The role of vitamin D deficiency and elevated inflammatory biomarkers as risk factors for the progression of diabetic nephropathy in patients with type 2 diabetes mellitus

1 Introduction

Diabetic nephropathy (DN) is generally characterised by chronic diabetic microvascular complications and is the main cause of end-stage renal disease. DN’s classical presentation is characterised by hyperfiltration and albuminuria in the early phases, which is then followed by a progressive loss in renal function [1].

Vitamin D deficiency has a great impact on human health, and it is becoming progressively significant [2]. In Saudi Arabia, vitamin D deficiency has been recognised in both genders and all age groups [3]. Recently, several studies determined that besides the principal function of vitamin D in skeletal health, it has anticancer, anti-inflammatory and antioxidant effects [4].

Vitamin D is a pleiotropic hormone that can be obtained from both sunlight and food sources. Vitamin D is hydroxylated in the liver to generate 25(OH)D, and in the kidney to form 1,25(OH)₂D₃ (active calcitriol) by CYP2R1 and CYP27B1 hydroxylases enzymes, respectively. Kidneys are identified as the major source of CYP27B1, and its gene has nine exons and is found on the long arm of chromosome 12 (12q14.1). The biological function of vitamin D is intimately linked to the vitamin D receptor (VDR), a well-known ubiquitous nuclear receptor. VDR binds 1,25(OH)₂D₃ and regulates vitamin D metabolism by activating gene transcription. The VDR gene has 11 exons and is found on the long arm of chromosome 12 (12 q12–14) [5,6].

Previous candidate gene investigations [7] and genome-wide association studies [8] have suggested that some frequent single nucleotide polymorphisms (SNP) in vitamin D metabolic pathway genes have been linked to the level of circulating 25(OH)D at common status. Several prevalent studies have found links between CYP2R1, CYP24A1 and CYP27B1 gene polymorphisms and vitamin D deficiency. The main metabolic enzymes, 25-hydroxylase (CYP2R1) and 1-αhydroxylase (CYP27B1), and VDR are encoded by these SNPs. Polymorphisms in the VDR gene may change their expression and mRNA stability, affecting VDR functionality and vitamin D action in the long term [9].

Vitamin D deficiency has been linked to the development of diseases such as type 2 diabetes mellitus (T2DM), heart disease, autoimmune disease and cancer. The most commonly used biomarker for measuring vitamin D status from both endogenous production and nutritional intake is the blood 25(OH)D level because it has a long half-life and more stability than the active form calcitriol [10,11,12].

Vitamin D deficiency is described as a potential risk factor for the progression of T2DM and is associated with its microvascular complications. Vitamin D deficiency is more common in diabetic patients with nephropathy [13].

Chemerin is a novel adipocyte-derived factor [14] and plays a role in inflammation regulation [15]. Chemerin also plays a crucial role in the development of T2DM and DN [16]. Serum chemerin levels are closely related to renal function [17], with markedly high levels recorded in type 2 diabetic patients with macroalbuminuria [18].

Chemerin is closely related to trans-differentiation stimulating factors, such as tumour necrosis factor-alpha (TNF-α), because of its role as a proinflammatory adipocytokine [19]. TNF-α is assumed to prompt insulin resistance through various mechanisms such as an increase in the serine phosphorylation of insulin receptor substrate-1 (IRS-1), which interrupts the insulin signalling cascade [20]. TNF-α plays a pivotal role in the development of microvascular diabetic complications, including nephropathy [21].

There are synergistic effects between chemerin and C-reactive protein (CRP). The levels of chemerin and CRP are clearly increased in DN patients. CRP and chemerin might participate in the occurrence and development of DN through adaptable insulin resistance, inflammation response, adipocyte differentiation and metabolism, as well as endothelial injury [22]. Elevated serum levels of CRP are closely associated with an increase in microalbuminuria and renal dysfunction in T2DM patients [23]. CRP can induce interleukin-6 (IL-6) via an NF-κB-dependent mechanism in the inflammatory cascade [24].

IL-6 has been identified as a key mediator in inflammation, immune responses and glucose metabolism. The IL-6 receptor blockade’s protective effects against diabetic renal injury could be because of lowered insulin resistance and inflammasome inhibition [25].

