SERUM CHEMERIN LEVEL IN RELATION TO RENAL FUNCTION IN EXPERIMENTALLY INDUCED DIABETIC RATS

By

Nawal K. Gerges and Doaa A. Abdel Moety

Physiology Department, Faculty of Medicine, Zagazig University

ABSTRACT

Background:  Chemerin, a recently discovered adipocytokine, regulates adipocyte development and metabolism as well as inflammatory and immune function. Data onto chemerin levels in diabetes and its associated renal affection is limited and controversial.

Objective: This work was designed to evaluate the serum chemerin level in acute and chronic experimentally-induced diabetes and its relation to some metabolic parameters, and renal function tests.

Materials and Methods: This study was done on 30 adult healthy male of local strain albino rats. Animals were divided into three equal groups, i.e.healthy group, acute diabetic group and chronic diabetic untreated group. Diabetes was induced experimentally by intra peritoneal injection of streptozotocin (STZ). In each of the three groups, each animal was put inside a cage  to collect 24h urine tomeasure albuminuria, then they were sacrificed, and  sera  were examined for levels of glucose, insulin, chemerin , cholesterol, triglycerides (TG), high density lipoproteins (HDL) , Low density lipoproteins (LDL), urea, creatinine, creatinine clearance, C-reactive protein (CRP), and tumor-necrosing factor alpha (TNF-α).

Results: There was a significant increase in serum chemerin levels in the chronic diabetic untreated group when compared to both control and acute diabetic groups. Also, there were significant positive correlations between serum chemerin levels and serum urea, creatinine, urine albumin, serum cholesterol, TG, LDL, CRP and TNF-α levels, as well as negative correlations between serum chemerin levels, and both serum creatinine clearance and HDL levels in the chronic diabetic untreated group.

Conclusion: Serum chemerin levels seemed to be related to impaired renal function test (albuminuria, serum creatinine, urea and creatinine clearance) rather than to serum insulin level. Serum chemerin levels elevated, without any concomitantsignificant change in serum insulin in chronic diabetic group, when compared to acute diabetic group. Accordingly, it can be hypothesized that the change of serum chemerin could be considered as a predicting marker of diabetic nephropathy rather than of diabetes.

Key words: Chemerin, Diabetes, Renal functions.

INTRODUCTION

     Chemerin regulates adipocyte differen-tiation and modulates the expression of adipocyte genes involved in glucose and lipid homeostasis, such as adiponectin, glucose transporter-4, and leptin (Bozoaglu et al., 2007 and Goralski et al., 2007).

     Tan et al. (2009) stated that insulin significantly increased chemerin produc-tion in adipose tissue explants, and there is a positive correlation between circulating insulin concentration and chemerin mRNA level in white adipose tissue. However, data about chemerin levels in diabetic patients is limited and controversial.

     Some reports demonstrated that the serum chemerin level was significantly elevated in both type1 (Verrijn Stuart et al., 2012 and Redondo et al., 2014) and type 2 diabetes (Stejskal et al., 2008; Bozoaglu et al., 2009; Weigertet al., 2010 and Bluheret al., 2012).

    On the other hand, other studies failed to find any significant difference in plasma chemerin levels between diabetic patients and healthy controls, and indicated also that serum chemerin may not be a significant marker of diabetes (Bozoaglu et al., 2007; Pfau et al., 2010 a; Weigert et al., 2010 and Hu &Feng., 2011)

     Diabetic nephropathy (DN) is consi-dered a serious microvascular complica-tion of diabetes. The pathological changes such as accumulation of extracellular matrix protein, expansion of mesangial cells, , thickening of tubular basement membranes, tubulointerstitial fibrosis, glomerulosclerosis, and renal endothelial disfunction are noted to occur in the diabetic kidney (Wagman and Nuss., 2001).

    Studies indicated a strong association of high chemerin level with decreased renal function, represented by serum creatinine and creatinine clearance in patients with diabetic nephropathy.(Yamamoto et al., 2010., Hu &Feng 2011; Hu et al., 2012, Rutkowski et al., 2012 and Lin et al., 2016).

     On the other hand, other investigators reported that serum chemerin levels remained significantly higher in hemo-dialysed patients compared with controls, suggesting that elevated circulating chemerin levels are not directly related to renal function (Pfauet al., 2010 a).

