Hepatocyte Growth Factor in Saliva of Patients with Renal Failure and Periodontal Disease

Abstract

Background: Hepatocyte growth factor (HGF), endogenous cytokine with pleiotropic repairing and regeneration properties in relation to most tissues and organs, contributes to the progression of periodontal disease (PD). Furthermore, PD is a significant health problem in patients with chronic renal failure (CRF). The role of HGF in the development of PD in this specific population was not a subject of research so far. Material and methods: The following groups were enrolled in the study: (1) 26 chronic hemodialysis (HD) subjects, (2) 26 patients treated by continuous ambulatory peritoneal dialysis (CAPD), (3) 28 predialysis CRF patients, (4) 26 subjects with advanced PD (without coexisting diseases), and (5) 20 healthy subjects without PDs. HGF level in saliva was measured using the immunoenzymatic method. Gingival index, papillary bleeding index, plaque index, and the loss of clinical attachment level were evaluated. Results: The HGF level in saliva of HD patients was twice higher than in that of subjects with healthy periodontium. Direct relationships between proper HGF level in saliva and the indices GI, PBI, and PI in CAPD-treated patients and with more severe PD were shown. It was found that PD is most advanced in HD patients, moderately in CAPD-treated patients and to the smallest extent in predialysis CRF patients. Conclusions: The HGF level in mixed saliva is the index of PD progression in subjects without renal failure and in CAPD-treated patients. PD is common in renal failure patients and is a significant problem concerning general health status.

• chronic renal failure
• dialysis therapy
• periodontal disease
• hepatocyte growth factor

INTRODUCTION

Hepatocyte Growth Factor

The hepatocyte growth factor (HGF) was identified for the first time as an endogenous substance responsible for activation and regeneration of animal liver cells.¹ Further research showed that HGF is produced by all cells of mesenchymal origin, including connective tissue fibroblasts. The mechanisms regulating the release and unique autocrine and paracrine repairing effects of HGF in reaction to tissue injury are not fully known yet. HGF is regarded now as one of the most important endogenous growth substances regulating the functions of human organism—its embryogenesis, morphogenesis, and mitogenesis as well as the ability to repair and regenerate injured tissues and organs.^2–4 Some of the pleiotropic properties of HGF may be of vital importance in the development and progression of oral and periodontal diseases (PDs). They include impact on bone metabolism, proliferation of epithelial cells, healing process, and local inflammatory condition. Experimental research carried out by the Yasushi Daikuhara group (Kagoshima University Dental School, Kagoshima, Japan) in the years 1993–2000 proved, for the first time, that gingival and dental pulp fibroblasts show the ability to HGF synthesis and secretion,^5–8 and this effect is particularly expressed during the exposure of fibroblasts in tissue culture to proinflammatory substances such as interleukin-1, tumor necrosis factor, and prostaglandin E₂. The connection between HGF and periodontitis was described for the first time by Ohshima et al.^9–11 (Nihon University School of Dentistry, Tokyo, Japan) in a series of articles published in the years 2000–2002. In clinical examination, they showed, for the first time, that HGF level in the fluid of gingival pockets in patients with PD is between 10 and 20 times higher than in subjects without PD.¹⁰ Close correlations between HGF level in gingival fluid,¹⁰ unstimulated mixed saliva,¹¹ and progression of pathological changes in periodontium were found as well. The HGF is a new factor in the pathogenesis of PDs.

PD in Patients with Chronic Renal Failure

There are not many studies concerning oral health of adults with chronic renal failure (CRF) so far.^12–22 They were conducted in small groups of patients, mainly chronic hemodialysis (HD) patients, and gave ambiguous results. However, a number of significant disorders (Table 1) indicating a worse condition of teeth and more advanced PD in HD patients compared to general population were found. Histopathological gingival examination in HD patients showed considerably intensified degenerative changes within epithelial cells, unusual in subjects with periodontitis without renal failure.²¹ The results of previous clinical researches showed that the PD is a significant health problem in CRF patients.^12–22 Risk factors of its accelerated development in this population are presence of uremic toxins, humoral and cell immunity disorders, anemia, malnutrition, vitamin D deficiency, secondary parathyroid gland hyperfunction, osteoporosis and other bone metabolism disorders, insulin resistance or diabetes, liver lesion in the course of frequent viral infections, as well as condition of general disability impairing the care for oral hygiene.^12–22 The role of growth factors in the pathogenesis of PD in CRF patients was not subject to research so far. Systemic activation of the HGF/c-Met system generally found in these patients,^8,21–27 in particular under the influence of heparin administrated for the purpose of anticoagulation of blood during HD treatments,^25–27 may be a significant element in the development and progression of PD.

