Background
==========

*Helicobacter pylori* is believed to be the major aetiological factor in the development of non-cardia gastric adenocarcinoma. Large-scale epidemiological studies have confirmed a strong association between *H. pylori* infection and both cancer \[[@B1]-[@B3]\] and the earlier histological stages, atrophy and intestinal metaplasia \[[@B4],[@B5]\]; both of which increase the risk of later neoplastic transformation. Animal models have also demonstrated the importance of *H. pylori* in gastric carcinogenesis \[[@B6],[@B7]\]. Increased rates of proliferation of the gastric mucosa are typical in *H. pylori* infection \[[@B8]-[@B11]\], and hyperproliferation within the gastrointestinal tract appears to be a marker for later malignant change \[[@B12]\]. The cause of the increased rate of proliferation is not clear, but the increased rates reduce to normal with clearance of the infection \[[@B8],[@B13]\]. Although hypeprproliferation is typical *in vivo,* studies testing the effects of *H. pylori* or its products *in vitro* have shown conflicting results, with both enhanced \[[@B14],[@B15]\] and diminished \[[@B16]-[@B18]\] proliferation reported. It is possible that other components of the inflammatory response typical of *H. pylori* infected mucosa could be at least partly responsible for driving the increased cell proliferation.

The pluripotent pro-inflammatory cytokine interleukin-1β has a central role in the pathogenesis of *H. pylori*-induced mucosal inflammation. IL-1β gene expression and protein production are increased in *H. pylori* infection and reduce with successful eradication \[[@B19],[@B20]\]. The presence of the IL-1β genotype polymorphism associated with enhanced IL-1β-production has been associated with a significant increased risk of gastric cancer and pre-cancerous lesions \[[@B21],[@B22]\]. Interleukin-1β is a potent inhibitor of gastric acid secretion and it is hypothesized that the enhanced IL-1β response alters the topography of the gastric infection and thus promotes inflammation and subsequent atrophy of the gastric corpus \[[@B23],[@B24]\]. The possibility that IL-1β itself drives the increased proliferation of gastric epithelial cells has not been fully investigated. Alteration of gastric proliferation by IL-1β might contribute to the carcinogenic process, in addition to effects on acid secretion. Therefore the direct effects of IL-1β on gastric epithelial proliferation have been assessed.

The mitogen-activated protein kinase (MAPK) cascades are well-characterised pathways transducing signals from the cell surface to the nucleus. The family includes distinct subgroups; extracellular signal-related kinases (ERKs), c-Jun NH~2~-terminal kinases (JNKs) and p38 MAPK \[[@B25]\]. The ERKs are activated by a variety of extracellular stimuli, and mediate the pro-proliferative effects of a number of hormones and growth factors \[[@B26],[@B27]\]. Activation by phosphorylation of a dual specificity protein kinase (MAP kinase kinase (MAPKK)), (also known as MEK), allows it in turn to activate a family of serine-threonine protein kinases, known as the ERKs. The ERKs in turn phosphorylate numerous cellular proteins including transcription factors and thus have a central role in propagation of mitogenic signals. Accordingly the role of the MAP-kinase pathway in mediating the responses to IL-1β has been assessed.

Methods
=======

Cell culture
------------

The human AGS gastric carcinoma cell line was purchased from the European Collection of Animal Cell Cultures (Porton Down, UK). Cells were grown in monolayer culture in RPMI 1640 medium supplemented with 100 μg/ml penicillin, 100 μg/ml streptomycin, 100 μg/ml gentamicin, 2.5 μg/ml amphoteracin B and 10 % foetal calf serum. Cells were grown in 75 cm^2^ tissue culture flasks at 37°C in an atmosphere of 5% CO~2~ and 95% air and passaged every 5--7 days.

Proliferation studies
---------------------

\[^3^H\]thymidine incorporation. Cells were grown in media containing 10% foetal calf serum, plated into 24-well plates at 10^5^ cells/well and allowed to attach overnight. After washing with serum-free media, cells were incubated in serum free medium containing 0.2 mM unlabelled thymidine for 24 hours in the presence of increasing concentrations of IL-1β, IL-8 or GM-CSF. DNA synthesis was estimated by measurement of \[^3^H\]thymidine incorporation into the trichloroacetic acid (TCA) precipitable material \[[@B28]\]. \[^3^H\]thymidine (0.1 μCi/ml, 10 Ci/mmol) was added 2 hours before the end of a 24 hour treatment period. Cells were washed twice with serum-free medium to remove unincorporated \[^3^H\]thymidine, and DNA was precipitated with 5% TCA at 4°C for 15 minutes. The precipitates were then washed twice with 95% ethanol, dissolved in 1 ml of NaOH, and analysed by liquid scintillation counting. Results are expressed as percent control unstimulated \[^3^H\]thymidine incorporation (mean ± SD) of 4--6 different experiments, each performed in triplicate. For the detection of growth inhibition, cells were incubated with either the specific MEK inhibitor PD 98059 (25 μM)\[[@B29]\], IL-1 receptor antagonist (500 ng/ml) \[[@B30]\] or the neutralising antibodies, anti-GM-CSF (5 μg/ml) or anti-IL-8 (10 μg/ml). Inhibitors or antibodies were added 30 minutes prior to cytokines.

Cell growth
-----------

Total viable cell numbers were assessed by a modified MTT (3-\[4,5-dimethylthiazol-2-yl\]-2.5 diphenyl tetrazolim bromide assay) \[[@B31]\]. Cells were plated into 24-well plates in medium containing 10% foetal calf serum. After attachment overnight, the medium was changed to 1% foetal calf serum-supplemented medium and increasing concentrations of IL-1β were added. Cells were cultured for 48 hours and then the medium was removed and fresh RPMI 1640 medium containing 0.5 ng/ml MTT was added. Cells were incubated at 37°C for 3 hours. The medium was then removed and 0.04 M HCl in isopropanol was added to extract the reduced formazan product. The resulting optical density at 550 nm was determined.

Chemicals and reagents
----------------------

Recombinant human IL-1β and IL-8 were purchased from Sigma (Poole, UK), recombinant human GM-CSF and IL-1 receptor antagonist, anti-GM-CSF and anti-IL-8 were from R and D systems (Abingdon, Uk). PD 95059 was from Calbiochem (Nottingham, UK). RPMI 1640 was from Gibco BRL (Paisley, UK) and all other reagents were from Sigma. Concentrations of inhibitors used were taken from manufacturers\' data and published data. The ability of 500 ng/ml IL-1RA to abolish 10 ng/ml IL-1β-stimulation of IL-8 secretion in AGS cells was confirmed. The efficacy of the anti-GM-CSF at 5 μg/ml antibody to abolish GM-CSF (1 ng/ml) induced AGS cell proliferation was confirmed.

Statistics
----------

Cytokine-stimulated results where compared with control unstimulated cells on the same 24-well plate. Data were compared by one-way analysis of variance and Student\'s t-test to determine statistical significance. Each experiment as performed in triplicate on 4--6 occasions. Results are expressed as mean ± standard deviation. Differences with P values of \< 0.05 were considered significant.

Results
=======

Effect of IL-1β on \[^3^H\]thymidine incorporation
--------------------------------------------------

Interleukin-1β caused a dose-dependent increase in DNA synthesis as measured by thymidine incorporation. As shown in figure [1](#F1){ref-type="fig"}, significant stimulation was seen with 1--100 ng/ml IL-1β. The maximal stimulation of 52 ± 6 % above control was seen with 10 ng/ml. The higher dose of 100 mg/ml was slightly less effective in stimulating proliferation.

![Effect of IL-1β on \[^3^H\]thymidine incorporation into gastric epithelial AGS cells Cells were treated with increasing concentrations of IL-1β for 24 hours and DNA synthesis assessed by \[^3^H\]thymidine incorporation. Results expressed as means ± standard deviation. ^\*^*P* \< 0.01 vs. control](1471-230X-2-7-1){#F1}

Effect of IL-1β on cell number
------------------------------

The increase in DNA synthesis by IL-1β was translated into an absolute increase in viable cell numbers. As shown in figure [2](#F2){ref-type="fig"}, IL-1β increased cell numbers in a dose-dependent manner similar to the effects on \[^3^H\]thymidine incorporation. The maximal stimulation was again seen at 10 ng/ml of IL-1β, which produced a 22 ± 5 % increase in total cell number.

