A Study On The Effect Of Chronic Periodontitis On Diabetic Kidney Disease And The Possible Mechanisms In OLETF Rats

BackgroundIn recent years, with the rapid progress of periodontal medicine, more dentists are beginning to pay much more attention to periodontal disease, especially the chronic periodontitis. Periodontitis is a common infection of the periodontal tissues and a major cause of tooth loss in adults. The most prevalent form is chronic periodontitis. The chronic periodontitis incidence rate in Americans is about47.6%in2010. Even the worse is the higher incidence in the Chinese population. As we all know, chronic periodontitis is a kind of non-specific inflammatory diseases caused by plaque microorganisms. That would result in periodontal tissue destruction, which is characterized by alveolar bone resorption and pathological periodontal pocket. Periodontitis is an inflammatory disease, but we would no longer regard it as a limited local inflammation. It might influence the whole body by the ways to increase the body’s systemic inflammatory burden and enhance the immune response.Currently, there are a large number of studies have revealed periodontitis and systemic disease associated, with type2diabetes (T2DM) is of great concern. T2DM is a kind of chronic diseases with high incidence and great harm. With the process of aging, diabetes and periodontitis patients have increased dramatically. It has been revealed that periodontitis and T2DM is the independent risk factor for each other. Chronic periodontitis may increase the body’s systemic inflammatory burden, increased systemic levels of inflammatory factors, and promote insulin resistance. And that was not conducive to blood glucose control, thereby affecting the occurrence and development of T2DM and its complications. In T2DM, the enhancement of the immune system to the body inflammation leads to local excessive infiltration of inflammatory cells in periodontal tissues, excessive secretion of inflammatory factors and proteases. That would cause the more destruction in local tissue and increase the degree of periodontal inflammation.The greatest hazards of T2DM are the long-term chronic complications. Diabetic kidney disease (DKD) is one of them and it is the leading cause of death of patients with T2DM. The main pathological changes of DKD are the renal extracellular matrix-type IV collagen (collagen) excessive accumulation, resulting in glomerulosclerosis and renal interstitial fibrosis. Mechanisms leading to the kidney lesions are very complex. Involving several key cytokines and proteases:TGF-β1plays a key role in renal interstitial fibrosis and glomerulosclerosis; matrix metalloproteinase MMP-2(gelatinase A), MMP-9(gelatinase B), and inhibitor TIMP-1were found to be the key factors to regulate renal ECM balance.Until now, there are few researches on the effect of Periodontitis on the DKD. Only a few available studies were the observation of clinical cases. Therefore, this study intends to composite type2diabetes and chronic periodontitis disease in an animal model. We aimed to discusse the the role of periodontitis in the development of T2DM and to determine whether periodontitis impact renal function and renal structure by excluding the interference of any other confounding factors. And through testing the above mentioned DKD closely related type IV collagen, TGF-betal, MMP-2, MMP-9, TIMP-1protein and Mrna expression, we attempted to further analyze whether periodontitis had an impact on DKD and the possible pathological mechanisms. And try to establish a reliable experimental basis for further study of periodontitis and T2DM or DKD relationship, diagnosis, treatment and prevention for clinical Multidisciplinary combination therapy.CHAPTER ONE The animal model of chronic periodontitis in OLETF ratsObjective:To establish experimental periodontitis in OLETF rats by ligature and periodontal pathogen infection for further evaluating the effects of periodontal inlammation on the diabetic kidney disease and the pathogenesis in OLETF rats.Methods:(1)30OLETF rats (O group),4-week-old, male, spontaneously type2diabetic;20LETO rats (L group), male, with the same germline and the same age but having normal glucose tolerance, as the control group. Every4weeks, oral glucose tolerance was determined with50%glucose solution2g/kg orally administered. Diabetes diagnosis is based on the results of the OGTT test, blood glucose peak>16.7mmol/L and glucose and blood glucose>11.1mmol/L in120min as the criteria. Meeting either one of the above criteria is recognized as impaired glucose tolerance (IGT). At36weeks old,(being IGT state)26OLETF rats were randomly divided into two groups:periodontitis group (O CP (+))(n=13) and non-periodontitis group (O CP (-) group)(n=13); LETO rats were also randomly divided into two groups, periodontitis group (L CP (+) group)(n=10) and non-periodontitis group (L CP (-) group)(n=10). (2) The bilateral maxillary first and second molars in the [L CP (+)] group and [O CP (+)] group were ligatured with3/0silks soaked with periodontal pathogens, in subgingival position for28weeks. The ligatures were checked regularly and re-done if necessary.