    On the face of this controversy, this study was designed to evaluate serum chemerin levels in both acute and chronic diabetes and its relation to some metabolic parameters, and renal function tests.

MATERIAL AND METHODS

Animals:

    This study was performed on a total number of 30 adult healthy male local strain albino rats (weighing 150–180 grams).  Animals were kept under hygienic conditions in steel wire cages (40x28x18 cm), in the animal house of the Faculty of Medicine, Zagazig University. All rats had free access to water and commercial rat standard chow which consisted of 25.8% protein, 62.8% carbohydrates and 11.4% fat (Ahren and Scheurink, 1998). Rats were kept at comfortable temperature (20 -24˚C) and   maintained on a normal light/dark cycle (Lesourd and Mazari, 1999). The rats were adapted to laboratory conditions for 1 week before starting the experimental regimen.

All the experimental procedures were conducted in accordance with the guiding principles for the care and use of research animals.

Rats were divided into three groups:

Group I (control group): Healthy rats fed on normal standard diet

Group II (acute diabetic group): Rats in which experimental diabetes was induced by intra peritoneal injection of streptozotocin.

Group III (Chronic diabetic non treated group): Rats in which experimental diabetes was induced by intra peritoneal injection of streptozotocin and left without any treatment of diabetes for 8 weeks.

Experimental diabetes was induced by intra peritoneal injection of streptozotocin (50mg/kg) dissolved in Na citrate solution adjusted at pH 4.5 (Hu et al., 2012). STZ was obtained from (Chemical, St. Louis, MO, Sigma-Aldrich,USA).

All animals were fasted overnight. Three days later, diabetes induction was confirmed by measurement of blood glucose level in each animal (samples were taken from the tail vein) with the Bionime- GM300 Glucometer (Yves and Theo, 2007). Rats with diabetes (blood glucose >160mg/dl) were selected for experiments (Palsamy et al., 2008).

In the acute diabetic group, rats were sacrified after 3 days of diabetes induction.

In the chronic diabetic group: rats were sacrified after 8 days of diabetes induction.

Because streptozotocin induces fatal hypoglycemia as a result of massive pancreatic insulin release, the rats were provided with 10% glucose solution after 6 hours of STZ administration for the next 48 hours to prevent hypoglycaemia.

In each of the three groups, each animal was put inside a cage to collect 24h urine to measure albuminuria (Hu et al., 2012), then they were sacrificed and blood samples were obtained at the time of scarification and were allowed to clot for 2 hours at room temperature and were centrifuged for 20 min. at approximately 500 rpm. The separated sera were stored at -20^oC. (Nishizawa et al., 2002), until its use for estimation of:

-Serum glucose levels (mg/dl) (Tietz, 1995).

-Serum insulin levels (µIU/ml) by enzyme – linked immunosorbent assay (ELISA) according to (Hu et al., 2012).

-Serum total cholesterol levels (mg/dl) (Tietz, 1995).

-Serum TG levels (mg/dl) (Fossati and Prencipe, 1982).

-Serum HDL levels (mg/dl) (Nauck et al. 1997).

-Serum LDL levels (mg/dl) (Friedewald et al. 1972).

LDL was calculated as follows: LDL = TC -HDL – TG/5

Kits for estimation of serum glucose, insulin, cholesterol,TG, LDL and HDL levels from Biosource Europe S.A., Belgium).

-Serum chemerin levels (ng/ml) by enzyme –linked immunosorbent assay (ELISA) (Hu et al., 2012) using kits from (New Test Company, Egypt).

 -Serum urea levels (mg/dl) (Kaplan, 1984).

-Serum creatinine levels (mg/dl) (Murray et al. 1984).       

  Kits for estimation of serum urea and creatinine levels were from S.A.U. Ctra.Santa Coloma, Spain.

-Serum creatinine clearance (ml/min.) from the following equation (Murray et al., 1984):

-Serum CRP levels (µg/ml) (Ridker et al., 1998), using Kits from Biosource International Inc. Road Camarillo, CaliforniaUSA.

-Serum TNF-α levels (Pg/ml)(Hosseni-Tabatabei et al., 2009), using kits from Monobind Inc.Lake forest,USA.

Statistical analysis: The data obtained in the present study were expressed as mean ±SD for quantitative variables and statistically analyzed according to the methods described by Kirkwood (1989).