In animals with DN, the rate of albumin elimination, the thickness of the glomerular basement membrane and the renal volume were lowered by 1,25 dihydroxy vitamin D supplementation [26].

In the current study, we aimed to explore the potential risk factors of albuminuria, a marker of DN, among diabetic patients.

2 Patients and methods

This case-control study was conducted at the College of Applied Medical Sciences, Taif University. Prior to starting the study, ethical approval was obtained from the ethical committee of Taif University (approval no. 42-0010), while written informed consent was obtained from each participant before they were enrolled in the study. Patients were recruited from Ministry of Health hospitals in Al-Ahsa City, Saudi Arabia, from October 2020 to January 2021. The study included 172 participants, and they were diagnosed with T2DM according to the American Diabetes Association 2010 [27]. The participants were divided into four groups: The control group comprised 38 healthy individuals (13 men and 25 women) not suffering from diabetes mellitus (DM), hypertension or any chronic disease, and the T2DM with normoalbuminuria group comprised 41 T2DM patients (18 males and 23 females) with normoalbuminuria without complications. The third group, T2DM with microalbuminuria, consisted of 43 patients (28 males and 15 females) with T2DM and microalbuminuria, and the fourth group, T2DM with macroalbuminuria, consisted of 50 patients (35 males and 15 females) with T2DM and macroalbuminuria. The age of the participants in the research ranged from 36 to 75 years. For the four groups, the control group, T2DM with normoalbuminuria, T2DM with microalbuminuria and T2DM with macroalbuminuria, the mean ages were as follows: 46.55, 41.41, 44.79 and 50.48, respectively. The mean values of body mass index (BMI) for the control, T2DM with normoalbuminuria, T2DM with microalbuminuria and T2DM with macroalbuminuria were as follows: 25.54, 26.82, 27.91 and 29.66, respectively. The mean values of the duration of DM for the groups T2DM with normoalbuminuria, T2DM with microalbuminuria and T2DM with macroalbuminuria were as follows: 6.3, 9.3 and 8.93, respectively.

3 Laboratory parameters

Five millilitres of venous blood was obtained from all fasting participants in serum-separating containers, placed at room temperature for 30 min, then centrifuged at 3,000 rpm. Sera were separated and stored at −20°C until use. Another 2 mL of blood samples were collected in EDTA-containing tubes for plasma chemerin and glycosylated haemoglobin (HbA1c) analysis.

DN was assessed according to the presence of microalbuminuria, which was evaluated using the immunoturbidimetric technique (Beckman Coulter IMMAGE, US). Microalbuminuria was considered if albumin excretion in the urine ranged between 30 and 300 mg/24 h, and macroalbuminuria was considered if the excretion exceeded 300 mg/24 h.

We utilised the BS-400 Mindray chemistry analyser and kits from Roche (Roche Diagnostics, Germany) to assess total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C), blood glucose, urea and creatinine. Kits and the High-Performance Liquid Chromatography Bio-Rad D-10 system (Bio-Rad Laboratories Inc., CA, US) were used to estimate HbA1c.

The estimated glomerular filtration rate (eGFR) was calculated using serum creatinine [28], and the albumin–creatinine ratio (ACR) in the urine was assessed using strips and laboratory methods.

To analyse the serum levels of 25(OH)D, the Abcam human vitamin D enzyme-linked immunosorbent assay (ELISA) kit, USA (Cat No: ab213966) was used, following the manufacturer’s instructions. If the serum level of 25(OH)D was less than 20 ng/mL, it was considered deficient. The parathyroid hormone (PTH) was assessed via chemiluminescent immunoassay technology using a Roche Cobas e-411 Immunoanalyser. Serum calcium was estimated using the calcium colourimetric assay kit, Abcam, USA (Cat No: ab102505).

Determination of the serum levels of IL-6, CRP and TNF-α was performed via a quantitative sandwich ELISA kit. An ELISA kit from R&D Systems (MN 55413, US) was used to estimate the plasma levels of chemerin.