Table 1.  Pathological changes within oral cavity and teeth of adult dialyzed patients.

Parameter	Change	Clinical symptom
Saliva pH	↑	↑ mineralization of dental deposits
Saliva secretion	↓	↑ dental plaque formation, ↑ gingivitis and caries
Dental plaque	↑	↑ gingivitis and periodontitis, ↑ caries
Loss of connective tissue attachment	↑/N	deepened gingival pockets, horizontal bone loss,↑ tooth mobility
Alveolar bone atrophy	↑/N	↑ tooth mobility and loss
Loss of permanent teeth	↑	Missing teeth, toothlessness
Caries rate	↑	Inflammatory diseases of the pulp and the bones, loss of teeth
Gingival bleeding	↑	Gingivitis and periodontitis

Note: Symbols: ↑ increase; ↓ decline; N (normal) normal value.^12–22

Direct aims of this article are

1.	to evaluate and compare HGF level in unstimulated mixed saliva of three groups of patients with CRF (chronic HD patients, continuous ambulatory peritoneal dialysis (CAPD) patients, and patients with chronic kidney disease undergoing conservative treatment).
2.	to compare HGF level in saliva of the above–mentioned patients in relation to patients with PD without CRF and other coexisting diseases and to healthy subjects without PD.
3.	to determine the correlation between HGF level in saliva and the progression of PD in the above–mentioned groups of patients with renal failure as well as in the general population of subjects with PD.

PATIENTS AND METHODS

Examined Subjects

We started the study with the general dental examination of all chronic HD patients (n = 105) under medical treatment at the Department of Nephrology and Transplantology with Dialysis Unit, Medical University of Bialystok. The subject of particular attention was the total number of teeth and consequently the fact whether the chewing function has been preserved. The entry condition for periodontal treatment was presence of at least two fully functional own teeth within at least one of the six (four lateral and two anterior) groups of teeth (sextants).²⁸ The general patients’ condition was also important. The data concerning coexisting diseases in HD patients were obtained on the basis of medical investigations and a detailed analysis of medical documentation. The procedure was carried out with the collaboration of specialists in nephrology, diabetology, and internal diseases. Criteria excluding dialyzed subjects and CRF patients treated conservatively from the participation in the study were as follows: neoplastic disease, liver cirrhosis or viral hepatitis B or C requiring specialized treatment, systemic inflammatory or infectious disease, injury or surgical intervention—currently or suffered in the month preceding the examination, viral or mycosel inflammations of the oral cavity and/or salivary glands, immunosuppressive therapy (including glucocorticosteroid), use of acenocoumarol, use of heparin (except for heparin administered during HD procedures), and regular use of nonsteroidal anti-inflammatory drugs. An additional aspect was obtaining a voluntary consent to the participation in the study. In this way, we managed to form a group of 26 HD patients. The other sample groups consisted, intentionally, of a comparable number of men and women. Furthermore, the natural history of PD and repairing role of HGF indicate that these effects intensify with age. Consequently, the enrolment of subjects to be examined aimed at the formation of a control group characterized by absence of PD with the most advanced age. It consisted of patients visiting successively the Department of Conservative Dentistry of the Medical University of Bialystok for the purpose of treatment of small cavities as well as selected healthy subjects.

Finally, periodontal examination and measurement of HGF level in unstimulated mixed saliva were conducted in 126 subjects in total. Examined subjects were classified into five groups:

1.	Chronic HD patients (n = 26)
2.	CAPD–treated patients (n = 26)
3.	Patients with CRF undergoing conservative treatment (n = 28)
4.	Patients with PD without clinically apparent coexisting diseases (n = 26)
5.	Healthy subjects (n = 20)

A demographic characteristic of examined subjects is presented in Table 2. Patients with CRF without coexisting diseases were enrolled out of individuals visiting successively, for the purpose of periodontal treatment, in the Department of Conservative Dentistry of the Medical University of Bialystok. The group of healthy subjects without PD, in whom HGF level in saliva was measured, consisted of volunteers: exclusion criteria as above. The investigation was conducted according to the principles of Good Clinical Practice in conformity with the Declaration of Helsinki, on prior approval of the report by the Bioethical Board of the Medical University of Bialystok. All persons were informed about the purpose of the investigation and gave their written consent to participating in it.