![Effect of IL-1β on cell numbers of gastric epithelial cells Cells were treated with increasing concentrations of IL-1β for 48 hours and total viable cell numbers assessed by MTT assay. Results expressed as means ± standard deviation. ^\*^*P* \< 0.01 vs. control](1471-230X-2-7-2){#F2}

Effects of cytokine inhibition or receptor antagonism on IL-1β-stimulation of proliferation
-------------------------------------------------------------------------------------------

Pretreatment of the cells with interleukin-1 receptor antagonist abolished the stimulatory effects of IL-1β on \[^3^H\]thymidine incorporation (Figure [3](#F3){ref-type="fig"}). Previous studies have shown that IL-1β can activate gastric epithelial cells, including AGS cells, to secrete other cytokines, particularly interleukin-8 and granulocyte-macrophage colony stimulating factor (GM-CSF)\[[@B31],[@B32]\]. Therefore further studies were undertaken to assess if the stimulatory actions of IL-1β were mediated by either of these two cytokines. Neutralising antibodies to either IL-8 or GM-CSF had no effect on unstimulated \[^3^H\]thymidine incorporation. Neutralisation of IL-8 had no effect on IL-1β-stimuated growth but the anti-GM-CSF antibody reduced IL-1β-stimulated proliferation by 31 ± 4 % (*P* \< 0.01) (figure [3](#F3){ref-type="fig"}).

![Effect of cytokine inhibition on IL-1β-stimulation of \[^3^H\]thymidine into gastric epithelial cells AGS cells were treated with 10 ng/ml IL-1β for 24 hours plus either interleukin-1 receptor antagonist (IL-1RA 500 ng/ml), neutralising anti-IL-8 antibody (10 μg/ml) or neutralising anti-GM-CSF antibody (5 μg/ml). DNA synthesis was assessed by \[^3^H\]thymidine incorporation. Results expressed as means ± standard deviation. ^\*^*P* \< 0.01 vs. IL-1β stimulation in absence of inhibitor.](1471-230X-2-7-3){#F3}

Effects of GM-CSF on proliferation
----------------------------------

In view of the results obtained with the anti-GM-SCF antibody, the direct growth-stimulatory actions of GM-CSF were examined. GM-CSF had a potent growth stimulatory action on AGS cells: significant enhancement of \[^3^H\]thymidine incorporation was seen at all concentrations (0.001--100 ng/ml) of GM-CSF. GM-CSF itself appeared to be a more potent stimulant than IL-1β ; maximal stimulation of 108 ± 17 % above control was seen with 100 ng/ml of GM-CSF (Figure [4](#F4){ref-type="fig"}). The inhibitory action of the anti-GM-CSF antibody was confirmed by abolition of the growth stimulatory action of 1 ng/ml GM-CSF (data not shown). Previous studies have shown that IL-1β-stimulated GM-CSF release under similar conditions in AGS cells to be approximately 10--20 pg/well/24 hours \[[@B32]\]. To confirm the results obtained with the anti-IL-8 antibody, \[^3^H\]thymidine incorporation was measured in response to IL-8. No enhancement of proliferation was seen at any concentration of IL-8 (0.001--100 ng/ml) (data not shown). Under similar conditions IL-1β-stimulated IL-8 release is approximately 3000 pg/well/24 hours \[[@B31]\].

![Effect of GM-CSF on proliferation of gastric epithelial cells. AGS cells were treated with increasing concentrations of GM-CSF for 24 hours. Cell proliferation was assessed by \[^3^H\]thymidine incorporation. Results expressed as means ± standard deviation. ^\*^*P* \< 0.05, ^\*\*^*P* \< 0.01 vs control](1471-230X-2-7-4){#F4}

Mechanism of IL-1β-stimulation of cell proliferation
----------------------------------------------------

The specific inhibitors genistein, which inhibits tyrosine kinases and PD 98059, which inhibits MAP kinase kinase (MEK), and thus inhibits the ERK-pathway, were used to assess the possible intracellular pathways mediating the effects of IL-1β. In order to examine the effects of IL-1β distinct from those of GM-CSF, these experiments were performed in the presence of the anti-GM-CSF neutralising antibody. As shown in figure [5](#F5){ref-type="fig"}, neither genistein nor PD 98059 altered unstimulated \[^3^H\]thymidine incorporation. Genistein completely abolished the IL-1β-stimulation of proliferation. Inhibition of MEK with PD 98059 reduced IL-1β-stimulated proliferation by 58 ± 5% (*P* \< 0.01) (figure [5](#F5){ref-type="fig"}) but did not completely abolish the growth stimulatory action of IL-1β. Further increases to supra-maximal concentrations of PD 98050 did not further inhibit IL-1β-stimulated proliferation (data not shown).

![Effect of inhibition of tyrosine kinase and MEK activity on IL-1β-stimulated gastric epithelial cell proliferation. AGS cells were treated with 10 ng/ml IL-1β for 24 hours in the presence of the tyrosine kinase inhibitor genistein (100 μM) or the MEK inhibitor PD 98059 (25 μM). Proliferation was assessed by \[^3^H\]thymidine incorporation. Studies were performed in the presence of anti-GM-CSF antibody (5 μg/ml). Results expressed as means ± standard deviation. ^\*^*P* \< 0.01 vs control](1471-230X-2-7-5){#F5}

Discussion
==========

This study demonstrated that IL-1β increased proliferation of AGS cells. This effect was reversed by the receptor antagonist, suggesting it was mediated via the interleukin-1 receptor. IL-1β stimulated both \[^3^H\]thymidine incorporation, as a measure of stimulation of DNA synthetic rate and also total cell numbers as measured by the MTT assay. This illustrates that the stimulation of DNA synthesis by IL-1β is translated into a real increase in cell numbers.

A portion of the stimulatory action of IL-1β appears to be indirect. Neutralisation of GM-CSF in the media led to a significant reduction of IL-1β-stimulated proliferation. AGS cells are known to secrete GM-CSF in response to IL-1β \[[@B32]\]. GM-CSF itself was a potent stimulant of cell proliferation. Thus it seems likely that part of the growth stimulatory actions of IL-1β are due to an autocrine intermediary action of GM-CSF. There are limited previous data suggesting that GM-CSF stimulates the proliferation of non-haematopoietic cells; Dippold *et al* showed that exogenous GM-CSF stimulated the growth of two out of two cultures derived from gastric carcinomas and two out of nine pancreatic carcinoma cell lines \[[@B33],[@B34]\]. However, unlike the current study, autocrine production of GM-CSF was no detectable.

Gastric epithelial cells also produce IL-8 in response to IL-1β \[[@B31]\]. However in this model system, IL-8 alone had no pro-proliferative action and neutralisation of IL-8 did not affect the stimulatory action of IL-1β. Therefore it is unlikely that IL-8 has an autocrine role in the growth-stimulatory action of IL-1β. It is possible that other cytokines produced by the gastric epithelial cells in response to IL-1β, *H. pylori or* other inflammatory insults could also play a role as autocrine or paracrine mediators of growth. It has recently been reported that another C-X-C chemokine, GRO/CINC-1, which is also upregulated in *H. pylori* infection stimulated proliferation in rat gastric epithelial cells \[[@B35]\]. The role of these other potential autocrine mediators deserves further study.

The results of the inhibitor studies strongly suggest that tyrosine kinase activity is essential for the growth promoting action of IL-1β in AGS cells. The tyrosine kinase inhibitor genistein abolished the stimulatory action of IL-1β. IL-1β is known to activate a plethora of intracellular signalling pathways \[[@B31],[@B36]-[@B39]\], but in the current situation there appears to be an absolute requirement for signalling via a tyrosine kinase, subsequent on receptor activation.

The mitogen-activated protein cascade is a well-characterised pathway mediating the cell growth-stimulatory actions of many growth factors and hormones. Inhibition of the ERK pathway, with the MEK inhibitor PD 98059, which prevents activation of ERKs by phosphorylation, had a significant inhibitory action against the stimulatory action of IL-1β. This suggests that activation of the ERK pathway is important in mediating the growth stimulatory actions of IL-1β. Activation of MAP kinase cascades, including the p42 and p44 ERK pathways and p46JNK and p55JNK c-Jun NH~2~-terminal kinases by IL-1β has been demonstrated in rat gastric epithelial cells \[[@B41],[@B42]\], but the functional importance of these pathways was not examined. Activation of ERKs and JNKs was inhibited by genistein \[[@B41]\], consistent with the inference of the current study that MAPKs lie downstream of tyrosine activity in IL-1β-induced signalling. However in the current study PD 98059 did not completely abolish the stimulatory action of IL-1β, suggesting that alternative pathways, activated subsequent on tyrosine kinase activity, also play a role in the signalling of the proliferative responses to IL-1β. Further studies are underway at present to examine these.