4 Statistical analysis

Our data were statistically evaluated using SPSS software version 20.0 (SPSS Inc., Chicago, US). The data were presented as mean values ± SD. For quantitative parameters, a one-way analysis of variance (ANOVA) was conducted to calculate the significance among all the groups studied, followed by a post hoc test to compare the groups with each other. A Chi-square test was used to compare proportions between qualitative parameters (sex and hypertension). Furthermore, a Pearson correlation was used to analyse the correlation between vitamin D and inflammatory markers, as well as between vitamin D and the ACR. p-values were considered statistically significant at < 0.05, while a p-value of <0.001 was considered highly significant. A logistic regression analysis model was used to analyse the relationship between the studied parameters and the risk of albuminuria. The logistic regression model relates the log of the odds to the explanatory variable log p 1 − p = β 0 + β 1 x , where p is a binomial proportion and x is the explanatory variable. The parameters of the logistic regression model are β0 and β1.

5 Results

As shown in Table 1, the macroalbuminuria group’s mean age (50.48 years) was significantly higher than that of the other three groups, and the microalbuminuria group’s mean age (44.79 years) was significantly higher than that of the normoalbuminuria T2DM group (p < 0.001). A comparison between the T2DM patients and controls showed that males were more likely to have micro- or macroalbuminuria (p = 0.033) than females. Additionally, a comparison between the normoalbuminurics and controls showed that albuminuric T2DM patients exhibited a substantially higher BMI (p < 0.001). The micro- and macroalbuminuric patients had significantly higher DM durations (9.3 and 8.95 years, respectively) than T2DM patients without albuminuria (6.3 years, p < 0.001).

A comparison between the groups showed that metabolic parameters such as FPG, HbA1c, TG, TC and LDL showed significant increase in T2DM patients with macroalbuminuria, followed by those with microalbuminuria, while the mean HDL was significantly lower in the micro- and macroalbuminuria groups compared to the other two groups (p < 0.001) for all tested parameters (Table 1).

When compared to the other three groups, T2DM patients with macroalbuminuria had a significantly higher statistical difference in the mean levels of ACR, PTH and urea, as well as a lower calcium level and eGFR; however, they had a significantly higher mean serum creatinine level compared to the controls and normoalbuminuria patients but showed no difference with the T2DM microalbuminuria patients.

The T2DM patients with microalbuminuria had substantially higher mean ACR and urea serum levels, as well as a lower total calcium level, compared to both the control and normoalbuminuria groups. In addition, they had significantly higher mean serum creatinine and PTH levels but no change in eGFR (Table 1).

As demonstrated in Table 1 and Figure 1, the T2DM patients had TNF-α, IL-6, CRP and vitamin D mean levels of 27.13 pg/mL, 32.03 pg/mL, 8.09 mg/L and 28.07 ng/mL for each biomarker, respectively, which showed significant statistical difference when their mean levels were compared with those of the control group (mean values: 4.27 pg/mL, 3.71 pg/mL, 1.89 mg/L and 36.16 ng/mL for each biomarker, respectively).

Figure 1

Vitamin D level and the inflammatory biomarkers in the four studied groups.

Among the T2DM patients, those with macroalbuminuria demonstrated statistically significant mean levels of TNF-α, IL-6, CRP, ACR, vitamin D, total calcium, PTH, urea and chemerin (65.61 pg/mL, 91.67 pg/mL, 53.20 mg/L, 457.98 µg/mg, 14.7 ng/mL, 7.56 mg/dL, 40.37 ng/L, 35.4 mg/dL and 247.72 ng/mL, respectively) compared to the controls and T2DM patients with normo- and microalbuminuria (Table 1 and Figure 1).

Moreover, patients with microalbuminuria demonstrated a significant difference in the mean levels of TNF-α, IL-6, CRP, ACR, vitamin D, calcium, PTH, urea, creatinine and chemerin (52.91 pg/mL, 60.61 pg/mL, 26.3 mg/L, 80.56 µg/mg, 28.07 ng/mL, 9.01 mg/dL, 37.12 ng/L, 31.78 mg/dL, 1.14 g/dL and 151.58 ng/mL, respectively) when compared to the control or normoalbuminuria T2DM groups (Table 1).