The statistical analysis was done by using SPSS program (19) (SPSS Inc. Chicago, IL, USA). P value < 0.05 was considered statistically significant.ANOVA test (Post hoc LSD test) was used to compare means among more than two groups.Corrélation coefficient (r): Pearson's correlation analyses were performed to screen potential factors related to serum concentrations of chemerin. Test was considered significant at P values <0.05.

RESULTS

     This study revealed a significant increase in fasting serum glucose levels (mg/dl) in group II (394.6±23.74) and group III (411.9±23.36) when compared to group I (82.8±3.97). [P<0.001, P<0.001 respectively],  but there was no significant change in serum glucose levels between group II and group III [P>0.05].

     Also, there was a significant decrease in serum insulin levels (µIU/ml) in groupII (0.882±0.18) and group III (1.26±0.4) when compared to group I (19.84±1.42) [P<0.001, P<0.001 respectively], but there was no significant change in serum insulin levels between group II and group III [P>0.05].

    There was asignificant increase in serum chemerin levels (ng/ml) in group III (263± 11.05) when compared to both group I (202.7±9.63) and GroupII(209.9 ±12.42) [P<0.001, P<0.001 respectively], but there was no significant change in serum chemerin levels between group I and group II [P>0.05].

    Significant increase in serum cholesterol levels (mg/dl) was found in groupIII (115.15± 2.72) when compared to group I (68.24±1.83) and group II(69.06± 1.83) [P<0.001, P<0.001 respectively], but there was no significant change in serum cholesterol levels between group I and group II [P>0.05].

     At the same time there was a significant increase in serum triglyceride levels (mg/dl) in groupIII (173.6± 5.07) when compared to group I (88.1±2.51) and group II (89.4 ±4.29) [P<0.001, P<0.001 respectively], but there was no significant change in serum triglyceride levels between group I and group II [P>0.05].

    Also, there was a significant decrease in serum HDL levels (mg/dl) in group III (36.64± 1.85) when compared to group I (49.16±2.42) and group II(48.7± 2.92) [P<0.001, P<0.001 respectively], but there was no significant change in serum HDLlevels between group I and group II [P>0.05].

    The study recorded a significant increase in serum LDL levels (mg/dl) in groupIII (133.97± 4.22) when compared to group I (42.2±1.81) and group II(44.5± 3.44) [P<0.001, P<0.001 respectively], but there was no significant change in serum LDL levels between group I and group II [P>0.05] (Table 1).

Table (1): Blood analysis and metabolic parameters in the three studied groups (mean ± SD).

*** = P < 0.001        a = versus group I          b = versus group II

     There was a significant increase in serum urea levels (mg/dl) in group III (68.8± 2.5) when compared to both group I (mean ± SD: 36.5±2.1) & group II (mean ± SD: 38.5±2.9) [P<0.001, P<0.001 respectively], but there is no significant change in blood urea levels between group I and group II [P>0.05].

     Also, the study recorded a significant increase in serum creatinine levels (mg/dl) in group III (2.9± 0.57) when compared to group I (0.37±0.02) and group II (0.41± 0.03) [P<0.001, P<0.001 respectively], but there was no significant change in serum creatinine levels between group I and group II [P>0.05].

     There was a significant decrease in serum creatinine clearance levels (ml/min.) in group III (0.26± 0.03) when compared to group I (0.78±0.05) and group II (0.76±0.05) [P<0.001, P<0.001 respectively], but there was no significant change in serum creatinine clearance levels between group I and group II [P>0.05].

    The present study showed a   highly significant increase in albumin levels in urine (µg/day) in group III (673.5± 52.49) when compared to group I (73±6.34) and group II(79.1±3.78) [P<0.001,P<0.001 respectively], but there was no significant change in albumin levels in urine between group I and group II [P>0.05].

    Significant increase in serum TNF-α levels (Pg/ml) was noticed in group III (84.36±4.36) when compared to group I (13.93±0.89) and group II (14.23±0.88) [P<0.001,P<0.001 respectively], but there was no significant change in serum TNF-α levels between group I and group II [P>0.05].

    In addition, there was a significant increase in serum CRP levels (µg/ml) in group III (2.79±0.56) when compared to group I (0.89±0.11) and group II(0.94±0.15) [P<0.001,P<0.001 respectively], but there was no significant change in serum CRP levels between group I and group II [P>0.05](Table 2).