There are conflicting data available concerning the direct effects of IL-1β on gastric epithelial proliferation. Although the current study and the study by Fan *et al* using stimulated-leukocyte conditioned media showed increased proliferation of the human AGS cell line \[[@B14]\], others have shown inhibition of serum, TGF-α and EGF-stimulated proliferation in RGM1 rat gastric epithelial cells by IL-1β \[[@B41],[@B42]\]. Tominaga *et al* showed that pre-treatment of RGM1 cells with IL-1β for 6 hours inhibited proliferation at 24 hours, but the inhibitory effect was lost at 48 hours, \[[@B41]\]. The reasons for these discrepancies are not clear. They may reflect intrinsic differences between the cell lines, differences in activation and involvement of paracrine growth-promoting pathways, species variability, an effect specific for certain growth factors, or underlying differences in the biology of cell lines derived from cancer (AGS) or normal tissue (RGM1). The studies demonstrating inhibition of proliferation by IL-1β were performed in the presence of powerful growth-promoting stimuli (high serum or specific growth factor concentrations in the culture media), whist the current studies were performed in serum-free or 1% serum media. It is possible that the multiple signalling pathways activated by IL-1β have differing effects on proliferation, the dominant effect depending on the complex inter-relationships of stimuli and signalling pathways under different circumstances. Pro-inflammatory cytokines such as IL-1β and TNF-α activate different signalling pathways with divergent results and different time-courses in gastric endocrine and parietal cells \[[@B37],[@B38],[@B43]-[@B46]\]. Further studies are underway examining the specific roles of the various signalling pathways in gastric epithelial cells under different conditions.

There are now strong epidemiological data linking *H. pylori* with gastric carcinoma. The carcinogenic process appears to involve a number of steps: *H. pylori*-induced inflammation progresses to atrophy, intestinal metaplasia, dysplasia and eventually carcinoma \[[@B47]\]. Similarly *H. pylori* infection in Mongolian gerbils readily induces gastric atrophy and cancer \[[@B6]\]. Whilst gastric carcinogenesis is undoubtedly a multifactorial process, involving pathogenic bacterial and host factors, including HLA status, diet and antioxidant status \[[@B47],[@B48]\], it is clear that increased gastric epithelial proliferation, especially when relatively increased compared to apoptosis, is an important part of the pathway \[[@B49],[@B50]\]. Increased gastric epithelial proliferation is typical of *H. pylori* infection; it is demonstrable throughout all stages of the infection and eradication of the infection reduces proliferation. Increased proliferation is an important marker of increased risk of gastrointestinal adenocarcinoma \[[@B12]\]. The mechanisms of the increased proliferation are not completely understood. *In vitro* studies testing cultures of *H. pylori or* constituents have given conflicting results in different systems, with different cell types and bacterial strains. Direct stimulation of gastric epithelial proliferation by *H. pylori* has been reported by some authors \[[@B14],[@B15]\], whilst either neutral effects \[[@B15]\] or increased apoptosis and decreased proliferation have been reported by others \[[@B16]-[@B18]\]. Fan *et al* reported that conditioned media from either *H. pylori or* mitogen activated lymphocytes directly stimulated AGS cell proliferation \[[@B14]\], suggesting that the inflammatory response could be partly responsible for the enhancement of epithelial proliferation. IL-1β production is enhanced in *H. pylori* infection and this cytokine is regarded as being central to the regulation of the pro-inflammatory response in *H. pylori* infection.

IL-1β is a profound inhibitor of gastric acid secretion *in vivo*\[[@B51]\] and in isolated parietal cells \[[@B37],[@B38]\]. Genetic polymorphisms of the IL-1β gene cluster causing increased transcriptional activity are associated with an increased risk of pre-cancerous and cancerous histological changes in *H. pylori* infection \[[@B21],[@B22]\]. The general hypothesis explaining this observation has been that enhanced IL-1β production consequent on *H. pylori* infection is responsible for greater suppression of acid secretion, which in turn allows greater colonisation of the acid-secreting body mucosa \[[@B24]\]. This greater colonisation provokes further inflammation, which ultimately leads to loss of specialised acid-secretory epithelium (atrophy) and further potentiates the vicious cycle of increased inflammation and diminished acid secretion \[[@B52]\]. Gastric atrophy significantly predisposes to cancer \[[@B3]\]. It is believed that atrophy in combination with mediators released in inflammation such as oxygen free radicals and nitric oxide, diet, anti-oxidant state and possibly bacterial overgrowth and generation of nitrosamines in the achlorhydric stomach \[[@B53]\] advance the histological changes, cause mutageneisis and drive progression to cancer.

An alternative, but not mutually exclusive, hypothesis is that the enhanced cytokine response directly enhances epithelial cell proliferation and itself predisposes to cancer, in addition to the effects on acid secretion. The increased cell turnover of this hyperproliferative response would itself make the mucosa more vulnerable to the mutagenic effects of free radicals and other toxic products generated in the achlorhydric inflamed stomach.

Elsewhere in the gastrointestinal tract, IL-1β has been shown to stimulate \[^3^H\]thymidine incorporation and increase cell number of cultured human colonic subepithelial myofibroblasts, which is thought to have importance in the remodelling of the mucosa in inflammation \[[@B54]\]. The pro-proliferative effects of IL-1β in the gastrointestinal tract deserve further study, given the importance of this cytokine in the regulation of the mucosal inflammatory response.

Conclusions
===========

The results of the current study suggest that IL-1β and GM-CSF can directly stimulate gastric epithelial proliferation. This might explain the proliferative responses to conditioned lymphocyte media demonstrated by Fan *et al*\[[@B14]\]. Enhanced epithelial proliferation due to IL-1β may contribute to the enhanced risk of gastric cancer and precancerous lesions in *H. pylori*-infected individuals with specific IL-1β alleles associated with higher levels of production. IL-1β stimulates proliferation via receptor-mediated activation of a tyrosine kinase pathway. Downstream signalling involves ERK-dependent and -independent pathways.

Further studies will be necessary to clarify the mechanisms involved in IL-1β-stimulation of gastric epithelial proliferation, as well as data correlating IL-1β genotype, IL-1β protein production and epithelial proliferation *in vivo.* These will compliment the current study and further enhance our understanding of *H. pylori*-induced gastric carcinogenesis.

List of abbreviations
=====================

EGF -- epidermal growth factor; ERK -- extracellular signal related kinase; GM-CSF -- granulocyte-macrophage colony stimulating factor; IL -- interleukin; JNK -- c-Jun NH~2~-terminal kinase; MAP -- mitogen activated protein; MTT -- 3-\[4,5-dimethylthiazol-2-yl\]-2.5 diphenyl tetrazolim bromide; TCA -- trichloroacetic acid; TGF-α -- transforming growth factor alpha.

Competing interests
===================

None declared.

Pre-publication history
=======================

The pre-publication history for this paper can be accessed here:

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Background
==========

The National Institutes of Health have recommended that patients with chronic progressive renal insufficiency be referred to a multidisciplinary pre-dialysis team in order to minimize patient morbidity and ensure a smooth transition to dialysis therapy. \[[@B1]\] Referral to a pre-dialysis clinic is associated with many desirable outcomes including longer dialysis-free time intervals, \[[@B2]\] enhanced employment opportunities, \[[@B3]\] better patient education, \[[@B4],[@B5]\] and greater participation in the selection of dialysis modality. \[[@B4],[@B5]\] Patients referred to a multi-disciplinary pre-dialysis program also demonstrate better metabolic profiles, are less likely to require central venous catheter insertion, and require fewer in-hospital emergency dialysis starts and hospital admission days compared to patients who receive standard care. \[[@B6],[@B7]\]

However, research exploring the benefits of pre-dialysis care from a patient-oriented perspective using self-reported quality of life measurements is limited. In a single Brazilian study, quality of life was compared between patients diagnosed with chronic renal failure less than one month before dialysis initiation (n = 53) and those patients diagnosed more than six months before starting dialysis (n = 60) using the Kidney Disease Questionnaire (KDQ). \[[@B8]\] Compared to patients diagnosed earlier, quality of life scores were significantly worse in the late-diagnosis group -- a finding that was particularly evident among elderly patients. However, statistical adjustment for covariates other that age and time of diagnosis was not performed. As well, only 28% of patients were over age 60, suggesting that the association between early diagnosis and improved quality of life may have been underestimated. Further study using a more representative sample reflecting the demographics of an aging Canadian population is therefore warranted.

The present study hypothesized that attendance at a pre-dialysis clinic is an independent predictor of improved quality of life scores measured within the first six months of dialysis initiation in an incident cohort of chronic hemodialysis patients. Quality of life, measured using the SF 36-item Health Survey, was compared between patients who attended a pre-dialysis clinic and those who did not, adjusted for clinical and biochemical covariates.

