short-living regulatory proteins plays an im-
pontant role in modulating their intracellular concentrations.
Besides those intracellular functions of ubiquitin, excreted
ubiquitin was isolated from the culture supernatant of oocytes
and shown to inhibit the cytotoxic properties of platelets (35).
A significantly increased level of ubiquitin was described in
the plasma of uremic patients and patients undergoing hemo-
dialysis treatment (36). At present, we cannot give exact in vivo
concentrations of the modified ubiquitin in uremic patients.
CIP has about the same size as ubiquitin as shown by
SDS-PAGE (Figure 2), but has a more acidic isoclcctnic point
of 5.2. Because ubiquitin did not show any chcmotaxis inhib-
itory activity, we conclude that CIP is a modified form of
ubiquitin and that this modification is responsible for its bio-
logical activity. Two types of modifications, i.e. , carbamoyla-
tion and glycation, could explain our findings, because both
reactions bead to a loss of positive charge by modifying lysine
residues in proteins. The molecular weight change caused by
either modification would not necessarily be big enough to be
detected by SDS-PAGE.
A small percentage (approximately 0.8%) of urea that accumu-
lates in the scra of urcmic patients is converted spontaneously to
cyanate (37). Cyanate can react with the epsilon-amino groups of
lysins, leading to a carbamoylated protein (38). Glucose can react
noncnzymatically with lysinc residues, leading to Schiff-base
intermediates that react to stable Amadori products, the glycated
proteins (39). There also are experiments indicating that soluble
proteins within the peritoneal cavity can be modified by glycation
during CAPD treatment. This seems not to be the case for CIP,
because we could isolate CIP from the ultrafiltrate of HD patients
as well. Furthermore, it could be shown that glycatcd proteins
preferentially accumulate in the CAPD effluent (40). Experiments
to identify the type of modification leading to CIP arc ongoing in
our laboratory.
We conclude from the results presented here that a modified
form of ubiquitin (CIP) exists in the pcnitoneab cavity of PD
patients and that this modification is responsible for the inhib-
itory effect of CIP on PMNL cheruotaxis and, hence, for the
increased risk of infection, leading, at least in part, to a
higher incidence of morbidity and mortality in this group of
patients.
References1 . Mailloux LU, Bellucci AG, Wilkes BM, Napolitano B, Mossey RT,
Lesser M, Bluestone PA: Mortality in dialysis patients: Analysis of
the causes of death. Am J Kidney Dis 18: 326-335, 19912. Korbet SM, Vonesh EF, Firanek CA: A retrospective assessment
of risk factors for peritonitis among an urban CAPD population.
Peril Dial mt 13: 126-131, 1993
3. Vanholder R, Van Loo A, Dhondt AM, Dc Smet R, Ringoir 5:Influence of uraemia and haemodialysis on host defence and
infection. Nephrol Dial Transplant I I : 593-598, 1996
456 Journal of the American Society of Nephrology
4. Himmelfarb I, Hakim RI: Biocompatibility and risk of infectionin heruodialysis patients. Nephrol Dial Transplant 9[Suppl 2]:
138-144, 1994
5. Haag-Weber M. H#{246}rlWH: Dysfunction of polymorphonuclear
leukocytes in uremia. Semi?l Nephrol 16: 192-201, 19966. Ho-Dac-Pannekeet MM, Krediet RT: Inflammatory changes in
vivo during CAPD: What can the effluent tell us. Kidney hit
50[Suppl 56]: 512-516, 1996
7. H#{246}rlWH, Haag-Weber M, Georgopoulos A, Block LH: Physi-
cochemical characterization of a polypeptide present in uremic
serum that inhibits the biological activity of polymorphonuclear
cells. Proc Natl Acad Sci USA 87: 6353-6357, 1990
8. Cohen G, Haag-Weber M, Mai B, Deicher R, H#{246}rlWH: Effect of
immunogbobulin light chains from hemodialysis and continuous
ambulatory peritoneal dialysis patients on polymorphonuclearleukocyte functions. J A,n Soc Nephrol 6: 1592-1599, 1995
9. Haag-Weber M, Mai B, H#{246}rlWH: Isolation of a granubocyte
inhibitory protein in uremia: Homology to beta,-microgbobulin.
Nephrol Dial Transplant 9: 382-388, 1994
10. Haag-Weber M, Mai B, H#{246}rlWH: Impaired cellular host defencein peritoneal dialysis by two granulocyte inhibitory proteins.