Table 2.  Basic demographic data of examined individuals.

	HD	CAPD	CRF	PD	Healthy
Number (n)	26	26	28	26	20
Women (n/%)	7/27	12/46	14/50	10/38	8/40
Men (n/%)	19/73	14/54	14/50	16/62	12/60
Age (years)	57 ± 12	51 ± 11	52 ± 17	49 ± 8	46 ± 10
Place of residence
Town/city (n/%)	23/88	18/69	18/64	21/81	15/75
Village (n/%)	3/12	8/31	10/36	5/19	5/25
Education
University (n/%)	2/8	6/23	3/11	3/11	6/30
Secondary (n/%)	8/31	7/27	15/53	16/61	9/45
Primary (n/%)	16/61	13/50	10/36	9/35	5/25

Note: HD, hemodialysis; CAPD, continuous ambulatory peritoneal dialysis; CRF, chronic renal failure; PD, periodontal disease.

Sampling and Preparation of Saliva

Samples of unstimulated mixed saliva were taken once only using the spit-out method in the morning hours (7–10 am) from patients with empty stomach or at least 30 min after a meal. In the case of HD patients, saliva was taken before the HD procedure (i.e., before heparin administration). Saliva was taken after rinsing the mouth with distilled water for 10 min. Afterward, the patients spat the saliva into a sterile vessel. The vessels with saliva were kept on melting ice no longer than 2 h. Then, the saliva was poured into glass test tubes and centrifuged at a speed of 2100 × g for 10 min at room temperature in order to separate impurities. The supernatant obtained was retrieved by a mechanical pipette (by placing its tip at one-third of upper homogeneous layer) and poured in portions of 500 μL into Eppendorf plastic test tubes. Saliva samples obtained in such a way were frozen and stored at −20°C until marking.

Methodology of Periodontal Examination

The examination was conducted by the guidelines of World Health Organization.²⁸ In the case of patients with renal failure, periodontal examination was conducted in the surgical room of the Department of Nephrology and Transplantology with Dialysis Unit of the Medical University of Bialystok using portable operating lamp. The following parameters of the dental and periodontal status were evaluated: gingival index (GI),²⁹ papillary bleeding index (Ger. Papillen-Blutungs-Index, PBI),³⁰ plaque index (PI),³¹ and loss of clinical attachment level (CAL).²⁸

Laboratory Tests

HGF levels in unstimulated mixed saliva were determined by the immunoenzymatic method (ELISA) using commercial kits Quantikine® human HGF (R&D Systems Inc., Minneapolis, MN, USA; cat. No. DHG00). The determinations were carried out twice, and then the means of the obtained results were calculated. Minimum HGF detection limit in body fluids using this kit amounts to 0.04 ng/mL. HGF levels in saliva were investigated according to instructions of the manufacturer of the Quantikine human HGF ELISA kit.

Statistical Analysis

The software Statistica for Windows version 6.0 PL (StatSoft, Tulsa, OK, USA) and a computer with the Windows XP operating system, Home Edition (Microsoft Corporation, Redmond, CA, USA), were used. The distribution of quantitative data was investigated using the Shapiro–Wilk test. Normal distribution data were shown as an arithmetic mean ± 1 standard deviation; skew distribution—as a median (minimum value–maximum value). HGF levels below the test detection limit (0.04 ng/mL) were shown as zero. Mainly nonparametric tests were used in order to compare data groups of quantitative character. Differences between two groups of unlinked data were investigated using the Mann–Whitney test. Differences within three and more groups were evaluated by means of the Kruskal–Wallis analysis of the variance test and, then in detail, using the Mann–Whitney test. Linked data were analyzed by means of the Wilcoxon matched pairs test. The chi-square (χ²) test or the method of difference analysis between two structure indices was used for the comparison of data in the nominal scale. In individual cases, when all data showed normal distribution, appropriate parametric tests were used. Correlations between two variables were evaluated by means of the nonparametric Spearman’s regression test or the logistic regression analysis. In all analyses, two-sided tests were used; as a statistically significant value, the level of probability (p) lower than 0.05 was assumed.