Methods
=======

Patient population
------------------

Kingston General Hospital is a tertiary care university teaching centre in Southeastern Ontario, Canada, providing pre-dialysis, hemodialysis, peritoneal dialysis and transplantation programs. The hospital is the only centre offering nephrology consultation in this region, serving a population of about 450,000 people. Referral patterns are therefore very centralized. Patients receive hemodialysis at either Kingston General Hospital or one of several affiliated satellite units that offer both self-care and full nursing care services.

Patients with renal failure are referred by primary care physicians to a nephrologist for initial assessment. The care of patients diagnosed with chronic and progressive renal failure who do not require imminent dialysis initiation is subsequently transferred by the nephrologist to the pre-dialysis clinic. Direct referral to the pre-dialysis clinic by a primary care physician is not permitted. The pre-dialysis clinic is scheduled weekly and staffed by nurse clinicians, dieticians, social workers, pharmacists, and nephrologists. The nephrologists at the hospital operate a group practice and staff the pre-dialysis clinic according to a rotating schedule.

The medical records of all patients beginning chronic hemodialysis at Kingston General Hospital or affiliated satellite dialysis units were reviewed by the primary investigator for study eligibility. Study inclusion criteria were first hemodialysis initiation from January 1 1998 to January 1 2000, history of chronic irreversible renal failure, and completion of the QOL survey within six months of dialysis initiation. Exclusion criteria included receipt of dialysis for less than four weeks and previous kidney transplantation. Reasons for non-completion of the SF-36 questionnaire within six months of dialysis initiation included death (16 patients from the pre-dialysis group and nine patients from the non-pre-dialysis group) and transfer to peritoneal dialysis (two patients from the non-pre-dialysis group). Five patients were excluded due to a significant number of missing data points. An incident cohort of 120 adult hemodialysis patients satisfying the above inclusion and exclusion criteria was identified, of whom 74 patients had attended the pre-dialysis clinic on at least one occasion, while 46 patients began dialysis without previous pre-dialysis clinic attendance.

Independent variables
---------------------

Possible predictors of outcome included age (at dialysis initiation), sex, diabetes (yes/no), pre-dialysis clinic attendance (yes/no), length of pre-dialysis clinic attendance, date of QOL assessment, history of ischemic heart disease, congestive heart failure, peripheral vascular disease, stroke, malignancy, and chronic lung disease, Kt/V (measured within three months of QOL assessment), residual creatinine clearance (at dialysis initiation), albumin and hemoglobin levels (measured within one month of QOL assessment). Positive diabetic status included a history of either Type I or II diabetes. Patients were classified as having attended the pre-dialysis clinic if they presented on at least one occasion. Length of pre-dialysis clinic attendance was measured from the date of first attendance to date of dialysis initiation. The date at which the QOL assessment was performed was included as an independent variable (calculated from the date of dialysis initiation to QOL assessment) in order to account for any possible confounding effect due to the different times at which the Health Survey was administered. Kt/V was estimated based on a single-pool model using the urea reduction ratio method. Residual creatinine clearance at the time of dialysis initiation was estimated using the Gault-Cockroft method and the serum creatinine level at the start of dialysis initiation. History of co-morbid disease was extracted from medical records using local chart review and the Canadian Organ Replacement Registry (a national dialysis database). Angina was defined as any previous or present history of ischemic symptoms (possibly but not necessarily requiring anti-anginal therapy) or any past history of coronary artery angioplasty or bypass surgery. Congestive heart failure was defined as any previous or present history of dyspnea, orthopnea or paroxysmal nocturnal dyspnea due to cardiac etiologies and not solely attributable to renal failure and volume expansion alone. Echocardiographic documentation of systolic dysfunction was not required. Peripheral vascular disease was defined as either a typical history of exertional leg discomfort or previous history of vascular angioplasty or bypass surgery. Stroke was defined clinically and did not require radiographic confirmation. Patients were primarily of Caucasian race, reflecting the demographics of the southeastern Ontario region. Race was therefore not included as an independent variable.

Dependent variables
-------------------

QOL was assessed using the SF 36-Item Health Survey. \[[@B9],[@B10]\] This health survey is routinely administered every three months to all dialysis patients at Kingston General Hospital as standard care in order to monitor patient well-being. The survey is a well-validated self-report questionnaire that assesses QOL in eight domains: physical function (phy), physical role limitation (rlp), emotional role limitation (rle), social function (soc), body pain (pai), emotional well being (ewb), energy (eng), and general health (gh). The maximum score in each domain is 100 -- higher scores being desirable and indicative of better well-being or less pain. Changes in score of 5 units have been shown to be clinically relevant. \[[@B9]\] Additionally, the final multivariate model was used to explore the association between selected independent predictors of outcome and two composite scores that summarize physical and emotional QOL. Composite physical (praw) and emotional (eraw) scores were calculated based on an aggregation of the eight separate domain scores using standard published equations (where each of the domain scores is standardized using age-stratified Canadian national means and standard deviations). \[[@B11],[@B12]\]

Statistical analysis
--------------------

The distribution of independent and dependent variables was examined using univariate analyses. Comparison of continuous and categorical independent variables was performed between patients who attended the pre-dialysis clinic and those who did not using t-test, Mann-Whitney, and chi-square analyses as appropriate (Tables 1 and 2; see [Additional file Table 1](#S1){ref-type="supplementary-material"} and [Additional file Table 2](#S2){ref-type="supplementary-material"}). QOL scores were compared between patients who attended the pre-dialysis clinic and those who did not using t-test and Mann-Whitney analyses as appropriate (Table 3; see [Additional file Table 3](#S3){ref-type="supplementary-material"}). The association between independent variables and QOL scores in each of the eight health domains was examined using bivariate linear regression analysis (Table 4; see [Additional file Table 4](#S4){ref-type="supplementary-material"}). Based on the results of bivariate analyses, clinically relevant and statistically significant independent variables were selected for multivariate linear regression analysis (Table 5; see [Additional file Table 5](#S5){ref-type="supplementary-material"}). The final model was examined for adherence to linear model assumptions. In the situation where linear model assumptions were compromised due to the non-parametric distribution of outcome variables, non-parametric bivariate and multivariate analyses were also performed using Spearman correlation and logistic regression analyses respectively. As such, non-parametric analyses were used to examine physical and emotional role limitation scores due to the non-parametric distribution of these two dependent variables. The non-parametric distribution of these two domains is attributed to the relatively small number of questions devoted to these two domains in the SF 36-item Health Survey and is therefore not unexpected. Recognizing that earlier research using the health survey has traditionally reported results using parametric analyses despite the non-parametric distribution of the data, the present paper will present both parametric and non-parametric results in order to conform to previous methodologies and permit comparisons.

Analyses were preformed using the SAS statistical analysis package for personal computers (version 6.12, Cary, NC). The study protocol was approved by the Queen\'s University Health Sciences and Affiliated Teaching Hospitals Research Ethics Board.

Results
=======

The distribution of categorical independent variables within the entire cohort and according to pre-dialysis clinic attendance is presented in Table 1 (see [Additional file Table 1](#S1){ref-type="supplementary-material"}). Overall, 55.8% of patients were male, while diabetes was identified in 43.6% of patients. Almost 40% of patients had a history of ischemic heart disease. The distribution of categorical independent variables was compared between the group of patients who attended the pre-dialysis clinic and those who did not. No statistically significant between-group differences were identified.

Table 2 (see [Additional file Table 2](#S2){ref-type="supplementary-material"}) shows the distribution of continuous independent variables within the entire cohort and according to pre-dialysis clinic attendance. Mean age for the entire cohort was 62.7 years. Mean residual creatinine clearance clearance at dialysis initiation was 0.18 ml/s. A comparison of continuous independent variables according to pre-dialysis clinic attendance demonstrated no statistically significant differences between the two groups, with the exception of residual creatinine clearance. Residual creatinine clearance at dialysis initiation was significantly higher among the group of patients attending the pre-dialysis clinic (p = 0.05).

Physical function, physical role limitation, emotional role limitation, social function, and general health scores were statistically higher among patients who attended the pre-dialysis clinic compared to patients who did not (Table 3; see [Additional file Table 3](#S3){ref-type="supplementary-material"}).