(3) OGTT was conducted every four weeks and measured body weight at the same time. The rat tail vein blood samples were obtained at36weeks and46weeks of age to evaluate the dynamic changes of serum insulin levels and HOMA-IR score.(4) At56weeks of age, rats were sacrificed under general anesthesia. Samples of the bilateral maxillary molar regions were resected from each rat and immediately fixed in10%neutral formalin for2days, and stored in70%ethanol for scanning by micro-CT. Maxillary samples were further decalcified with10%tetrasodium-EDTA equeous solution (PH7.0) for4weeks at4℃after scaning. The periodontal tissue samples were embedded in paraffin and sections (thickness:5um) were stained with hematoxylin and eosin (HE). Serum samples were also collected and stored in-80℃.The overall comparisons between the four groups were done by using a2x2factorial design analyzes. Single factor data of the two groups were compared using independent samples t test. Independent samples t test were also used to compare the same factors at two different levels and the same level of different factors. Multiple indicators measured in different times between general groups or within groups were compared by by analysis of variance with repeated measurements. With or without the intervention of comparison in the same time within the same group was compared by the two independent samples t test.ResultsThe spontaneous T2DM rats-OLETF rats and normal control LETO rats were used as research objects in this study. OLETF reached the IGT period at36weeks age vividly simulating human T2DM chronic disease process. OLETF rats showed typical clinical features of the T2DM, just like polyphagia, polydipsia and polyuria, rapid weight gain. Blood glucose AUC of the OLETF rats increase statistically significant with ageing (F=5.952P=0.029). However, at any weeks of age, AUC slightly increased in O CP (+) group than in the O CP (-) group without significant difference. Serum triglyeride (TG) and cholesterol (CHOL) in O group were significantly higher than those in Group L. TG and CHOL in O CP (+) group were both higher than those in O CP (-) group, while serum CHOL difference with significance.Serum FI and HOMA-IR in O group was significantly higher than that in Group L. O CP (-) group FI level was slightly higher than O CP (+) group. O CP (-) group and O CP (+) group had significantly increased HOMA-IR values with increasing age.Intraoral Periodontal examination in CP (+) group show:food residue around tooth neck, swollen gums, alveolar bone resorption, periodontal pocket. O CP (+) group had heavier periodontal inflammation. Micro-CT three-dimensional reconstruction of the rat alveolar bone in four groups display that the alveolar bone loss in O CP (+) group and L CP (+) group were significantly higher than that in O CP (-) group and L CP (-) group, respectively. Moreover, the rats in O CP (+) group have more bone loss than L CP (+) group.Through HE staining of decalcified rat mandible, we found periodontal attachment loss, destruction of collagen fibers, inflammatory cell infiltration in the end the periodontal pocket, alveolar bone resorption significantly. In the OCP (+) group, inflammation was more heavier, with junctional epithelium migrate to root direction, a large number of diffuse inflammatory cell infiltration in periodontal pockets and gingival connective tissue, and even migrate into the periodontal pocket. ConculusionOLETF rat is a very typical T2DM animal model which simulates human T2DM characteristics in glucose and lipid metabolism and insulin resistance vividly.Periodontitis promote the degree and progress of insulin resistance in T2DM. It could deteriorate glycemic control and lead to compensatory high serum insulin. Thereby deteriorate the T2DM state. T2DM could aggravate periodontal tissue destruction conversly. The degree of periodontal inflammation in T2DM is much heavier than in normal periodontitis group. Chaper2Effect of Chronic periodontitis on rat renal function and renal morphologyObjectiveWe aimed to investigate whether chronic periodontitis affect the OLETF kidney function and the morphology kidneys by measuring kidney function