Table (2): Kidney function testsandinflammatory mediators in the three studied groups (mean ± SD)

*** =P<0.001a = versus group I            b = versus group II

    Significant +ve correlations were recor-ded between serum chemerin levels and serum urea levels, serum creatinine levels, urine albumin levels, serum cholesterol, triglycerides, LDL,TNF-α and CRP levels,as well as –ve correlations between chemerin and serum creatinine clearance &HDL levels in group III. But, there were no correlations between chemerin and any of the previous parameters in either group I or group II. Also, there were no significant correlations between serum chemerin levels and serum glucose or serum insulin levels in any of the three studied groups (Table 3).

Table (3): Different correlations between serum chemerin levels (ng/ml) and all studied parameters in the three studied groups

DISCUSSION

    Although chemerin may be linked to obesity and related diseases, relatively little informations about the factors affec-ting regulation of serum chemerin  are known (Stelmanska et al., 2013). Data about chemerin levels in diabetic patients is limited and controversial. Therefore, this study was designed to evaluate serum chemerin levels in both acute and chronic diabetic rats and its relation to some metabolic parameters and renal function test.

     The result of the present study revealed that there was no significant change in serum chemerin level in acute diabetic group when compared to control group. There was a significant decrease in serum insulin level, which was associated with a significant increase in serum glucose level in acute diabetic group, when compared to control group. In addition, there were insignificant changes in lipid profile (TG, cholesterol, HDL, and LDL), renal function tests (serum urea, serum creatinine, creatinine clearance, and albumin in urine), and inflammatory markers (CRP& TNF alpha) in acute diabetic group, when compared to control group

     These results were in agreement with some investigators who can not find any significant difference in plasma chemerin levels between diabetic patients and healthy controls, and also indicated that serum chemerin may not predict diabetes(Bozoaglu et al., 2007; Hu and Feng, 2010;Pfau et al., 2010b and Weigert et al., 2010).

    On the other hand, other investigators found thatrecent diagnosed autoimmune type 1 diabetes had significantly higher serum chemerin level in both lean (Verrijn Stuart et al., 2012), and obese children (Redondo et al., 2014), when compared to healthy control group.

    This controversy could be explained by that circulating TNF-alpha was elevated  in autoimmune type 1 diabetes in both obese and lean children compared with healthy control group. This proinflamma-tory cytokine was reported to stimulate chemerin production by 3T3-L1 cells and primary adipocytes (Parleeet al., 2010).

Relatively little information is available concerning the regulation of chemerin gene expression in adipose tissue by insulin. Tan et al. (2009) found that there was significant increase in chemerin production by insulin in human adipose tissue explants. Furthermore, Bauer et al. (2012a) observed that insulin enhanced chemerin release from human adipocytes, while serum chemerin levels were not changed.

     In the present study, changes in serum insulin did not seem to be a determinant factor for serum chemerin level because acute diabetic group presented with significant decrease serum insulin level, while there was an insignificant change in serum chemerin level. Therefore, it seemed that other factor beside insulin may affect serum chemerin level.

    In chronic diabetic untreated group, the present study found that there was a significant increase in serum chemerin level when compared with both acute diabetic and control groups. In addition, there was a significant increase in albuminuria, serum urea and creatinine, and a significant decrease in creatinine clearance levels in chronic untreated diabetic group when compared withbothcontrol and acute diabetic groups.

     These results were in agreement with Hu and Feng (2011) who demonstrated that serum chemerin level markedly increased in diabetic patient with macroalbuminuria compared with healthy subjects and diabetic patients with normoalbuminuria and microalbuminuria. This was supported by other studies that indicated a strong relationship between high chemerin level and decreased renal function, represented bya significant increase in serum creatinine, as well as the significant decrease in creatinine clearance and eGFRin patients with diabetic nephropathy.(Yamamoto et al., 2010; Hu et al., 2012 and Yu et al., 2015). Moreover, Rutkowski et al., (2012) reported that elevated serum chemerin concentrations in patients with end-stage renal disease (ESRD) were normalized after kidney transplantation, which supports the hypothesis that serum chemerin levels are related to renal functions.