Results of a series of bivariate linear regression analyses examining the association between each independent variable and each of the eight QOL domains are presented in Table 4 (see [Additional file Table 4](#S4){ref-type="supplementary-material"}). A positive parameter estimate denotes a positive correlation between independent variable and QOL score, while a negative parameter estimate indicates an inverse relationship. Younger age, male gender, pre-dialysis clinic attendance, longer attendance at the pre-dialysis clinic, higher residual creatinine clearance at dialysis initiation, absence of ischemic heart disease, stroke, and peripheral vascular disease, and higher albumin level were associated with a significantly higher physical function score. Higher scores in the physical role limitation domain were associated with younger age, pre-dialysis clinic attendance, and higher albumin level. Because physical role limitation scores did not follow a normal distribution, the bivariate analysis was re-examined using a nonparametric Spearman correlation analysis. Using a nonparametric analysis, pre-dialysis clinic attendance still predicted higher QOL score. Younger age (p = 0.02), pre-dialysis clinic attendance (p = 0.03), absence of ischemic heart disease (p = 0.04) and stroke (p = 0.03), and higher albumin level (p = 0.03) were associated with higher physical role limitation scores. Linear regression analysis suggested that higher emotional role limitation score was predicted by longer pre-dialysis clinic attendance. Due to the non-parametric distribution of the emotional role limitation score, a non-parametric analysis was also preformed, which demonstrated that pre-dialysis clinic attendance and longer pre-dialysis clinic attendance were the only predictors of better emotional role limitation score (p = 0.04 and p = 0.01 respectively). Significant predictors of better social function were pre-dialysis clinic attendance, absence of lung disease, and higher albumin level. Male gender, absence of ischemic heart disease, peripheral vascular disease and malignancy, and higher albumin level were significant predictors of higher pain scores (where higher score is indicative of less pain). Better emotional well-being was associated with male gender alone. Younger age, higher residual creatinine clearance at dialysis initiation, absence of peripheral vascular disease and lung disease, and higher albumin level predicted better energy level. Male gender, pre-dialysis clinic attendance, absence of ischemic heart disease, peripheral vascular disease and lung disease, and higher albumin level were significant predictors of higher general health scores.

A total of nine independent variables were selected for inclusion in the final multivariate analysis: pre-dialysis clinic attendance, residual creatinine clearance, Kt/V, age, albumin, sex, ischemic heart disease, peripheral vascular disease, and diabetes (Table 5; see [Additional file Table 5](#S5){ref-type="supplementary-material"}). Age, sex, pre-dialysis clinic attendance, ischemic heart disease, peripheral vascular disease, and albumin were included as covariates because of their significant associations with QOL scores in bivariate analyses. Despite the absence of statistically significant associations with QOL scores, diabetes and Kt/V were included as covariates in the final multivariate model because of their perceived clinical relevance. Since residual renal function at dialysis initiation was significantly higher among patients attending the pre-dialysis clinic compared to those who did not, residual creatinine clearance was also included in the final model in order to permit statistical adjustment. The final multivariate model suggested that pre-dialysis clinic attendance was an independent predictor of four of eight health domains: higher physical function, emotional role limitation, social function, and general health scores. Physical function score was predicted by pre-dialysis clinic attendance, younger age, and absence of ischemic heart disease and peripheral vascular disease, while emotional role limitation and social function scores were predicted by pre-dialysis clinic attendance alone. Male gender and absence of peripheral vascular disease were independent predictors of higher pain score (reflecting less pain). Higher emotional well-being score was predicted by male gender. Pre-dialysis clinic attendance, male gender, and absence of peripheral vascular disease were independent predictors of higher general health score. Because of the non-parametric distribution of the physical and emotional role limitation scores, repeat analyses were performed for these two QOL domains using multivariate logistic regression analyses. The conclusions of logistic and linear regression analyses were identical.

Multivariate analysis using the final model suggested that pre-dialysis clinic attendance, younger age, and absence of ischemic heart disease and peripheral vascular disease were independent predictors of the composite physical health score (Table 5; see [Additional file Table 5](#S5){ref-type="supplementary-material"}). Pre-dialysis clinic attendance was not an independent predictor of the mental health composite score.

Adjusted r-squared values (Table 5; [Additional file Table 5](#S5){ref-type="supplementary-material"}) indicate the extent to which the variability of each health survey domain score can be explained by the final multivariate model. The final model accounts for 42% and 28% of the physical function score and composite physical score variability respectively, suggesting that the model is most effective in predicting physical function scores.

Discussion
==========

Although previous research has suggested that pre-dialysis clinic attendance is associated with better objective outcomes compared to standard care, research examining the benefits of pre-dialysis care with respect to patient-oriented outcomes such as QOL is limited. The results of the present study suggest that attendance at a predialysis clinic is an independent predictor of improved quality of life measured within the first six months of dialysis initiation in an incident cohort of chronic hemodialysis patients. The benefits of pre-dialysis clinic attendance are therefore not limited to objective measures of patient health such as decreased hospitalization and better biochemical profiles at the time of dialysis initiation. Rather, these results suggest that the benefits of pre-dialysis clinic attendance can be extended to include more subjective measures of patient health.

The evaluation of medical therapies using patient-oriented outcomes has acquired a larger research profile in recent years. Nonetheless, to our knowledge, only one study has formally evaluated the benefits of pre-dialysis clinic attendance using patient-administered QOL questionnaires. \[[@B8]\] The present study confirms the conclusions of Sesso et al, who suggested that pre-dialysis clinic attendance improved QOL as measured by the Kidney Disease Questionnaire. However, only 28% of patients were over age 60 in the Brazilian study. Recognizing that the benefits of pre-dialysis clinic attendance were particularly pronounced among elderly patients, the value of pre-dialysis clinic attendance may have been underestimated by this earlier study. The mean age of 62.7 years in the present cohort is a closer representation of North American dialysis populations. The study is also benefited by a publicly funded and universally accessible Canadian healthcare system in which the importance of confounding variables such as socioeconomic status has been minimized. Furthermore, whereas the results of the Brazilian study were adjusted for age and time of diagnosis, differences in residual renal function and other covariates were not included. The present study has controlled for several covariates including age, sex, ischemic heart disease, peripheral vascular disease, diabetes, albumin, residual renal function, and Kt/V.

Interestingly, pre-dialysis clinic attendance was associated with improved QOL scores in a wide range of domains that included both physical and emotional outcomes. Attendance was an independent predictor of better QOL score in four of eight domains: higher physical function, emotional role limitation, social function, and general health. Indeed, pre-dialysis clinic attendance was the only independent predictor of QOL score in the case of emotional role limitation and social function. Importantly, the difference in QOL scores between patients who did and patients who did not attend the pre-dialysis clinic is not only statistically significant but clinically significant as well. Previous research has suggested that a difference in SF-36 scores of five or more points is considered clinically relevant. \[[@B9],[@B11]\]

The means by which a pre-dialysis clinic leads to better physical and emotional QOL is likely multi-factorial, resulting from opportunities for patient education, dietary counseling, modality selection, dialysis access creation, and management of co-morbid illnesses. The importance of early pre-dialysis correction of anemia using erythropoietin has also been emphasized in recent years. Several studies have suggested that earlier initiation of erythropoietin may improve patient morbidity and mortality by retarding or preventing the development of cardiomyopathy secondary to anemia. \[[@B13]\]

The association between several covariates and QOL score seems intuitive in many cases. For example, in addition to pre-dialysis clinic attendance, younger age and absence of ischemic heart disease and peripheral vascular disease were independent predictors of better physical function score. Absence of peripheral vascular disease was also an independent predictor of better pain and general health scores. Although bivariate analysis suggested that higher serum albumin level predicted better QOL in many domains (including physical function, physical role limitation, social function, pain, energy, and general health), a statistically independent relationship between serum albumin and QOL was not demonstrated by multivariate analysis.

Both pre-dialysis clinic attendance and increasing length of time attending the pre-dialysis clinic were predictors of better physical function and emotional role limitation scores in univariate analysis, but not multivariate analysis, thus suggesting statistical redundancy between these two covariates. The absence of an independent association between length of pre-dialysis clinic attendance (and presumably severity of chronic renal failure) and QOL outcome parallels the conclusions of Harris and colleagues, who found that, in a cohort of 360 non-dialysis patients with chronic renal failure, functional status was correlated with socioeconomic status and comorbid illness, but not severity of renal function. \[[@B14]\]

Interestingly, multivariate analysis suggested that male gender was an independent predictor of better pain, emotional well-being, and general health scores. Although a significant association between male gender and QOL scores may seem doubtful, this finding has in fact been demonstrated previously in non-dialysis populations in which males consistently score slightly higher than females in all QOL domains. \[[@B12]\]

QOL scores in the present study are comparable to earlier reports and underscore the fact that QOL is severely compromised among dialysis populations. \[[@B15],[@B16]\] In a prospective Dutch study involving 13 dialysis centres, QOL measured three months after dialysis initiation using the SF-36 questionnaire was significantly impaired compared to a general non-dialysis population, particularly with respect to physical role limitation and general health status. \[[@B15]\] Mean physical role limitation and general health scores for hemodialysis patients corresponded to the lowest 8% and 12% of the reference group respectively. Independent predictors of poor quality of life included a higher number of co-morbid illnesses, lower hemoglobin level, and lower residual renal function. Pre-dialysis attendance was not included as a covariate.

