Key Words

Diabetes; Apical Periodontitis; Periodontal Disease; Creatinine Level.

Introduction

DOral infections such as pulpal and periodontal disease are frequently associated with diabetes mellitus [1-3]. Periodontal disease is one of the most prevalent complications in diabetic patients [3-5]. It is also evident in the increased severity of chronic periodontitis, periodontal abscess, and predisposition to infections in these individuals [6, 7]. The reverse is also true, as periodontal infection is capable of exacerbating a diabetic condition, which is another important aspect to be considered [8].

The current literature is replete with studies linking diabetes mellitus to periodontal disease alone [7, 9-11]. However, information on pulpal disease, which is characterized by apical periodontitis due to infectious necrosis of the pulp, is still lacking [1, 12, 13].

The effect of diabetes mellitus in apical periodontitis has been investigated in animal models [14, 15]. It was observed that diabetes accelerates the development and progression of periapical periodontitis in rats, as well as the relationship of periodontal disease [16]. However, information on the association between pulpal and periodontal disease with diabetes is limited to a few studies [1, 17], and the effects on systemic conditions have not been studied in detail [1].

One of the systemic complications for diabetic patients is renal dysfunction [18], which can be diagnosed by measuring urea and creatinine levels in the blood [19]. Diabetic nephropathy, a major long-term complication of diabetes mellitus, is the most common cause of end-stage renal disease worldwide requiring dialysis, and it is becoming a challenge to public healthcare systems [20, 21].

Creatinine is the preferred marker for renal dysfunction and is readily available in clinical laboratory settings [22]. Being an indicator of decreased kidney function, serum creatinine levels are largely unaffected by a typical diet, and are thus used as markers of renal function [23, 24]. Creatinine is a byproduct of muscle metabolism and typically remains in a steady state balanced out by renal elimination [22]. Despite their limiCreatinine is the preferred marker for renal dysfunction and is readily available in clinical laboratory settings [22]. Being an indicator of decreased kidney function, serum creatinine levels are largely unaffected by a typical diet, and are thus used as markers of renal function [23, 24]. Creatinine is a byproduct of muscle metabolism and typically remains in a steady state balanced out by renal elimination [22]. Despite their limitations, creatinine levels are currently used in clinical practice to estimate the glomerular filtration rate because it is inexpensive, harmless, and easy to perform in patients [25].

High creatinine levels can relate to other infections such as acute infectious meningitis [25]. It has been suggested that creatinine levels increase due to glomerular hyperperfusion [26]. Experimental studies have shown that high creatinine levels are observed during the initial hyperkinetic phase of sepsis [27, 28].

Lower serum creatinine represents a greater risk for developing diabetes [29]. The relationship between chronic kidney disease, diabetes, and oral health status has been reported [30]. Nephropathy is a chronic complication of diabetes mellitus and may be related to periodontal disease [31, 32]. It is known that periodontal disease causes changes in serum creatinine levels [33]; however, to our knowledge, the association of this condition with diabetes or pulpal disease has not been explored thus far.

Thus, it seemed pertinent to evaluate the relationship between pulpal and/or periodontal disease and serum creatinine levels in a rat model of diabetes mellitus.

Methods

Eighty male Rattus norvegicus albinus Wistar rats, each weighing 250–280 g, were used in the study. The animals were housed in temperature-controlled rooms and given ad libitum access to water and food. The institutional ethical committee approved the experimental protocol, which was conducted in accordance with the institutional ethical committee guidelines. The animals were divided into 8 groups of 10 animals each,as shown in Table 1.

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Induction of experimental diabetes

The animals were intramuscularly anesthetized with ketamine (87 mg/kg; Francotar; Virbac do Brasil Ind. e Com. Ltda., Roseira, Brazil) and xylazine (13 mg/kg; Rompun; Bayer S.A., Sao Paulo, Brazil). The rats were randomly assigned into groups and were endovenously injected in the penile vein with either citrate buffer solution (0.01 M, pH 4.5; groups 1–4, n = 40) or with streptozotocin (Sigma-Aldrich Corp., St. Louis, MO, USA). Streptozotocin was dissolved in citrate buffer solution at 65 mg/kg body weight for experimental induction of diabetes (groups 5–8, n = 40) [34].

Six days after diabetes mellitus was induced, blood samples were collected from each animal to determine their blood glucose levels. Rats with blood glucose levels exceeding 250 mg/dL were used in the study [35].

Induction of oral infections

For the development of apical periodontitis, the pulps of the right upper first molars were exposed on the mesial surface using surgical round burs (Broca LN Long Neck; Maillefer, Dentsply Ind. e Com. Ltda, Petrópolis, Brazil) (groups 2, 4, 6, and 8) [36]. To induce periodontal disease in rats from groups 3, 4, 7, and 8, sterile 4/0 silk ligatures (Ethicon; Johnson & Johnson, São Paulo, Brazil) were placed around the left second maxillary molar [1].

Assessment of serum creatinine levels

To determine serum creatinine levels, venous blood samples (1 mL) were collected via cardiac puncture after 30 days, and the animals were killed with an overdose of the anesthetic solution.

The samples were placed in EDTA and immediately transferred to a technician who was blinded to case status. Serum creatinine was measured by the Jaffe method [19, 24, 25, 29]. The Jaffé reaction is based on the yellow-orange color produced by creatinine reacting with alkaline picrate, which is measured at 492 nm. The amount of color formed is proportional to the concentration of creatinine in the sample. The sensitivity of the Jaffé reaction is 0.5 mg/dL creatinine in samples, and the entire detection time spans approximately 30 min (Creatinina K Labtest®, Labtest Diagnóstica S.A., Brazil).