Logistic regression analysis was performed to determine the risk factors of albuminuria in the studied groups of patients, as shown in Table 2. The duration of DM, BMI, glucose, GFR, TC, LDL, TNF-α, IL-6, CRP, ACR, vitamin D, PTH and chemerin were significant risk factors for albuminuria (p-values were 0.012, <0.001, 0.025, 0.004, 0.008, 0.003, <0.001, <0.001, 0.011, 0.014, <0.001, 0.010 and 0.021, respectively). Insignificant albuminuria risk factors were age, sex, HbA1c, TG, creatinine, HDL, total calcium and urea (p > 0.05 for all).

Regarding the correlation between vitamin D and markers of inflammation, a significant negative correlation was found between vitamin D levels and TNF-α, CRP, IL-6 and chemerin levels (p < 0.001 for all). Moreover, a significant negative correlation between vitamin D levels and ACR (p < 0.001) was found, as shown in Table 3 and Figures 2–6.

Figure 2

Scatter plot with regression line between vitamin D and TNF-α.

Figure 3

Scatter plot with regression line between vitamin D and CRP.

Figure 4

Scatter plot with regression line between vitamin D and IL-6.

Figure 5

Scatter plot with regression line between vitamin D and chemerin.

Figure 6

Scatter plot with regression line between vitamin D and ACR.

6 Discussion

DN has been described as diabetes with a gradual increase in urinary albumin, hypertension and compromised renal function [29]. Albuminuria has two stages known as micro- and macroalbuminuria. However, not all patients with microalbuminuria will develop DN, but patients with macroalbuminuria, which is a more advanced stage, have a bad prognosis [30].

To our knowledge, this is the first study to evaluate the relationship between vitamin D status, a combination of proinflammatory cytokines (IL-6 and TNF-α), chemokine (chemerin) and general inflammatory markers (CRP) in the development of DN in T2DM Saudi patients in Al-Ahsa City, Saudi Arabia.

In our included patients, an increase in TC, TG and LDL-C and a decrease in HDL-C were noted relative to the controls. Stadler et al. [31] reported that dyslipidaemia is involved in the development and progress of DN. The association between dyslipidaemia, hyperglycaemia and the progress of diabetic kidney disease (DKD) was explained through the enhancement of glomerulosclerosis by dyslipidaemia in diabetic patients [32]. Moreover, in diabetic patients, increased LDL-C and decreased HDL-C were observed due to the impairment of lipoprotein metabolism [33].

Several prevalent studies have found links between CYP2R1, CYP24A1 and CYP27B1 hydroxylase gene polymorphisms and vitamin D deficiency. Vitamin D has an ameliorative effect on the inflammatory process via the anti-prostaglandin effect, p38 stress kinase signalling inhibition, cytokine release reduction and lymphocyte production [34]. Timms et al. [35] reported that a reduction in serum vitamin D participates in microangiopathy by affecting the formation of CRP and matrix metalloproteinase. Moreover, Jablonski et al. [36] reported that lower levels of 25(OH)D enhanced by inflammation related to the NF-κB leads to impairment of the vascular endothelium.

In the current study, there was a significant difference in ACR (µg/mg) between diabetic patients with micro- and macroalbuminuria and T2DM patients. Our results were in agreement with the results of Chida et al. [37], who concluded that higher urinary ACR levels in adults with T2DM are associated with a greater incidence of macroalbuminuria, and urinary ACR is a reliable predictor of the progression of DN.

Macroalbuminuria is caused by glomerular damage and enhanced macromolecule permeability in the glomerulus. Several factors can influence protein transport across the filtration membrane, including the haemodynamic pressure gradient across the glomerular basement membrane and features unique to the filtration membrane, such as pore size and anion charge distribution. Macroalbuminuria and increasing renal function loss indicate obvious DN due to several mechanisms that change the protein-selective glomerular basement membrane. Many clinical investigations have shown a link between the degree of albuminuria or proteinuria and the progression rate of DN [38].

Prolonged, uncontrolled hyperglycaemia enhances the formation of glycated substances and the activation of macrophages, which enhance the production of inflammatory cytokines. Glycaemic management is, thus, important in preventing the progression of normoalbuminuria to microalbuminuria and microalbuminuria to proteinuria [39,40].