RESULTS

Hepatocyte Growth Factor in Saliva

The level of HGF in unstimulated mixed saliva differed significantly among five investigated groups (Table 3). The level of HGF in saliva of patients with PD was nearly three times higher than in that of healthy subjects (p = 0.004, Figure 1). At the same time, it exceeded significantly the HGF level in CAPD patients (p = 0.023) and CRF patients (p = 0.020); no difference between subjects with PD and HD patients was shown (p = 0.567). Higher HGF level (p = 0.047) in saliva of HD patients compared with that of healthy subjects was found. No difference of HGF level among three groups of patients with renal failure was shown (χ² = 0.91, p = 0.634). The results did not change significantly after adjusted analysis considering the age of examined groups. The results are shown schematically in Figure 1. Correlations between HGF level in saliva and investigated clinical and laboratory variables in particular groups of patients with renal failure are shown in Table 4. In the groups of patients with PD and healthy subjects, no direct relationship between HGF level and age (p = 0.279 and p = 0.720), sex (p = 0.094 and p = 0.826), place of residence (p = 0.486 and p = 0.112), and education (p = 0.395 and p = 0.471) was found.

Figure 1.  Hepatocyte growth factor in unstimulated mixed saliva—individual values, central measures, and differences among individual groups.

Table 3.  Level of hepatocyte growth factor in saliva.

HGF (ng/mL)
HD	CAPD	CRF	PD	Healthy	χ^2, p
1.06* (0.02–9.65)	0.85* (0.04–3.03)	0.83* (0.02–9.09)	1.87** ± 1.32	0.68* (0–7.33)	10.5, 0.032

Notes: HD, hemodialysis; CAPD, continuous ambulatory peritoneal dialysis; CRF, chronic renal failure; PD, periodontal disease. The bold values mean statistical significance.* and **Denote a median (minimum–maximum) and an arithmetic mean ± standard deviation, respectively.

Table 4.  Significance (p) of correlation between HGF level in saliva and some parameters in patients with renal failure.

	HD	CAPD	CRF
Age	0.896	0.216	0.581
Sex	0.245	0.772	0.216
Place of residence	0.441	0.845	0.174
Education	0.938	0.932	0.776
Dialysis therapy period	0.186	0.801	–
Viral hepatitis B/C^a	0.295	0.727	–
Cardiovascular disease	0.955	0.297	0.249
Diabetes	0.609	0.002	0.069
Arterial hypertension	0.391	0.547	0.847
Erythropoietin therapy	0.218	0.626	–
Antithrombotic drugs^b	0.927	0.730	0.121
Creatinine clearance	–	–	0.602
Leukocytes	0.122	0.897	0.381
Hemoglobin	0.544	0.588	0.993
Thrombocytes	0.605	0.588	0.855
C-reactive protein^c	0.018	–	0.422
Fibrinogen	0.513	0.386	0.888
Albumins	0.503	0.360	0.899
Alanine aminotransferase	0.084	0.929	0.482
Phosphates	0.030	0.090	0.388
Blood pH	0.847	0.313	0.259
in blood	0.862	0.777	0.204

Notes: p Spearman’s regression (two continuous variables) or logistic regression of the type quasi-Newton or Rosenbrock pattern search (dichotomic variable vs. continuous variable: presence of property = 1, male sex = 1, place of residence in a village = 1, university education = 1 vs. primary or secondary = 0). HD, hemodialysis; CAPD, continuous ambulatory peritoneal dialysis; CRF, chronic renal failure; PD, periodontal disease. The bold values mean statistical significance.

^aNot found in patients with CRF.

^bAcetylsalicylic acid or ticlopidine.

^cAll CRP values in CAPD-treated patients below detection ability of the test (6 mg/L).

GI, PBI, and PI

Examined groups differed significantly in the case of GI and PBI (Table 5). The value of GI in the group of subjects with PD (Figure 2) was significantly higher than in patients with kidney diseases (vs. HD p = 0.021, vs. CAPD p < 0.0001, vs. CRF p < 0.0001). As related to PBI, similar differences were found (Figure 3) (PD vs. HD p = 0.004, PD vs. CAPD p < 0.0001, PD vs. CRF p < 0.0001). PI values did not show significant difference between the groups (Table 5). At the same time, GI (Figure 2) as well as PBI values (Figure 3) in the group of HD patients were significantly higher than in patients treated with CAPD (GI p = 0.004, PBI p = 0.012) or conservatively (GI p = 0.014, PBI = 0.018). In the case of PI, all these differences remained statistically insignificant (p > 0.455).

Figure 2.  Gingival index (GI)—individual values, central measures, and differences among individual groups.

Figure 3.  Papillary bleeding index (PBI)—individual values, central measures, and differences among individual groups.

Table 5.  Periodontal status indices and intergroup differences.