Nephrol Dial Transplant 9: 1769-1773, 1994
1 1 . Tschcsche H, Kopp C, H#{246}rlWH, Hempelmann U: Inhibition of
degranulation of polymorphonuclear leukocytes by angiogenin
and its tryptic fragments. J Biol Chein 269: 30274-30280, 1994
12. Balke N, Holtkamp U, H#{246}rlWH, Tschesche H: Inhibition of
degranulation of human polymorphonuclear leukocytes by com-
plement factor D. FEBS Lett 371: 300-302, 1995
13. Haag-Webcr M, Cohen G, H#{246}rlWH: Modulation of granubocyte
(PMNL) function by complement factor D isolated from perito-
neal dialysis effluent [Abstract]. JAm Soc Nephrol 7: 1480, 1996
14. Vanholder R, Dc Smet R, Waterloos MA, Van Landschoot N,
Vogeleere P, Hoste E, Ringoir 5: Mechanisms of uremic inhibi-
tion of phagocyte reactive species production: Characterizationof the role of p-cresol. Kidney mt 47: 510-517. 1995
15. Metcalf IA, Gallin II, Nauseef WM, Root RK: Laboratory Man-
ual of Neutrophil Function, New York, Raven, 1986, pp 2-5
16. Nelson RD. Quie PG. Simmons RL: Chemotaxis and spontane-
ous migration of human polymorphonuclear leukocytes and
monocytes. J Immunol 1 15: 1650-1655, 1975
17. Schlesinger DH, Goldstein G, Niall H: The complete amino acid
sequence of ubiquitin, an adenylate cyclase stimulating polypep-
tide probably universal in living cells. Biochemistry 14: 22 14-
2218, 1975
18. Vijay-Kumar 5, Bugg CE, Cook WI: Structure of ubiquitinrefined at 1.8 A resolution. J Mol Biol 194: 5 13-544, 1987
19. Hans AL, Murphy KE, Bright PM: The inactivation of ubiquitin
accounts for the inability to demonstrate AlP, ubiquitin-dependent
proteolysis in liver extracts. J Biol C’hem 260: 4694-4703, 1985
20. Vanholder R, Van Loo A, Dhont AM, Dc Smet R, Ringoir 5:
Influence of uraemia and haemodialysis on host defence andinfection. Nephrol Dial Transplant 1 1 : 593-598. 1996
21 . Vanholder R, Lameire N, Waterloos MA, Van-Landschoot N,
De-Smet R. Vogeleere P. Lambert MC, Vijt D, Ringoir 5:
Disturbed host defence in peritoneal cavity during CAPD: Char-
acterization of responsible factors in dwell fluid. Kidney mt 50:
643-652, 199622. Porter Cl, Burden RP, Morgan AG, Daniels I, Fletcher I: Im-
paired polymorphonuclear neutrophil function in end-stage renal
failure and its correction by continuous ambulatory peritoneal
dialysis. Nephron 71: 133-137, 1995
23. Leypoldt 1K, Cheung AK: Removal of high-molecular-weightsolutes during high-efficiency and high-flux haemodialysis.
Nephrol Dial Transplant 1 1: 329-335, 1996
24. Downey GP: Mechanisms of leukocyte motility and chemotaxis.
Curr Opin bminunol 6: 1 13-124, 199425. Baggiolini M, Walz A, Kunkel SL: Neutrophil-activating peptide
1/interleukin 8, a novel cytokine that activates neutrophils. J Cliii
invest 84: 1045-1049, 1989
26. Marasco WA, Phan SH, Krutzsch H, Showell Hi, Feltner DE,
Nairn R, Becker EL, Ward PA: Purification and identification of
formyl-methionyl-leucyl-phenylalanine as the major peptide
neutrophil chemotactic factor produced by E.s-cherichia coli.
J Biol Chem 259: 5430-5439, 1984
27. Goldstein IM: Cheruotactic factor receptors on leukocytes:
Scratching the surface. J Lab Clin Med 97: 599-601, 1981
28. Bos HI, Boorsma DM, Tuk CW. Dc Veld IC, Van Der Muysen-
berg AJC, Helmcrhorst TIM, Struijk DG, Van Bronswijk, Beelen
RHI: Chemotaxis of the penitoneal cells and the detection of a
chemo-attractant in the effluent from penitoneal dialysis patients.
Eur J Cliii bnvest 20: 555-562, 1990
29. Schlesinger DH, Goldstein G: Molecular conservation of 74
amino acid sequence of ubiquitin between cattle and man. Nature
255: 423-424, 1975
30. Goldstein G, Scheid M, Hammerling U, Boyse EA, Schlesinger
DH, Niall HD: Isolation of a polypeptide that has lymphocyte-
differentiating properties and is probably represented universally
in living cells. Proc NatI Acad Sci USA 72: 1 1-15, 1975
3 1 . Hershko A: Ubiquitin-mediated protein degradation. J Biol Chem
263: 15237-15240, 1988
32. Chau V. Tobias 1W, Bachruair A, Marriott D, Ecker Di, Gonda
DK, Varshavsky A: A multiubiquitin chain is confined to spe-
cific lysine in a targeted short-lived protein. Science 243: 1576-
1583, 1989
33. Mitch WE, lurkovitz C, England BK: Mechanisms that cause
protein and amino acid catabolism in uremia. Am J Kidney Dis
21: 91-95, 1993
34. Mitch WE, Goldberg AL: Mechanisms of muscle wasting: The
role of the ubiquitin-proteasorue pathway. N Engl J Med 335:
1897-1905, 1996
35. Pancr#{233}V. Pierce RI, Foutnier F, Methali M, Delanoye A, Capron
A, Auriault C: Effect of ubiquitin on platelet functions: Possibleidentity with platelet activity suppressive lymphokine (PASL).
Ear J Immunol 2 1 : 2735-274 1 , 1991
36. Okada M, Miyazaki 5, Hirasawa Y: Increase in plasma concentra-
tion of ubiquitin in dialysis patients: Possible involvement in beta2-
microglobulin amyboidosis. Cliii Chi,n Ada 220: 135-144, 1993
37. Roxborough HE, Millar CA, McEneny I, Young IS: Carbamy-
lation inhibits the ferroxidase activity of caeruboplasmin. Bio-
chein Biophys Res Coinmun 2 14: 1073-1 078, 1995
38. Stark GR, Stein WH, Moore 5: Reactions of the cyanate present
in aqueous urea with amino acids and proteins. J Biol Chem 235:
3177-3181, 1960
39. Schliecher E, Wieland OH: Protein glycation: Measurement and
clinical relevance. J Cliii Chem Cliii Biochem 27: 577-587, 1989
40. Lamb E, Cattel WR, Dawnay A: Glycated albumin in serum and
dialysate of patients on continuous ambulatory penitoneal dialy-
sis. Cliii Sci 84: 619-626, 1993