indices and observing the kidney morphology. To supply evidence for the Pathological basis of whether periodontitis affecting the development of T2DM complications.MethodsThe biochemical markers in the four rat groups including serum albumin (ALB), total protein (TP), urea (UREA) and creatinine (Cr) were analyzed by automatic biochemical analysis instruments. In order to do a comprehensive analysis of renal pathological changes, routine HE staining and PAS, MASSON, PASM special staining were adiministered. Glomerulosclerosis index (GSI) was calculated by analyzing the PAS stained sections. MASSON staining was used to calculate the index renal interstitial fibrosis (RIFI). Factorial design analysis of variance and two independent sample t-test were used for statistical analysis of the results. ResultsThe levels of serum TP and ALB decreased significantly in O group than in the group L (t=11.131, P=0.002). Consistently, serum levels of ALB and TP decreased significantly in O CP (+) group than in O CP (-). In the L group or without CP intervention, there is no significant difference. Serum creatinine and urea levels were significantly higher in O gourps than in L groups (t=9.099, P=0.005).The histological features in L CP (+) group were close to L group showing normal renal histological features. The glomerular diameter increased and cortex narrowed and medulla widened in OLETF rats. Bowman’s capsule wall became thickened significantly, glomerular balloons were adhered partly and cysts increased. There are moderate to severe hyperplasia in glomerular mesangial matrix diffusively or nodularly, basement membrane thickening of glomerular capillary, focal glomerulosclerosis in some areas, glomerular capillary plexus lobulation, and vascular wall thickening of arteryies in and out of glomerular surrounding. There were hyaloid and vacuolar degeneration in cortical tubular epithelial cells, dilatation of some tubular, protein-like substances or protein casts in lumen. It was also found that there were tubular epithelial cell desquamation, tubular degeneration and atrophy in some areas, dilation or atrophy of hylic tubules, scattered or gathering infiltration of lymphocytes and mononuclear cells and fibrosis in interstitial areas.Concerning the GSI and RIFI, their results are similar. The GSI and RIFI in OLETF rats are significantly higher than Leto rats (P<0.01). They are slightly higher in L CP (+) group than in L CP (-) group, no significant difference. However, they are significantly higher in OCP (+) rat than in (P<0.01). There is an interaction effect between T2DM and periodontitis.It is noteworthy that obvious inflammatory infiltration was found in the O group. Among O CP (-) group, a large number of scattered inflammatory cell infiltration or large focal inflammatory cell infiltration were found in three of eight rats (3/8). In O CP (+) group, a large number of scattered inflammation cell infiltration or large focal inflammatory cell infiltration in rat patients was found in six of eight rats (6/8) and most of the inflammatory cells are monocytes and macrophages.ConclusionChronic periodontitis may not cause significant changes in kidney function and histopathology in normal rats. However, chronic periodontitis may significantly increase the renal interstitial inflammatory cell infiltration risk in T2DM. Moreover, the chronic periodontitis promote kidney glomerulosclerosis and renal interstitial fibrosis in T2DM, aggravating kidney disease and deteriorate kidney function thereby. The factorial analyzes show:T2DM and the chronic periodontitis had interacted synergy in DKD. Chaper3Effect of Chronic periodontitis on expression of peroteins and their mRNA expression involved in renal lesions in OLETF ratObjectiveTo detecte the changes of proteins and the corresponding mRNA expression of type IV collagen, and the expression of its four major regulators:TGF-β1, MMP-2, MMP-9, and TIMP-1of kidneys in the four groups for exploring whether chronic periodontitis affect the expression in the presence of T2DM. And To investigate the possible pathological mechanisms of the effects of chronic periodontitis on T2DM renal damage.MethodsThe expression of TGF-β1, MMP-2, MMP-9, TIMP-1, type IV collagen in the kidney tissue of four groups of rats were detected by immunohistochemistry ENVISION two-step semi-quantitative analysis. The rat serum concentration of TGF-β1was determined by ELISA assay. Meanwhile, using the SYBR Green real-time quantitative PCR (Real-Time PCR) technology (fluorescent dye incorporation) we tested type IV collagen, TGF-betal, MMP-2, MMP-9, TIMP-1, mRNA differences in the rats’kidney tissue. The experimental results analyzed by factorial design analysis of variance and two independent samples t-test.ResultsType IV collagen was found in the glomerular basement membrane and mesangial matrix, blood vessel walls, tubular basement membrane etc. It was significantly higher in O group than that in the L group. The expression in O CP (+) group was significantly higher than that in the O CP (-) group.