The present study’s results revealed that the serum 25(OH)D level was lower in T2DM and DN patients than in the controls and is related to the stage of DN. These findings comply with the results of Xiao et al. [41]. Moreover, Huang et al. [30] concluded that deficiency in 25(OH)D is associated with microalbuminuria, and Mao et al. [42] reported that a low serum 25(OH)D level is associated with increased serum and urinary markers of TNF-α and IL-6 in DN patients.

Previous studies have been done on the role of vitamin D in DN, but the combination of inflammatory markers included in this research has not been investigated before in DN. These markers may be studied separately in DN or collectively in other diseases. In the current study, a significant negative correlation between serum vitamin D and the studied inflammatory markers (IL-6, TNF-α, chemerin, CRP and ACR) was reported. Further, a deficiency in serum 25(OH)D was linked with elevated levels of all studied inflammatory markers.

Chemerin is an adipokine regulator of metabolism and energy balance [43]. The plasma chemerin was significantly increased in our patients, especially in those with macroalbuminuria. Hu and Feng [17] also reported elevated chemerin in their T2DM with macroalbuminuria patients compared to normo- and microalbuminuria patients. In our group of patients, CRP was significantly elevated. Wang et al. [22] described a positive synergistic association between serum chemerin and CRP in the inflammatory process of DKD. On the other hand, several reports attributed the elevated levels of chemerin in DKD to diminished renal excretion rather than enhanced formation, while haemodialysis decreased plasma chemerin levels [44–46].

An evaluation of the serum levels of TNF-α in our patients showed a significant elevation in T2DM patients and patients with T2DM and micro- and macroalbuminuria compared to the controls. The increase in type 2 diabetic nephropathy (T2DN) reflected an excessive inflammatory load in DN [47]. In Lampropoulou et al.’s [48] study, microalbuminuria was not significantly associated with serum TNF-α levels. Meanwhile, other investigators described higher serum TNF-α levels in macroalbuminuric and microalbuminuric patients [49].

TNF-α, IL-1 and IL-6 were increased in DN with dominant proinflammatory activities. These data provide a comprehensive overview of DN pathophysiology and support the idea that inflammatory pathways play a vital role therein [42]. Further, previous studies have reported increased levels of serum IL-6 in DN patients that showed a positive correlation with the degree of proteinuria [50,51]. In addition, Ebrahim et al. [52] revealed that the significant elevation of the serum IL-6 level was considered a marker for DN progression. In Pojskić et al.’s [53] study, increasing levels of serum hs-CRP in patients with T2DM and microalbuminuria were detected in comparison to those with normoalbuminuria. On the other hand, Cao et al. [54] detected increased plasma CRP in diabetic subjects but did not associate it with the status of albuminuria.

CRP is a ring-shaped, pentameric serum protein that the liver produces. As an acute phase inflammation marker released in response to IL-6 signalling by T-cells, macrophages and adipocytes, CRP levels increase in a wide range of acute and chronic inflammatory conditions. These include infection, inflammatory disease, tissue injury, cardiovascular disease and malignancy. The early stage of DN is associated with endothelial dysfunction. In insulin resistance, TNF-α and IL-6 levels increase, which can further increase liver function and consequently lead to an increase in CRP. The oxidative stress triggered by CRP that performs the inflammatory reaction causes direct damage to the glomerular endothelial cells [22].

Himansu et al. [55] concluded that endothelial function in early DN patients significantly improves following vitamin D supplementation. Furthermore, vitamin D supplementation positively affects the cardiovascular outcome, which is reflected by an improvement in flow-mediated dilation (FMD) and a decrease in the inflammatory mediators as CRP level.

From the current results, we concluded that vitamin D deficiency and the related inflammatory mediators such as chemerin, TNF-α, IL-6 and CRP are the most essential risk factors for albuminuria in T2DM patients and may help in the early diagnosis and prediction of the prognosis of DN.

Moreover, we suggest that vitamin D supplementation in early diagnosed DN is essential to improve the condition and prevent its progression. Therefore, further studies are needed to optimise the dose of vitamin D supplementation that will be effective in early diagnosed DN.