	HD	CAPD	CRF	PD	χ^2, p
GI	1.37 (0–2)	0.95 (0–2)	1 (0–2)	2 (0–2.75)	29.3, <0.0001
PBI	1.45 (0–2.75)	0 (0–2.50)	0 (0–3)	2.20 (0–3.20)	23.0, <0.0001
PI	2.05 (0–3)	1.61 ± 0.91	1 (0–2)	2 (0–3)	2.50, 0.475
CAL (mm)	5.24 ± 2.21	3.65 ± 2.24	2.9 (0–9.4)	4.68 (1.14–10.6)	5.22, 0.156
CAL 0 mm (n/%)	48/12	138/35	225/46	76/14	6.58, 0.086
CAL 1–2 mm (n/%)	34/9	54/13	51/10	89/16	3.22, 0.361
CAL 3–4 mm (n/%)	114/28	115/29	47/9	133/24	18.4, 0.004
CAL ≥ 5 mm (n/%)	203/51	91/23	168/35	258/46	11.3, 0.010

Notes: GI, gingival index; PBI, papillary bleeding index; PI, plaque index; n, number of teeth with particular CAL values; x, number of all teeth examined in the group during the measurement of CAL values (HD x = 399, CAPD x = 398, CRF x = 490, PD x = 556). HD, hemodialysis; CAPD, continuous ambulatory peritoneal dialysis; CRF, chronic renal failure; PD, periodontal disease. The bold values mean statistical significance.

Loss of CAL

In four examined groups, no statistic difference between the values of CAL loss was found (Table 5). The groups differed among themselves only with respect to the percentage of teeth with CAL = 3–4 mm and CAL ≥ 5 mm (Table 5). HD and CAPD patients as well as subjects with PD had significantly more teeth with CAL = 3–4 mm than patients with CRF (all p < 0.0001). The percentage of teeth with CAL ≥ 5 mm in HD patients exceeded 50% and was comparable to the percentage found in subjects with PD (p = 0.127). The values CAL ≥ 5 mm in both the above-mentioned groups were significantly higher than in CAPD patients and subjects with CRF (all p < 0.009). At the same time, the percentage of teeth with CAL ≥ 5 mm was higher in patients with CRF compared to that in CAPD patients (p = 0.001).

HGF and Periodontal Status among HD Patients

No significant correlations between HGF level in saliva and PI (r = 0.022, p = 0.913), PBI (r = 0.196, p = 0.336), and GI values (r = 0.062, p = 0.762) were found. No correlations between HGF level in saliva and mean CAL loss values in individual patients were found (r = −0.151, p = 0.462).

HGF and Periodontal Status among CAPD-Treated Patients

In this group of patients, significant, positive correlations between HGF level in saliva and PI (r = 0.440, p = 0.025), PBI (r = 0.566, p = 0.003), and GI (r = 0.406, p = 0.039) were found (Figure 4). No significant correlation between HGF level in saliva and CAL loss (r = −0.063, p = 0.759) was found.

Figure 4.  HGF level in saliva and periodontal status indices in CAPD-treated patients. (A) PI; (B) PBI; and (C) GI.

HGF and Periodontal Status among Patients with CRF

The HGF level in saliva did not correlate significantly with PI (r = 0.140, p = 0.477), PBI (r = 0.216, p = 0.270), or GI (r = 0.145, p = 0.462). The loss of CAL did not correlate with HGF level in saliva (r = 0.047, p = 0.814, respectively).

HGF and Periodontal Status in Patients with PD

Like in CAPD-treated patients, statistically significant, direct correlations between HGF level in saliva and the indices PI (r = 0.590, p = 0.001), PBI (r = 0.394, p = 0.046), and GI (r = 0.550, p = 0.004) were observed (Figure 5). HGF level did not correlate with CAL loss (r = 0.276, p = 0.172).

Figure 5.  HGF levels in saliva and periodontal status indices in patients with PD. (A) PI; (B) PBI; and (C) GI.

DISCUSSION

This article describes, for the first time, the HGF level in unstimulated mixed saliva in three groups of patients with CRF compared to patients with advanced PD and to healthy subjects. Moreover, it is the first attempt to evaluate the correlation between HGF level in saliva and the progression of PD in chronic HD patients, patients treated with peritoneal dialysis, and predialysis CRF patients.