Compared to more objective measures of patient well-being, the importance of QOL, not only as an outcome, but also as a predictor of patient morbidity and mortality, is probably under-estimated. For example, DeOreo has demonstrated that dialysis patients with a physical component summary score below the cohort median were twice as likely to die and 1.5 times more likely to be hospitalized compared to patients with scores above the median. These findings suggest that patient-reported QOL is as much a significant predictor of mortality as more well-established markers of patient care such as normalized protein catabolic rate and Kt/V. \[[@B17]\] Patient morbidity and mortality is significant even prior to dialysis initiation. \[[@B14],[@B18]\] Therefore, interventions that can potentially improve QOL should be pursued early and aggressively during the pre-dialysis phase of chronic renal insufficiency. The present study suggests that pre-dialysis care can improve QOL measured six months after dialysis initiation, while the conclusions of DeOreo argue that such improvement could lead to lower patient mortality.

The inability of the present study to identify any predictors of the mental health composite score may be due to small sample size causing a Type II statistical error. However, other researchers have demonstrated that, while hemodialysis patients report low physical scores, mental and emotional scores are less affected, suggesting that the absence of any statistically significant predictors of poor mental health composite score in the present study may be valid. \[[@B15],[@B16]\]

Surprisingly, diabetes did not predict QOL scores, even in bivariate analysis. This also could be due to effect of small sample size. Alternatively, failure to identify an association between diabetes and QOL scores may be due to short follow-up time in which the more long-term complications of diabetes could not be realized. Furthermore, the results of studies that have reported an association between diabetes and functional status are possibly limited by the use of cross-sectional study designs and prevalent cohort populations. \[[@B19]\] In a prospective cohort of dialysis patients over age 60, Kutner and colleagues showed that diabetic nephropathy was not an independent predictor of survival when controlled for functional status in multivariate analysis. \[[@B16]\]

Kt/V was not a predictor of QOL outcomes in this study or previous investigations. \[[@B15]\] Some investigators have suggested that the absence of an association between Kt/V and QOL may be due to the effect of residual renal function and the possibility that clearance achieved by native kidneys is superior to that obtained by dialysis. \[[@B15]\] However, residual creatinine clearance was not a predictor of QOL outcomes in the present study. The absence of a statistically significant association between Kt/V and QOL scores could also be attributed to short follow-up time in which QOL assessments were performed soon after dialysis initiation. Alternatively, since Kt/V values were confined to a relatively narrow range in which the majority of patients achieved adequate dialysis dosing, a Type II statistical error simply due to lack of variability of the independent variable may account for the absence of any statistically significant association.

The present study is limited by the retrospective design in which the possibility of selection bias cannot be definitively excluded. Indeed, previous research has demonstrated that referral of patients by primary care physicians to nephrology programs is influenced, either consciously or unconsciously, by patient-related factors including age, gender, diabetes, and other co-morbid illnesses. \[[@B20],[@B21]\] Consequently, referral bias is currently a feature of primary care practice and is likely unavoidable regardless of study design. Not surprisingly, the existence of referral bias in the present study is suggested by the lower residual renal function at the time of dialysis initiation seen in patients who did not attend the pre-dialysis clinic compared to those patients who did attend the clinic. Patient self-selection is also a consideration. For example, it is possible that non-compliant patients are less willing to attend the pre-dialysis clinic, and as such, the poorer QOL scores reported by patients who did not attend the pre-dialysis clinic may be related to issues of self-selection and compliance rather than pre-dialysis clinic attendance itself. However, other than residual renal function, no statistically significant demographic or clinical differences between those patients who did attend the pre-dialysis clinic and those who did not were identified in the present study. Notwithstanding the possibilities of referral and self-selection biases, the decision to examine an incident population restricted to new dialysis patients has avoided the other biases associated with earlier investigations using cross-sectional study designs and prevalent populations. \[[@B19]\]

The retrospective study design also causes difficulty in isolating the independent influences of confounding covariates. For example, pre-dialysis clinic attendance and early dialysis initiation are separate yet potentially confounding variables, both of which are likely related to improved QOL scores. In the absence of randomized prospective studies, the effect of each covariate is difficult to examine independently since patients referred to a pre-dialysis clinic are probably less likely to require emergent dialysis initiation due to more regular follow-up. Consequently, it can be argued that the benefits of pre-dialysis clinic attendance may be the result of either selection biases (related to patient age and co-morbid illness) or confounding variables (such as timing of dialysis initiation and residual renal function) rather than pre-dialysis clinic interventions in and of themselves. However, a comparison of continuous and categorical independent variables between patients who did and did not attend the pre-dialysis clinic failed to demonstrate any statistically significant difference between the two groups apart from residual creatinine clearance at the time of dialysis initiation (which was therefore included as a covariate in multivariate analysis).

The results of the present study support the NIH recommendation suggesting that patients with chronic progressive renal insufficiency should be referred to a pre-dialysis team in order to minimize patient morbidity and mortality. While previous research has demonstrated that pre-dialysis clinic attendance is associated with favourable objective outcomes (including better biochemical profiles and decreased hospitalization at the time of dialysis initiation), the results of the present study suggest that the benefits of a pre-dialysis program can be extended to include more subjective measures of health status. The importance of this finding is underscored by earlier research demonstrating that QOL scores are themselves independent predictors of patient mortality. However, it remains unclear to what extent the benefits of pre-dialysis clinic attendance are sustainable over time. Longitudinal studies evaluating the effects of pre-dialysis care with respect to prospective repeated-measure QOL assessments and objective outcomes are therefore required.

Competing interests
===================

None declared.

Authors\' contributions
=======================

Authors 1 (CW) and 2 (RP) designed the study and carried out data entry. Authors 3 (ML) and 4 (DH) performed the statistical analyses. Author 4 (DH) drafted the manuscript. All authors read and approved the final manuscript.

Pre-publication history
=======================

The pre-publication history for this paper can be accessed here:

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Supplementary Material
======================

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Acknowledgements
================

The authors grateful acknowledge the assistance of the Canadian Organ Replacement Registry and medical record staff of Kingston General Hospital. We also appreciate the assistance of Ms. Wilma Hopman, Mckenzie Health services Research Group, Queen\'s University, Kingston, Ontario, Canada.


Background
==========

The primary cause of senile cataract development is still unclear. To date the involvement of α-crystallin, a molecular chaperone for β- and γ-crystallin, has principally been considered in senile cataract development \[[@B1],[@B2]\], the decrease in the chaperone ability of α-crystallin with age being implicated. Xanthurenic acid is produced from a metabolite of tryptophan (3-hydroxy-kynurenine) \[[@B3]\] in the presence of 2,3-dioxygenase \[[@B4]-[@B7]\]. While 3-hydroxykynurenine \[[@B3]\] is photochemically inert \[[@B8]\] and serves as a protective UV filter of retina, xanthurenic acid is a photosensitizer \[[@B9],[@B10]\]. Xanthurenic acid accumulates with aging in mammalian lenses \[[@B11],[@B12]\] and is involved in the increased fluorescence of the lens with aging \[[@B13]\]. The glucoside of xanthurenic acid is also present in aged lenses \[[@B14]\]. Xanthurenic acid is an example of an endogenous ER stressor provoking an accumulation of unfolded proteins which in turn leads to an overexpression of Grp 94 and calreticulin in the lens epithelial cells of young mammals \[[@B15]\]. We have previously reported that porcine lens epithelial cells in culture respond to xanthurenic acid exposure by an overexpression of stress chaperone proteins, Grp 94 and calreticulin, in \[[@B15]\]. Here, we report that xanthurenic acid leads to human lens epithelial cells (HuLEC) death associated with caspase-3 activation, intracellular Ca^2+^ increase and calpain Lp82 induction. Previously, lens epithelial cell apoptosis was observed in an *in vitro* model of the cataract \[[@B16]-[@B18]\].