(T=11.099P=0.000<0.01). While there was no significant difference between the L CP (+) group and the L CP (-) group (t=2.032P=0.062).TGF-β1was expressed in proximal convoluted tubule and distal convoluted and the expression was weak in renal glomerulus. It was found TGF-β1was positively expressed strongly in the kidneys of O CP (+) group. It’s expression in O CP (+) group was significantly higher than CP (-) group (t=12958, P=0.000). It was significantly enhanced in L CP (+) group than that in L CP (-) expression (T=8.923, P=0.000). The expression significantly increased after the intervention of the CP. Group and periodontitis both has a significant impact on the expression of TGF-β1protein. Periodontitis and diabetes had corresponding interaction on the expression of TGF-betal protein (F=5.729P=0.024). TGF-β1mRNA expression trend is similar with and protein expression trend. Its expression increased significantly in L group after adding CP.MMP-9protein was found mainly expressed in the proximal tubule and distal convoluted tubule epithelial cells and a small amount of expression was found in mesangial cells.In Normal control group L, the expression of MMP-9significantly higher than that of group O. the expression reduced significantly in L CP(+) group than in L CP (-)(t=11.274, P=11.274). There was an interactive effect between T2DM and periodontitis (F=66.397P=0.000). Renal MMP-9mRNA Expression of the overall trend in four groups was also agreed with the protein expression trend. In normal rat kidney of L group had the highest expression levels and the O group was significantly lower than that of group L.MMP-2protein was expressed in the proximal tubule, distal convoluted tubule and glomerular visible trace. The little expression can also be found in the area of inflammatory infiltration of inflammatory cells. The highest expression level is still found in the L two groups, It was significantly decreased in L CP (+) group compared with L CP (-) group (t=9.030, P=0.000). O group was significantly reduced than in the L group. In O CP (+) group MMP-2positive staining area was not obvious. The main effect of group and periodontitis both had a significant impact on on expression of MMP-2protein. There is an interaction between group and periodontitis (F=13.529P=0.001). The MMP-2mRNA expression had the opposite trend in the four groups compared with its protein expression trend. It was significantly higher in group O than in group L. And it was significantly higher in O CP (+) group than that in O CP (-) group (t=2.475P=0.027).The expression of TIMP-1protein was found in the proximal tubules, distal convoluted tubule, and renal interstitial and glomerular matrix region. The expression of TIMP-1in O group was significantly higher than that of group L (t=1721.404, P=0.000<0.01). The expression significantly enhanced in O CP (+) group than in the O CP (-) group (t=9.007P=0.000<0.01). The expression in L CP (+) group and L CP (-) group was close and found no statistically significant difference (t=1.245P=0.234). The expression trend of TGF-beta1, and collagen type IV, TIMP-1mRNA in the four groups was consistent with the protein expression of respectively. Namely, L group have normal low expression of the above three gene. And the expression in O group was significantly increased than in L group (P<0.01). The mRNA expression in the O CP (+) group is slightly higher than the O CP (-) group without statistically difference.ConclusionIn the presence of diabetes, periodontitis not only promoted fibrogenic core factor TGF-β1expression in kidney tissue but also increase TGF-β1serum level. In this combinding model, periodontitis also contributed to the high expression of TIMP-1which is the inhibition of collagen degradation enzymes. Futhermore, it inhibits extracellular mechanisms degrading enzymes MMP-2, MMP-9normal expression and thus led to an increase of type Ⅳ collagen deposition in kidney tissue.This suggests the metabolic balance of extracellular matrix in the kidney has been broken with the increase of collagen production and the decreased collagen degradation in T2DM. This imbalance was exacerbated in the presence of periodontitis, resulting in the accelerated glomerulosclerosis and renal interstitial fibrosis. And finally the kidney function was impaired.