HGF as a PDI

he study of Ohshima et al.¹¹ published in 2002 showed, for the first time, that HGF level in unstimulated mixed saliva can be an index of PD intensity. The presence of significant, positive correlations between HGF level in saliva and the number of periodontal pockets deeper than 4 mm (Pearson’s r = 0.541, p < 0.001), pockets deeper than 6 mm (r = 0.683, p < 0.001), the greatest probing depth of a periodontal pocket (r = 0.558, p < 0.001), and the number of bleeding spots during probing (r = 0.511, p < 0.001) was found.

An earlier study showed that HGF level in fluid taken from gingival pockets of patients with PD is 15 times higher than in subjects without PD and more than 300 times higher than HGF level in the blood serum of healthy subjects.¹⁰ Furthermore, direct correlations between HGF level in gingival fluid and clinical PD progression indices, as probing depth and GI, as well as the levels of interleukin-1β and prostaglandin E₂, as indices of the activity of local inflammatory condition, were found.^8,10 A prospective study confirmed that periodontal treatment causes the reduction of the total number of HGF in gingival pocket fluid.¹⁰ On the basis of the above-mentioned clinical investigation ^18–20 and the results of earlier in vitro experiments,^5–11 Ohshima et al. and Daikuhara et al. formulated a new PD progression hypothesis. According to this theory, the HGF, being one of the strongest endogenous growth factors of epithelial cells, stimulates the proliferation and migration of gingival epithelial cells deep into the pathological periodontal pocket. Moreover, this effect is strengthened by new formation and accelerated regeneration of blood vessels within the gingiva under the influence of HGF. Then, in growing epithelium covers, prematurely, the tissue loss of tooth root as a result slows down the regeneration of collagen fibers of the connective tissue attachment. The effect of HGF action in this clinical situation is inhibiting of the reconstruction of deep periodontal tissues. In light of this theory, stimulation of HGF production and secretion in response to local inflammatory condition and destruction of periodontal tissues may be a significant factor of PD progression.

Our research confirmed, for the first time, direct correlation between HGF levels measured in unstimulated mixed saliva and the progression of periodontitis in the general population described previously by Ohshima et al.¹¹ Positive correlations between HGF level in saliva and GI and PBI—indices of periodontal status comparable to parameters investigated by Japanese dentists—were found in a group of 26 patients with PD.¹¹

Moreover, our research showed direct, positive correlation (r = 0.590, p = 0.001) with logarithmic adjustment type (Figure 5) between HGF level in unstimulated mixed saliva of subjects with PD and PI. The correlation was not investigated so far. The correlation between HGF level and CAL loss remained positive as well; the absence of statistical significance may partly result from considerable progression of periodontitis in the examined group. Furthermore, our research showed, for the first time, that HGF level in saliva of patients with PD is nearly three times higher than in the saliva of subjects with healthy periodontium; examined groups were comparable with respect to age, sex, place of residence, and education. Results of this article confirm and consistently complement the correlation between HGF level in unstimulated mixed saliva and PD intensity indices found in the earlier Japanese study.¹¹

HGF level in saliva of patients with chronic kidney disease was not subject to research so far. This article shows that the level of HGF is increased in HD-treated patients with terminal renal failure compared with that in subjects with healthy periodontium and comparable to values found in subjects with advanced PD (Figure 1). At the same time, HGF levels in saliva of patients treated with peritoneal dialysis and predialysis CRF patients did not differ from levels observed in healthy subjects. Moreover, no statistically significant difference in HGF level among the three above-mentioned patient groups with renal failure was found. In view of the above results, the necessity of selection of chronic HD patients (resulting from the assumptions of the study), and the statistical analysis considering the age of examined subjects (as a parameter differentiating the above five groups), it may be assumed that HGF level in unstimulated mixed saliva of HD patients is higher than in the remaining two groups of CRF patients. The observation requires further confirmation.

Correlations between HGF level in saliva and periodontal status indices in examined groups of patients with renal failure were not homogeneous. Only in the group of patients treated with the CAPD method, direct, significant correlations between the HGF level and the indices PI, PBI, and GI were observed (Figure 4). The correlations were similar to those found in subjects with PD and did not differ even in statistical significance of particular correlation coefficients.