The total assessed values were tabulated for each experimental group, and a single calibrated operator analyzed the data in a blinded manner. One-way analysis of variance and Tukey’s test were used for statistical analysis, and a significance level of 5% (p < 0.05) was used to compare the mean values.

Histopathological analysis

Maxillae were removed and immediately placed in 10% bbuffered formalin. Specimens were rinsed in sterile water and decalcified in 10% EDTA. They were rinsed again in sterile water, dehydrated in ethanol, cleared in xylene, and embedded in paraffin [1]. Six micron-thick serial sections of the molars and surrounding tissues were prepared and stained with hematoxylin and eosin (HE). We made serial 6-μm thick paraffin sections of the mesial–distal aspects of all upper molars, and these were stained with HE. Morphological studies of the periapical and periodontal area were conducted.

Results

Thirty days after pulp exposure, pulps exhibited total necrosis and periapical lesions were established. Moderate to severe inflammatory reaction composed primarily of neutrophils (polymorphonuclear cells) and mononuclear cells was observed (Fig. 1a, b).

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N, total necrosis, AB, alveolar bone, C, cementum, D, dentin; arrowheads, inflammatory infiltrate,*, root resorption

Periodontal disease was observed based on the presence of inflammatory cells and by measuring the distance from the cemento-enamel junction to the alveolar bone crest at the palatal surface of the midmesiopalatal root region of the second molar under ×100 magnification. We observed evidence of aggressive periodontitis with substantial periodontal bone loss (Fig. 1c, d).

Blood glucose levels were significantly increased in diabetic rats as compared to that in control rats, indicating development of hyperglycemia (Table 2). The changesin the serum creatinine levels of non-diabetic and diabetic rats are shown in Table 2.

Total serum creatinine levels were similar in the nondiabetic(p > 0.05) and in diabetic groups (p > 0.05).Conversely, diabetic rats had significantly higher serum creatinine levels than the control rats (p = 0.000) (Table2).

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Diabetic rats with pulpal and periodontal disease (G8)had high levels of serum creatinine, which was statistically significant compared to all non-diabetic rat groups (G1–G4). Normoglycemic rats with pulpal and periodontal disease (G4) had serum creatinine levels similar to those of diabetic rats without oral infection (G5) (Table 2).

Discussion

There are a number of different rodent models for diabetes, particularly type 1 diabetes. Two models are commonly used: streptozotocin-induced diabetes in rats and non-obese diabetic (NOD) mice [37]. The advantages of the streptozotocin model are that diabetes can be induced in a controlled manner in all animals, and animals in the diabetes and non-diabetes groups are of the same genetic background [38]. In this study,blood glucose levels were higher in rats in the diabetic model group than in those in the normal control group, indicating that hyperglycemia persisted in the diabetic rats.

Two models of oral infection were used [1]. Our results are in agreement with that of other studies [14, 39], which demonstrated histologically that induced periradicular lesions in diabetic rats were larger than that in non-diabetic rats. The induction of experimental periodontitis stimulated inflammatory infiltrate formation in both diabetic and normoglycemic rats. In summary, diabetic rats had a higher degree of inflammation and greater periodontal bone loss through enhanced resorption. These results also agree with that of previous studies [40, 41].

We measured creatinine levels in the rat serum with the Jaffé method, similar to other studies [19, 24, 25, 29]. The traditional method for detecting creatinine is based on a modified Jaffé reaction, which is widely used in both laboratory and clinical detection. The entire detection time requires approximately 30 min. The reaction is carried out for a fixed period in order to minimize the interference of other substances reacting with the picric acid [42].

Creatinine is transported by the bloodstream to the kidney. There is little to no tubular reabsorption of creatinine by the kidney tubules. Therefore, creatinine levels in the blood may be used to calculate creatinine clearance, which reflects the glomerular filtration rate. Any condition that impairs the function of the kidney will likely increase the creatinine level in the blood. Changes in creatinine levels have been linked with diabetes [29, 43, 44], periodontal disease [33], acute infectious meningitis [25], hyperkinetic phase of sepsis [23, 24], kidney disease, kidney transplant, and oral health status [30, 45, 46].

In the present study, serum creatinine levels were significantly elevated in the diabetic group (0.73 ± 0.10 mg/dL) compared with that in the control group (0.56 ± 0.09 mg/dL). These results are also in agreement with that of previous studies [43, 47]. These results confirm that streptozotocin-induced diabetes is a factor for changes in creatinine levels. Conversely, lower serum creatinine levels increased the risk of type 2 diabetes [29].

Some studies have shown that periodontal conditions may affect renal function [33, 48-50]. However, severe periodontitis was not correlated with renal dysfunction in a studied population [51]. This difference between these studies may be related to methodology.

There is no related study regarding the relationship to pulpal infection. In the present study, a higher mean serum creatinine level was associated with pulpal and periodontal disease in normal and diabetic rats (Table 2). In addition, a recent report demonstrated that pulpal and periodontal disease increase triglyceride levels in diabetic rats, confirming the hypothesis that endodontic infections influence systemic health when associated with periodontal disease [1].

We found that the presence of pulpal and periodontal disease increased serum creatinine levels in normoglycemic and diabetic rats; however, no statistical differences were found (p > 0.05). In conclusion, the present results suggest that serum creatinine levels are higher in diabetic rats and may be related to the presence of oral infections.

Acknowledgements

This study was supported by PROPe/UNESP (005/2011).

Conflict of Interest

The authors declare that they have no conflicts of interest to declare.