Introduction

Diabetic retinopathy (DR), seen in approximately 80% of patients with 10 or more years of type 2 diabetes mellitus (T2DM), is a major microvascular complication. Possibly, it is responsible for a large proportion of vision problems and blindness in the population (1). In addition to the duration of diabetes, DR development is strongly linked with chronic hyperglycemia; dyslipidemia, diabetic nephropathy (DN), hypertension and mitochondrial dysfunction accompanied by induced oxidative stress and is associated with abnormal adiponectin (ADPN) levels (1,2). Mitochondrial dysfunction is also associated with renal tubular epithelial cell injury and the occurrence of DN, and ADPN is involved in promoting mitochondrial biogenesis and functional renal tubular epithelial cells (3). The mechanisms in the development of DR and DN seem very similar in terms of inflammation and oxidative stress.

ADPN secreted by adipocytes exists as a trimer and three multimer forms: low molecular weight, medium molecular weight, high molecular weight (HMW). It has effects such as antidiabetic, anti-inflammatory, regulating endothelial functions, antioxidant, antiapoptotic, antiatherogenic, antithrombotic, inhibiting smooth muscle proliferation and facilitating vasodilation (4). In studies conducted in different ethnic groups, plasma ADPN levels were found to be low in patients with obesity and T2DM. In contrast, the degree of hypoadiponectinemia was closely related to the degree of insulin resistance and hyperinsulinemia rather than the degree of adiposity (5). There was a relation between CRP, ADPN and quantitative insulin resistance check index to detect microvascular measurements [capillary density, urine albumin creatinine ratio (UACR) and endothelial measurements] in non-diabetic and normotensive healthy subjects (6).

The development of DR or albuminuria/proteinuria is accepted as findings in favor of endothelial dysfunction in the organism. The impact of UACR and estimated glomerular filtration rate (eGFR) on serum ADPN is clearly observed. As is known, the risk of cardiovascular disease (CVD) increases with endothelial dysfunction. As a result, ADPN has a positive effect on cardiovascular health. Its protective role against atherosclerosis is mediated by inhibiting vascular smooth muscle and endothelial cell proliferation. ADPN has been shown to be a good marker for metabolic control and atherosclerotic risk (7).

In this study, we investigated the relationship between serum ADPN or related indices and DR degree with or without albuminuria/proteinuria. Demographic/clinical/anthropometric findings, smoking status, blood pressure values, inflammatory blood markers and other routine laboratory findings were also considered.

Methods

A total of 122 patients, followed up in out-patient clinics of ophthalmology and internal medicine, were included our study. Patients with known thyroid dysfunction, urinary infection, nephropathy (of the non-diabetic causes), malignancy, diabetic neuropathy or eGFR <30 mL/min were all excluded. An approval of the research protocol was received by the University of Health Sciences Turkey, İstanbul Training and Research Hospital Ethics Committee (approval number: 117, date: 08.04.2022) in accordance with international agreements (World Medical Association Declaration of Helsinki) was received.

All the cases were divided into four different groups; group 1 (non-diabetics, n=27), group 2 (T2DM without retinopathy, with/without albuminuria and/or proteinuria n=45), group 3 (mild to moderate DR, without any albuminuria or proteinuria, n=26) and group 4 (severe non-proliferative or proliferative retinopathy, with albuminuria and/or proteinuria n=24). DR grades were defined by the same ophthalmologist based on clinical examination and angiographic findings.

Serum ADPN was run via immunoturbidimetric method (catalog no: AO 2999, Randox Laboratories Limited, Crumlin, UK) using AU2700 (Beckman Coulter Inc, Brea, Ca, USA). We found a coefficient of variation (within-run precision) of 1.45% at a serum level of 5.85 μg/mL (n=17), and 1.00% at a serum level of 12.05 μg/mL (n=19).

Anthropometric measurements of waist circumference (WC), height, weight and blood pressure were recorded. Data from the patient records were retrieved, the measurements were performed as follows; chemistry/immunochemistry assays using AU 2700 and Image 800 (Beckman Coulter Inc.), fibrinogen levels using BCS XP coagulometer (Siemens Healthcare Diagnostics Inc.), hemoglobin A1c (HbA1c) using (ADAMS HA-8180V (Arkray Inc.) and complete blood counts using BC 6800 (Mindray Medical International Ltd.). Spot urinalysis and some definitions were used as albuminuria (albumin/creatinine: ≥30 mg/g) and proteinuria (protein/creatinine >0.2 g/g).

Low-density-lipoprotein cholesterol (LDL-C) levels were calculated using the Friedewald equation. In eGFR values were estimated by the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation by Levey et al. (8); as follows, eGFR_CKD-EPI=141 x minimum (Scr/k,1)^a x maximum (Scr/k, 1)^-1.209 x0.993Age x 1.018 [if female].