Reasons for the absence of cross-sectional relations between the indices PI, PBI, and GI and the HGF level in saliva of HD patients, and pre-dialysis CRF patients, as distinguished from CAPD-treated patients and patients with periodontitis, were not explicitly explained in this study. It was shown that there are no consequence of differences in age, sex, place of residence, and education. The analysis of possible influence of some clinical variables and biochemical parameters of blood reflecting the presence of main coexisting diseases and metabolic disorders on HGF level in saliva revealed a range of statistical relationships. However, the relationships seem to be incidental and without coherent clinical significance. The results certainly require further verification and investigations involving a larger number of patients. However, they may suggest that the HGF concentration in unstimulated mixed saliva of patients with renal failure can be independent of systemic diseases and disorders. It is rather a measure of activity of a local inflammatory condition. The lack of correlation between the HGF concentration and the PD progression in the groups of HD and CRF patients can be a consequence of a different composition of unstimulated mixed saliva. Apart from inflammatory gingival fluid being the primary cause for the increase of HGF level in oral cavity,¹⁰ other sources may also be the cause for the increase: saliva, nasal mucosal secretion, paranasal sinuses, and bronchi (in case of even subclinical inflammatory condition) as well as blood coming from small cuts within oral cavity.^32,33 We must also not forget a possible influence of the HD treatment itself on the HGF concentration in saliva. It is known that during a HD procedure the HGF concentration in the blood plasma increases considerably as a result of HGF release from proteoglycan complexes under the influence of exogenous heparin.²⁷ Our observations indicated that such increase was not followed by a similar increase in HGF concentration in saliva; furthermore, we noticed a tendency to the normalization of HGF concentrations in saliva during a dialysis procedure.³⁴ The final evaluation of usability of the measurement of HGF level in unstimulated mixed saliva as a screening test for detection of PD in chronic HD patients and CRF patients undergoing conservative treatment requires further research in appropriately numerous groups.

Periodontal Status of Patients with Renal Failure

Results of this study point to considerable progression of PD in patients with chronic kidney disease. Values of some periodontal status indices evaluated in these patients were comparable to those found in subjects with fully symptomatic form of periodontitis visiting a specialist center for the purpose of treatment. The groups of HD patients, CAPD patients, and predialysis CRF patients as well as the control group of subjects with periodontitis (without coexisting diseases) did not differ among themselves with respect to age, sex, place of residence, and education. The progression of PD in the group of patients with renal failure constitutes here a very significant risk factor of chronic inflammatory condition, atherosclerosis and its complications, ischemic heart disease, and cerebral stroke.^35,36 The rate of mortality of patients with kidney disease (particularly HD patients) due to cardiovascular diseases is several dozen times higher than that of general population.³⁷ It turned out that the PD not only occurred in individuals with the kidney disease more frequently than in general population but also had a more rapid course. The causes of such status have been described in the introduction hereto. Complex analysis of all presently investigated indices of periodontal and indirectly dental status indicates that PD is most intensified in HD patients, moderately in CAPD treated patients and to the relatively smallest extent in patients with CRF treated conservatively. The conclusion was based in most cases on statistically significant differences, and partly on clinically grounded and logical tendencies, taking into consideration the relatively small size of investigated groups. It was found that the indices GI and PBI were lower in all groups of patients with renal failure compared to that in individuals with advanced periodontitis. At the same time, significantly higher values of these indices in the group of HD patients compared to other patients with renal failure was observed; the significance of statistical difference between HD patients and CAPD-treated patients was in both cases greater than that found between HD patients and CRF patients (Figures 2 and 3). At the same time, values of the PI did not differ significantly among the four examined groups (Table 5). It proves that the accumulation of plaque—the basic factor of PD development—is similar in examined patients with renal failure and in subjects with advanced form of periodontitis. Consistently to the sequence of PD intensity in examined groups suggested above, PI values were highest in HD patients and comparable to those observed in subjects with PD; at the same time, they showed the tendency to decline successively, in CAPD-treated patients and CRF patients. Identical regularities were also observed in case of absolute value of CAL loss (Table 5), being a sensitive measure of PD progression. Moreover, it was shown that the percentage of teeth with CAL = 3–4 mm was statistically significantly smallest in CRF patients compared to that in all other groups (Table 5). On the other hand, the greatest PD intensity in HD patients is confirmed by a considerable percentage of teeth with CAL loss ≥5 mm, prevalent in this group and exceeding 50%. The percentage is comparable to the one observed in persons with advanced periodontitis (46%) and significantly higher than in CAPD (23%) and conservatively (35%) treated patients. Statistical difference regarding higher percentage of CAL loss ≥5 mm among patients with CRF compared to CAPD patients (p = 0.001) is a fact that can be evidence against greater PD intensity in CAPD patients than in conservatively treated patients. The above comparisons of classical periodontal status indices such as GI, PBI, PI, and CAL are considered separately, and particularly in inter-relationship, appear to prove considerable PD progression in patients with renal failure. The best status was found in CAPD patients and the worst in HD patients. It is difficult to interpret such situation, especially as no clear data comparing the oral condition in groups similar to our study can be found in the literature. The only similar study was our paper published 5 years ago,³⁸ containing the results of investigations conducted in similar groups of patients with kidney disease, and that data corresponded to the present findings. A Brazilian study of Brito et al.³⁹ described results where, similar to our research, a worse periodontal status in HD patients compared to that in CAPD patients was found. In their article, the Brazilian authors stressed the diversity of methodology in studies on this subject. Different authors (including those whose research was cited herein) investigated different indices. To sum up, however, one conclusion can be drawn, namely that most researches indicate a better periodontal status of CAPD patients compared to that of HD patients and outpatients.^38,39 Some authors did not find any significant differences even in comparison to general population.^39–41 Certainly, based mainly on clinical observations, CAPD patients are usually statistically better educated, mostly more responsible and receive a better care from the family. A better physical and manual fitness results in a better care of oral hygiene and more frequent dental visits. Not insignificant is the fact of supply in these patients of a considerably more liberal diet and liquids compared to HD patients. The investigations of the oral condition and the radiological images of the alveolar bone in these patients proved that despite a worse oral hygiene no differences in these patients compared to that in general population were found.⁴¹ Presented results of our research are difficult to compare with data from the literature also due to the fact that all previous studies concerned only chronic HD patients; a considerable part of them was of subjective and just qualitative character, related to patients outside of the European population and was conducted between 10 and 20 years ago. In most cases, examined groups were characterized by a considerably younger age, probably better clinical status and rarer coexistence of diabetes, atherosclerosis, and cardiovascular complications.^12–22 Erythropoietin therapy was not taken into consideration in previous investigations of periodontal and dental status of HD patients, leading not only to considerable improvement of anemia but also to general and local immunity.