Materials and Methods
=====================

Reagents
--------

We used the following polyclonal antibodies from Santa Cruz Biotechnology Inc. CA,  against cytochrome c, secondary antibodies IgG-fluoresceine (FITC)-conjugated. Primary antibody against active caspase-3 p17 was from Promega, Madison, USA. Secondary IgG-Texas Red-conjugated antibodies and Mitotracker CMXRos, DiOC~18~, Calcium Orange™, Hoechst 33342, propidium iodide were from Molecular Probes, Leiden, The Netherlands. Antibody against GPIP1 peptide of gelsolin was prepared as described previously \[[@B24]\]. Other reagents were from Sigma if not specified. Antibody against calpain Lp82 was from Dr. T. R. Shearer (University of Oregon, Oregon, USA).

Preparation of human lens epithelial cells primary culture
----------------------------------------------------------

Lenses were obtained after transplantation of cornea from 58, 59, and 63 years old donors from Eye Bank University Hospital, Bern. The primary cells cultures were prepared separately from every donor using two lens capsules. The lens capsules from one donor were treated with 1.5 mg/ml of collagenase 1A and 4 overnight at 37°C. Thereafter, 1 ml of MEM medium with 10% FCS was added, and cells were centrifuged for 10 min at 300 g. The supernatant was discarded and the cells were re-suspended in 1.5 ml the growth medium, as described below, for 14 days in one well of 12-well plate. The cells were cultivated in the dark, in Minimal Essential Medium (MEM) with Earle\'s salts (Gibco BRL). Cells were grown under a humidified atmosphere of 5% CO~2~ in air at 37°C in MEM supplemented with 10% fetal bovine serum, penicillin (10 U/ml), streptomycin (10 μg/ml) and fungizone (250 ng/ml). When confluent, they were incubated in MEM or MEM supplemented with xanthurenic acid. A 20 mM stock solution of xanthurenic acid was prepared in 0.5 M NaHCO~3~, and diluted in 0.01 M PBS pH 7.4.

Cytotoxicity and apoptosis assay
--------------------------------

Cells were observed with differential interference contrast and phase contrast optics on a Zeiss Avionert 405 M inverted microscope, and images recorded with a Matsumoto 3-chip CCD cooled camera with images stored using Adobe Photoshop 4. Cell viability was determined by staining the cells with Hoechst 33342 and propidium iodide (PI) (Juro, Switzerland) using 50 μg/ml of each dye. Fragmented, apoptotic, nuclei were observed with excitation at 350 nm, and necrotic nuclei at 530 nm.

Cell lysis and immunobloting
----------------------------

Cells were washed twice with cold 0.01 M PBS, pH 7.4. For Western blotting, cells were lysed in buffer containing 50 mM Tris (pH 8.0), 150 mM NaCl, 1 %Triton X-100, and the following protease inhibitors: 1 mM phenyl-methylsulfonyl fluoride, and leupeptin, aprotinin, and pepstatin, each at 1 μg/ml. The concentration of proteins was calculated from the absorption maximum at 280 nm, as described previously \[[@B15]\], and the concentration of xanthurenic acid from its absorption maximum at 342 nm (ε~M~ 6500). The lysate was centrifuged for 10 min at 14 000 g, and the supernatant was boiled in loading-buffer for 5 min. Proteins (50 μg per lane) were separated by SDS-PAGE containing 10 or 12.5% acrylamide. After transfer to Hybond ECL membrane (Amersham Pharmacia Biotech AB, Uppsala, Sweden) the proteins were probed with the appropriate antibodies. Chemilunimescence ECL system (Amersham Pharmacia Biotech AB, Uppsala, Sweden) was used for the detection of peroxidase-conjugated secondary antibody.

Immunofluorescence studies
--------------------------

Cells grown on glass coverslips were fixed for 10 min at room temperature in 4% paraformaldehyde in 0.1 M PIPES, pH 6.8, washed in PBS and permeabilized for 5 min in PIPES containing 0.05% saponin (65 μl per coverslip), washed in PBS, incubated for 10 min in cold aceton for additional fixing and permeabilisation, and again washed in PBS. Cells were incubated for 1.5 hour with the first antibody diluted in PBS containing 1% bovine serum albumine, and after washing incubated for 1.5 hour with the secondary antibody. The coverslips were then washed in PBS and incubated for 10 min with 65 μl of solution containing 1μl of Hoechst 33342 dye (1 mg/ml), washed in PBS, and incubated with Antifade Kits (Molecular Probes, Leiden, The Netherlands) according to the supplier\'s instruction. Staining of mitochondria was performed using Mitotracker CMXRos, as follows: confluent cells cultures were pre-incubated without or with xanthurenic acid in MEM medium for 72 hours. The medium was removed and replace with medium containing 100 nM Mitotracker CMXRos. After an incubation for 1 hour Mitotracker CMXRos was removed, coverslips were washed twice with PBS, and mounted on the slides using as antioxidative solution 9% w/v of Mowiol (Calbiochem) in 22% glycerol buffered with 0.2 mM Tris/HCl to pH containing 3.5% (w/v) of 1,4-diazabicyclo (2.2.2) octane (Sigma). Membranes were stained using overnight incubation in DiOC~18~ at concentration of 12 μM in MEM medium.

Results
=======

Xanthurenic acid activates caspase-3 and translocates gelsolin from the mitochondrial region to the cytoskeleton
----------------------------------------------------------------------------------------------------------------

We observed that 10 μM xanthurenic acid in the HuLEC cell culture medium activate caspase-3 (Fig. [1B](#F1){ref-type="fig"}). In the same cells the translocation of gelsolin from perinuclear region to cytoskeleton was observed (Fig. [1C, 1D](#F1){ref-type="fig"}). Previously it was reported that caspase-3 and -9 are associated with mitochondrial membranes \[[@B19]\]. Gelsolin is the cytoskeletal protein responsible for the disintegration of F actin during apoptosis induced by Fas \[[@B20]\]. It was also suggested that gelsolin keeps caspases in the inactive state \[[@B21]\]. We reported previously that gelsolin is abnormally cleaved during apoptosis induced by xanthurenic acid. This abnormal gelsolin cleavage could be a reason of absence of cytoskeleton breakdown in the presence of active caspase-3 (Fig. [1B, 1D](#F1){ref-type="fig"}).

![Caspase-3 activation and gelsolin translocation in HuLEC in the presence of xanthurenic acid for 96 hours. Cells stained for active caspase-3 (A, B), and gelsolin (C, D): (A) control cells stained with Hoechst 33342 and for cleaved caspase-3, (B) cells exposed to 10 μM xanthurenic acid and stained for cleaved caspase-3, (C) control cells, (D) in the presence of 10 μM xanthurenic acid.](1471-2415-2-1-1){#F1}

Xanthurenic acid leads to mitochondrial migration, cytochrome c release, and destruction of mitochondrial structure
-------------------------------------------------------------------------------------------------------------------

In the control cells mitochondria occupy the perinuclear region (Fig. [2A](#F2){ref-type="fig"}). In the presence of 10 μM xanthurenic acid mitochondrial migration was observed (Fig. [2B](#F2){ref-type="fig"}). However, at higher concentrations (20 and 40 μM) xanthurenic acid led to the destruction of mitochondria. An intrinsic apoptotic pathway is activated by cytochrome c release and apoptosome formation with APAF-1 and ATP \[[@B22]\]. The apoptosome leads to activation of caspase-9, which activates caspase-3. We observed that in the presence of 10 μM xanthurenic acid cytochrome c was release from mitochondria (Fig. [3](#F3){ref-type="fig"}). APAF-1 is present in the HuLEC and its level is independent from xanthurenic acid concentration (not shown). Thus, release of cytochrome c is responsible for the observed caspase-3 activation, and nucleus cleavage.

![HuLEC, grown in the presence of xanthurenic acid for 96 hours, stained with Mito-Tracker Red CMXRos: (A) control cells; (B-D) in the presence of xanthurenic acid: (B) 10 μM, (C) 20 μM, (D) 40 μM.](1471-2415-2-1-2){#F2}

![Cytochrome c release in HuLEC grown in the presence of xanthurenic acid for 72 hours. (A) control cells, (B) cells grown in the presence of 10 μM xanthurenic acid.](1471-2415-2-1-3){#F3}

Mitochondrial damage in the presence of xanthurenic acid is associated with nuclear cleavage
--------------------------------------------------------------------------------------------

In control cells about 5 percent are apoptotic (Fig. [4A](#F4){ref-type="fig"}, Fig. [5](#F5){ref-type="fig"}). In the presence of 10 μM xanthurenic acid condensed and/or cleaved nuclei were observed, which a mostly stained only with Hoechst 33342, and not with propidium iodide. This indicated that the observed cell death was apoptotic and not necrotic (Fig. [4B, 4C](#F4){ref-type="fig"}). With concentrations of xanthurenic acid of 20 μM (Fig. [4D,4E,4F](#F4){ref-type="fig"}) and 40 μM (Fig. [4G,4H,4I](#F4){ref-type="fig"}) the destruction of the mitochondrial structure, was associated with nuclear cleavage. Cell death depended on the xanthurenic acid concentration (Fig. [5](#F5){ref-type="fig"}). In the presence of 10 μM xanthurenic acid about 40 percent of cells were dead, and an increase of xanthurenic acid to 20 μM provoked about 70 percent of cell death.