     In the same context, the present study found that there was a significant   positive correlation between serum chemerin level and albuminuria, serum urea and creatinine, while there was a significant negative correlation between serum chemerin level and creatinine clearance.

    On the other hand, other investigators reported that serum chemerin levels remained significantly higher in hemodialysed patients compared with control levels, suggesting that elevated circulating chemerin levels are not directly related to renal function (Pfau et al., 2010 a). This controversy could be explained by Yamamoto et al. (2010) who reported that higher levels of serum chemerin in hemodialysed patients were positively correlated with markers of dyslipidemia and inflammation.

   The present study tried to explore the precise mechanism that leading to increase in serum chemerin level in chronic diabetic group.

    The first possible explanation of elevated chemerin levels may be a consequence of impaired clearance or catabolism of chemerin in kidney that may lead to accumulation of chemerin in plasma. In line with this hypothesis, other investigators concluded that renal filtration and/or renal degradation plays a critical role in the elimination of serum chemerin (Bozaoglu et al., 2007; John et al., 2007; Hu &Feng 2011, and Rutkowski et al., 2012). Unfortunately, in the present study urine chemerin level were not estimated. So, further studies of urine chemerin concentrations and analysis are required.

     The second possible explanation was that elevated chemerin levels may be the outcome of disturbed lipid profile (increased cholesterol, triglycerides and LDL). In agreement with this hypothesis, the present study found that there was a significant positive correlations  between serum chemerin levels and cholesterol, triglycerides, and LDL levels

     This was supported by other investiga-tors who demonstrated that in chronic diabetes, elevated serum chemerin levels were  positively correlated with markers of dyslipidemia (Goralski et al., 2007; Yamamoto et al., 2010; Lehrke et al., 2009; Lee et al., 2013 and Stefanov et al., 2013).  Moreover, another study demonstrated that serum FFA and TAG together significantly affected chemerin gene expression in WAT and serum chemerin concentrations (Bauer et al., 2012 b).

     The third possible explanation was that chemerin was seemed to be related to many inflammatory factors including CRP and TNF- α , which  have been shown to be involved in both the development and progression of diabetic nephropathy (Lehrke et al., 2009; Sell et al., 2010 and Weigert et al., 2010).

    This in accordance with the result of the present study that also found that there was increasein inflammatory factors CRP and TNF-α in chronic diabetic group, when compared to acute diabetic, and control groups.In addition, serum chemerin levels were also positively correlated  with  inflammatory factors including CRP and TNF-α in chronic diabetic group.

    Chemerin is considered a pro-inflammatory chemokine (Zabel et al., 2005), and inflammation is associated with chronic renal disease (Stenvinkel , 2005). Thus, one can suppose that chemerin is mediating in inflammation of renal failure by stimulating macrophage adhesion to extracellular matrix proteins, and adhesion molecules (Hart and Greaves, 2010).

    This was supported by other studies that illustrated that the expression of chemerin and chem. R23 in the kidney of diabetic rats noticeably increased than that of control group (Hu et al., 2012 and Yu et al., 2015). This overexpression of local chemerin levels in renal tissues may directly contribute to the inflammation and metabolic dysfunction of this tissue (Nishimura et al., 2009 and O'Rourke, 2009)

    Although renal production of chemerin and its contribution to circulating chemerin is not well known.Some data suppose that renal synthesis of chemerin may contribute to elevated serum concentrations of this protein (Bozaoglu et al., 2007; Roh et al., 2007 and Rutkowski et al., 2012).

     However, the results of other study suggested that chemerin is an anti-inflammatory protein (Ernst and Sinal, 2011). Moreover, another   study suggested that elevated concentrations of chemerin are associated with a survival advantage in dialysed patients (Yamamoto et al., 2010). Consequently, its role in systemic inflammation requires further study.

     In conclusion, serum chemerin level seemed to be related to impaired renal function test (albuminuria, serum creatinine, urea and creatinine clearance) rather than to serum insulin level. Serum chemerin level elevated without any concomitant significant change in serum insulin in chronic diabetic group, when compared to acute diabetic group. Accordingly, it can be hypothesized that the change of serum chemerin could be considered as a predicting marker of diabetic nephropathy rather than of diabetes.

     Further studies are needed to determine the pathophysiologic significance of chemerin in chronic kidney disease, and also to determine whether elevated chemerin might be the cause behind, or simply mirror of a reduced renal function.

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