Indices of ADPN were estimated as ADPN/BMI, ADPN/WC, ADPN/fibrinogen, ADPN/CRP.

Statistical Analysis

We used MedCalc (MedCalc Software, Broekstraat, Mariakerke, Belgium). The Kolmogorov-Smirnov tests investigated Gaussian distribution. Non-gaussian variables were given as median (25^th percentile-75^th percentile), or else mean ± standard deviation. In comparison, One-Way ANOVA or Kruskal-Wallis H test was used in multiple group comparisons. Tukey HSD and Tamhane’s T² test for One-Way ANOVA, or Mann-Whitney U test for Kruskal-Wallis H test were used in post hoc comparisons. Pearson’s chi-square test, Pearson’s correlation coefficient (r) or Spearman’s rank correlation coefficient (rs) was used. All statistical tests were two-sided, and p-values less than 0.05 were considered to indicate significance, except p values less than 0.008 when the Mann-Whitney U test was used for post-hoc test.

Results

No significant difference in the male-to-female ratio was found among all the groups (groups 1-4), as shown in Table 1. Also, no statistically significant difference was found among the age, body mass index (BMI), and WC among the diabetics (groups 2-4).

Each diabetic-subgroup presented a mean of HbA1c over 8.0% and dyslipidemia with high triglyceride levels, as shown in Table 2. Also, diabetics had significantly decreased iron, hemoglobin levels and increased white blood cell values. CRP was higher in group 2, 3 than the controls. Serum urea and creatinine were higher in group 4 compared to group 1. However, eGFR values and serum albumin levels decreased in group 4 compared with group 1.

In healthy subjects (group 1), we had higher ADPN/CRP median values compared to group 2 and group 3 (Table 3). However, ADPN, ADPN/WC, ADPN/BMI and ADPN/fibrinogen were higher in group 4, compared with group 2, except ADPN/CRP.

ADPN/CRP was correlated with high-density cholesterol (HDL-C) in group 1, 2, 4 (Table 4). There was a negative correlation between ADPN/CRP and triglycerides in group 3, 4. In group 4, ADPN/CRP was also correlated with hypertension.

Discussion

The results demonstrated in this work provide a new perspective on understanding ADPN pathophysiology in different grades of DR. ADPN/CRP was significantly decreased in group 2, 3 (in diabetics), according to the healthy subjects. Theoretically, the terminal stage of various chronic diseases can occur in different ways in humans and experimental models; the ADPN paradox is alive among them. One possibility is that the persistence of the terminal stage of chronic diseases for which medical care is sought in humans contributes to the loss of ADPN function and may be related to metabolic syndrome (9). Although a significant negative correlation was shown between serum ADPN levels and smoking in women by Persson et al. (10), we had no difference in smoking percentage among all the groups in our study.

ADPN and indices of ADPN/BMI, ADPN/WC, ADPN/fibrinogen values significantly increased in group 4, in which microvascular complications increased mostly (both DR ad DN), compared with group 2. We also showed that UACR and eGFR values had a significant effect on serum ADPN levels. ADPN levels increasing with DR degree may be associated with its anti-inflammatory protective effect. ADPN/CRP indices did not differ significantly, although there was no any increase in CRP levels decreasing ADPN/CRP ratio in group 4, compared with group 2. There was a negative correlation between ADPN/CRP and triglycerides in patients with DR, group 3, 4. Also, ADPN/CRP was correlated with hypertension as a macrovascular complication in group 4. The prominence of the ADPN/CRP index highlights the importance of both inflammation and neovascularization. ADPN influences endothelial adhesion and transmigration of leukocytes and macrophages (11).

Both positive and inverse associations between ADPN and DR progression have been reported in meta-analysis studies that combined various ethnic groups (1). ADPN plays a critical role in retinal oedema and neovascularization, and it’s a potential therapeutic target for treating diabetic macular oedema, proliferative DR, and retinal vein occlusion (12) or DN (13); in an observational study, Kuo et al. (14) showed that ADPN levels increased with DR, and ADPN was seen positively correlated with DR progression.

In diabetic animal models, ADPN was upregulated in damaged muscle tissue (15), and it was shown that after myocardial injury ADPN levels increase to be a part of the revascularization process (16). Therefore, it is possible in the DR neovascularization process to have increased ADPN levels.

In a cross-sectional study, there was an association between plasma ADNP level and the variance of ADPN related genes on the DR status, individually and in combination (17). Moreover, genes associated with CVD are the ADPN gene and apolipoprotein E polymorphism gene with the x2 allele (18).