![Nuclear DNA degradation in HuLEC in the presence of xanthurenic acid. (A) control cells stained with Hoechst; (B) cells grown in the presence of 10 μM xanthurenic acid for 96 h and stained with Hoechst; (C) as (B) but stained with Hoechst and propidium iodide; (D-F) cells in the presence of 20 μM xanthurenic acid: (D) stained with Mitotracker, (E) stained with Hoechst, (F) merge of (D and E); (G-I) cells in the presence of 40 μM xanthurenic acid: (G) stained with Mitotracker, (H) stained with Hoechst, (I) merge of (G) and (H).](1471-2415-2-1-4){#F4}

![Apoptotic death of HuLEC in the presence of xanthurenic acid.](1471-2415-2-1-5){#F5}

Xanthurenic acid leads to damage of the cell membrane
-----------------------------------------------------

The cell membranes were stained with DiOC~18~ and co-stained with Mito-Tracker Red CMXRos, and the nucleus was stained with Hoechst 33342 (Fig. [6](#F6){ref-type="fig"}). In the control cell mitochondria were in the perinuclear region and the cell membranes were uniformly stained with DiOC~18~ (Fig. [6A,6B,6C](#F6){ref-type="fig"}) In the presence of 10 μM xanthurenic acid the mitochondria migrated to the cell periphery and the cell membranes were not uniformly stained (Fig. [6D,6E,6F](#F6){ref-type="fig"}). In the presence of 20 μM of xanthurenic acid the mitochondrial structure was destroyed, nuclear DNA was degraded and membranes were not stained with DiOC~18~ (Fig. [6G,6H,6I](#F6){ref-type="fig"}).

![Staining of the mitochondria (Mito-Tracker Red CMXRos), nucleus (Hoechst) and cell membranes (DiOC~18~) in HuLEC grown in the presence of xanthurenic acid for 96 hours: (A-C), control cells: (A) mitochondria, (B) nucleus, (C) membranes; (D-F) with 10 μM xanthurenic acid: (D) mitochondria, (E) membranes, (F) merge of D, E, and nucleus stained by Hoechst; (G-I) with 20 μM xanthurenic acid: (G) mitochondria, (H) merge of (G) and Hoechst staining, (I) membranes](1471-2415-2-1-6){#F6}

Xanthurenic acid causes an increase of free intracellular Ca^2+^ and an induction of the lens calpain Lp82
----------------------------------------------------------------------------------------------------------

Ca^2+^ increases are associated with cataract development. We investigated intracellular Ca^2+^ by loading the cells with acetometoxyl ester of Calcium Orange™. This dye becomes fluorescent when hydrolysed in the cell by esterases and conjugated with free Ca^2+^ Cells were incubated without xanthurenic acid or with xanthurenic acid at concentration of 0.125; 0.25, 0.5; 1; 2; 5, 10, and 20 μM. A presence of xanthurenic acid in the cell culture medium higher then 2 μM provokes an increase of intracellular Ca^2+^ in comparison with control. In the presence of xanthurenic acid at concentration of 10 μM and 20 μM an intensive staining with Calcium Orange™ was observed indicating an increase of free Ca^2+^ in the cell in a xanthurenic acid concentration-dependent manner (Fig. [7](#F7){ref-type="fig"}).

![Increase of free Ca^2+^ in HuLEC after growth in the presence of xanthurenic acid for 96 hours. HuLEC were stained with Calcium Orange™: (A) control cells, (B) in the presence of 10 μM xanthurenic acid, (C) in the presence of 20 μM xanthurenic acid.](1471-2415-2-1-7){#F7}

The lens specific calpain Lp82 \[[@B23]\] was not detectable using the Western blot analysis in the lens epithelial cell culture cultivated in the absence of xanthurenic acid (Fig. [8](#F8){ref-type="fig"}, lane 1). In the presence of xanthurenic acid the calpain Lp82 was induced (Fig. [8](#F8){ref-type="fig"}, lanes 2-4).

Discussion
==========

Our previous studies in cell culture showed that xanthurenic acid is a potent endogenous pathological substance in retinal pigment epithelium and smooth muscle cells \[[@B24]\]. In this study, we observed that xanthurenic acid leads to lens epithelial cells death associated with an induction of caspase-3 and calpain Lp82. Apoptosis was observed in models of cataract upon lens treatment with staurosporine, diamide, and ionophore \[[@B16]-[@B18]\]. In the selenite cataract model caspase-3 and calpain were induced \[[@B25]\]. In the normal apoptosis, such as observed with development, cells disappear because of caspase-3-dependent cleavage of DNA and the cytoskeleton \[[@B26],[@B27]\]. The caspase remodeling of the cytoskeleton was indicated as a possible mechanism leading to the aging of lenses \[[@B28],[@B29]\]. Apoptosis is considered as a common cellular basis for non-congenital cataract in mammals \[[@B30]\]. Recent data indicate that senile cataract is a result of proteolysis of crystalins by calpains \[[@B31]\]. Calpains are activated by Ca^2+^\[[@B32]\].

Previously, it was reported that ER Ca^2+^ homeostasis affects the cells\' sensitivity to apoptosis \[[@B33]\]. Lens epithelial cells overloaded by Ca^2+^ showed vimentin cleavage and opacification \[[@B34]\] Thapsigargin, a plant alkaloid, which depletes Ca^2+^ from the ER, was used to stop lens epithelial cell growth \[[@B35]\]. Here, we show that xanthurenic acid, an endogenous molecule, lead to induction of calpain Lp82 and caspase-3 activation. The simultaneus activation of caspase and calpain may lead to an abnormality of apoptosis because calpain cleaves caspases \[[@B36]\]. The calpain Lp82 is involved in cataract formation in connexin α-3 knockout mice \[[@B37]\]. The induction of calpain Lp82 leads to the cleavage of crystalins in the lenses and is involved in the senile cataract development \[[@B38]\]. Xanthurenic acid leads to formation of unfolded proteins \[[@B15]\].

An accumulation of unfolded protein can lead to Ca^2+^ release from intracellular stores as well to caspase induction \[[@B39],[@B40]\]. The observed death of the lens epithelial cells has apoptotic characteristics because release of cytochrome c and caspase-3 activation were observed. However, the apoptosis-like process does not lead to a collapse of the cytoskeleton driven by caspase-3 cleaved gelsolin in the presence of Fas-induced apoptosis \[[@B20]\]. In our study, in the presence of xanthurenic acid cells look normal when observed by the light microscopy. However, when visualized by fluorescence microscopy with Hoechst and propidium iodide a nuclear dysfunction is evident. We have previously reported that xanthurenic acid leads to an abnormal cleavage of gelsolin. The gelsolin stains cytoskeleton of the dead cells. Ca^2+^ and bis-phosphatidylinositol (PPI2) regulate, respectively, association and dissociation of gelsolin to actin \[[@B41]\]. Thus, also the increase in Ca^2+^ may play a role in the association of gelsolin to cytoskeleton.

The mitochondrial damage observed in the presence of xanthurenic acid associated with cytochrome c release could lower energy necessary for the lens enzymes activation observed in senile cataract development.

In this study we observed that xanthurenic acid accumulation can be an upstream event leading to an induction of the lens proteases: caspase and calpain. An accumulation of xanthurenic acid in the human lenses with aging can change the intracellular Ca^2+^ homeostasis. In summary, xanthurenic acid can induce the pathology of the lens epithelial cells without participation of light.

Author contributions
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All authors contributed equally to realize this work

Competing interests
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None declared

![Induction of calpain Lp82 in HuLEC growing 24 hours in controlled cells (lane-1) and in the presence of 1,2,4 mM xanthurenic acid (lanes-2,3,4, repectively).](1471-2415-2-1-8){#F8}

Pre-publication history
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The pre-publication history for this paper can be accessed here:

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Acknowledgements
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This work was supported by grant awarded to H. Z. M. by the Swiss National Foundation (32-59183.99) and MSE-Pharmaceutika GMBH, Bad Homburg, Germany. We thank Dr. T. R. Shearer for the kind gift of the antibody against calpain Lp82, Mr. R. Fischer and Mrs A. Marrero Nodarse for cell culture facilities, and Mrs D. Zuercher for lens epithelial cell culture preparation.