Representative investigation of periodontal status of HD patients was conducted in 1999 by Gavaldá et al.¹⁴ from the University Hospital in Valencia, Spain. It concerned 105 patients aged 59 ± 14 years and 53 appropriately selected healthy subjects. In the examined group, PI amounted to 2.1 ± 0.8 and CAL loss to 4.9 ± 2.1 mm; the values were significantly higher than in the control group and comparable with values found in own research [2.05 (0–3) and 5.2 ± 2.2 mm, respectively]. Moreover, the calculus index (according to Greene–Vermillion) was over 50% higher in HD patients than in healthy subjects; the groups differed with respect to the DMF index. At the same time, DMFT (decayed, missed, filled teeth) index in the Spanish population (14.9 ± 8.7) remained by 35% lower than in the presently examined group of HD patients (23.7 ± 7.4) and was comparable with the index found in the group of American patients.¹³

Researches of Frankenthal et al.²⁰ and Marakoglu et al.²² provided quantitative data comparable with results obtained earlier by Gavaldá et al.¹⁴ and at present in own research. A group of 25 patients (mean age, 44 years) dialyzed in Rambam Medical Centre, Haifa, Israel, found the following values of periodontal status indices: PI = 1.76 ± 0.17, GI = 0.97 ± 0.01, CAL = 4.43 ± 0.29 mm, and PD = 2.92 ± 0.14 mm.³² In 36 chronically dialyzed patients (mean age 50 years) in the city of Sivas, Turkey, the indices amounted to PI = 2.2 ± 0.6, GI = 1.5 ± 0.3, and PD = 1.8 ± 0.6 mm.²² When comparing results obtained in the four above-mentioned methodically similar studies, additionally the fact that the patients treated in Israel were more than 13 years younger ²⁰ than patients in Valencia¹⁴ and Bialystok has to be considered. The above review of not numerous, so far, scientific reports as well as the results of own research indicate considerable PD progression in the population of HD patients and the necessity of further, in particular, multicenter and nationwide research.

CONCLUSIONS

1.	The level of HGF in unstimulated mixed saliva is a direct index of progression of PD in subjects without renal failure and in patients treated with peritoneal dialysis. The examination may become a new screening test in these patient groups.
2.	HGF is a probable cause of considerable intensity of PD in chronic HD patients.
3.	PD occurs commonly in the population of patients with chronic kidney disease and is a problem important for general health. Its progression and the level of HGF in saliva diminish successively in HD patients, peritoneal dialysis patients, and conservatively treated patients.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.