In another study, serum ADPN levels decreased in DN, but higher levels were found in DR or diabetic neuropathy (19), these findings support our results. In a meta-analysis, ADPN levels were higher in T2DM patients with microvascular complications (20). Plasma ADPN levels increased significantly with the severity of DN; they were associated with eGFR and UACR. The relative risk of impaired renal function requiring dialysis was found to be independent of ADPN levels (21). Additionally, a cohort study showed that ADPN levels were significantly increased in patients with chronic kidney disease (22), however when we consider that clearance of ADPN is mainly processed in the liver, renal function loss itself doesn’t contribute to ADPN elevation (23).

According to some studies, no correlation was found between ADPN and serum lipids in the form of triglycerides, LDL-C or total cholesterol (24,25). In another study, ADPN correlated positively with total cholesterol and HDL-C (21). In our study, ADPN/CRP was negatively correlated with triglycerides in group 3 and group 4. Additionally, there were varying degrees of positive correlations between ADPN/CRP and HDL-C levels in group 1, group 2, and group 4. Still a stronger correlation was found in non-diabetics, group 1. Patients with DR were included in groups 3 and 4; also, there was DR progression, especially in the proliferative phase in group 4. In group 4, a weak negative correlation was found between ADPN/CRP and hypertension. The fact that the parameters of eGFR or albumin in group 4 did not differ from the diabetic control group (group 2) strengthened our study to see the pure effect of the severity of DR progression, especially in the proliferative phase.

Blood levels of irisin, another adipokine, decreased with increasing stage of chronic kidney disease (UACR ≥300 mg/g, eGFR <60 mL/min 1.73 m²) and insulin resistance. It was also significantly associated with sarcopenia and carotid atherosclerosis in patients receiving peritoneal dialysis (26).

Recently, increased HMW level or HMW/total ratio has been associated with visceral fat type obesity, diabetes, glucose tolerance and insulin resistance, CVD, metabolic syndrome. Urinary ADPN was shown to be useful as a surrogate marker for DN risk performed with ultra-sensitivity by employing a two-site immune complex transfer enzyme immunoassay fluorescently after gel filtration of immunoreactivity; urinary concentrations of HMW- ADPN (size, >250 kD) increased as the disorder progresses in the glomerular molecular barrier (27). Urinary ADPN levels were much better than that of UACR, as a reliable indicator of proteinuria (28). Also, it was suggested that the increase in urinary ADPN was associated with the decreased renal function (UACR ≥30 mg/g or eGFR <60 mL/min/1.73 m²) (29). Total and HMW-ADPN levels correlated moderate to highly with UACR and eGFR within a fully automated immunoassay system (30).

Study Limitations

The most important limitation was the number of patients and having no sub-groups at variable stages of diabetes. Moreover, it should be better to include diabetic neuropathy. For technical reasons, we could not reach the patient group with isolated or accompanying diabetic neuropathy, another microvascular complication. The second limitation was that the control group was younger. And data including diabetic age was confidential.

Conclusion

We had increased serum ADPN and indices of ADPN/BMI, ADPN/WC, ADPN/fibrinogen values in the DR neovascularization process among diabetics, so clinicians can be encouraged to benefit from this immunoturbidimetric assay for diabetics via personalized medicine approach. But, further loss of kidney function itself prevented the increase in serum ADPN/CRP levels. To estimate progression in the advanced stages of DR, serum ADPN/CRP was a valuable marker, if there was no urinary loss of ADPN.

Meanwhile, more expanded studies should be performed where ADPN isoforms (molecular weight and immunoreactivity) or indices evaluated together with hepatic steatosis determined sonographically, and endothelial dysfunction or heart status determined radiologically for monitoring other vascular complications of diabetes. Besides biomarkers and imaging findings, life-style conditions, including exercise and dietary options/habits, should be recorded.

Ethics Committee Approval: An approval of the research protocol by the University of Health Sciences Turkey, İstanbul Training and Research Hospital Ethics Committee (approval number: 117, date: 08.04.2022) in accordance with international agreements (World Medical Association Declaration of Helsinki) was received.

Informed Consent: It wasn’t obtained.

Peer-review: Externally peer-reviewed.

Authorship Contributions: Surgical and Medical Practices - Ö.A.O., H.G., E.A., Ş.K.; Concept - M.G.M., H.A., S.K.; Design - M.G.M., H.A., M.U., E.S., S.K.; Data Collection or Processing - M.G.M., M.U., E.S.; Analysis or Interpretation - M.G.M., H.A., Ö.A.O., H.G., E.A., M.U., Ş.K., E.S., S.K.; Literature Search - M.G.M., H.A., S.K.; Writing - M.G.M., H.A., M.U., S.K.

Conflict of Interest: No conflict of interest was declared by the authors.

Financial Disclosure: The authors declared that this study received no financial support.