Perspective Chapter: The Level of Lipocalin-2 in Patients with Early Stages of Chronic Kidney Disease against the Background of Obesity

Abstract

Chronic kidney disease (CKD) is an important contributor to morbidity and mortality from noncommunicable diseases. To date, other markers of early diagnosis of kidney disease have been used, in particular those that do not depend on their filtration function and indicate tubulointerstitial damage. Lipocalin-2, as a new marker of immunity and acute phase response to infectious lesions, is actively involved in fatty acid transport, inflammation modeling, and metabolic homeostasis. Neutrophil-gelatinase-associated lipocalin (NGAL) is also considered an early marker of tubulointestinal damage. Studies have shown an increase in the secretion of lipocalin-2 in the urine, especially with a higher body mass index of patients with CKD. NGAL is positively correlated with cystatin C levels and may be a significant predictor of albuminuria. A close correlation between NGAL levels and markers of systemic inflammation, such as C-reactive protein and tumor necrosis factor-alpha (TNF-α), and an increase in microalbuminuria has been shown to indicate a progressive decline in renal function.

1. Introduction

Chronic kidney disease (CKD) is an important factor, contributing to morbidity and mortality from noncommunicable diseases. Large-scale screening research in different countries around the world has shown that the common causes of CKD are hypertension, diabetes, obesity, gout, and, more rarely, exposure to toxins, heavy metals, HIV, etc. [1]. To date, a lot of epidemiological research studies have shown that indicators such as glomerular filtration rate (GFR) and the ratio of albuminuria/creatinine, which are the markers of decreased renal function progression [2], are used for the diagnosis of CKD. However, a proper renal function test, especially in the presence of risk factors and comorbidities, has primary importance for recognizing the initial signs of kidney damage, therapy supervision, and deciding on prevention measures in patients with CKD [3]. Therefore, other markers of the early diagnosis of kidney damage are currently used, in particular those that do not depend on their filtration function and indicate tubulointerstitial damage [4]. One such marker is lipocalin-2 or neutrophil-gelatinase-associated lipocalin (NGAL).

Lipocalin-2 is produced in many tissues, such as kidneys, liver, immune cells, uterus, and chondrocytes [5], and according to other research studies, its main source is white adipose tissue [6]. Currently, NGAL in the blood is described as a new component of immunity and acute phase response to infectious lesions [7]. Lipocalin-2 is also involved in fatty acid transport, hematopoietic cell apoptosis, inflammation modeling, and metabolic homeostasis [8]. In recent years, it has been found that serum lipocalin and urinary lipocalin are the early markers of acute renal damage [9]. However, recent publications and research show that NGAL levels increase against the background of inflammation and increased immune activity, which are the main pathogenetic factors in the pathogenesis of CKD. The change of this marker indicates that acute kidney damage is a prelude to its chronic disease, and lipocalin-2 is a mediator of CKD progression [10]. Research comparing serum lipocalin (sNGAL) and urinary lipocalin (uNGAL) has shown that urinary lipocalin has a negative correlation with GFR at all stages of CKD in patients with GFR < 90 mL/min with different etiologies of CKD, and in patients with proteinuria, it correlated more with its severity compared with serum lipocalin [11]. Based on this research, it was concluded that uNGAL level is a better biomarker of CKD than serum lipocalin.

Endothelial dysfunction also plays an important role in the development and progression of CKD [12]. The impairment of endothelium vasomotor function and a disequilibrium toward vasoconstriction leads to the formation of vasospasm, impairment of microcirculation, and local renal ischemia. According to current data, reduction in the formation of nitric oxide (NO), increase in the tubular reabsorption of water, and increase in the concentration of sodium ions help reduce the glomerular filtration rate (GFR), which ultimately leads to glomerular sclerosis [13]. It is also known that the decrease in NO synthesis due to chronic decreased blood flow in the kidneys increases the level of proinflammatory cytokines, endothelin-1 (ET-1), and other vasoconstrictors [14]. According to Khan and Pandey, chronic hypoxia of renal tissue is the main cause of the tubulointestinal system damage and leads to the progression of CKD [15].

The high prevalence of obesity today is a serious medical and social problem. Research in recent years has shown that the risk of chronic kidney disease, cardiovascular disease (CVD), and diabetes depends not only on the volume of adipose tissue, but also on its hormonal and metabolic activity [16]. It is proved that an increase in body mass index (BMI) by 10% enhances the probability of a steady decrease in GFR by 1.27 times [17]. The appearance of microalbuminuria (MAU) is an early marker of glomerular disorders and an indicator of endothelial dysfunction [18]. Thus, the diagnosis of glomerular and tubular dysfunction is insufficiently studied in patients with the early stages of CKD against the background of obesity and will assess the kidney damage in the early stages.

The purpose of the research is to evaluate the level of lipocalin-2 as a marker of the early diagnosis of tubular lesions and its relationship with the endothelial dysfunction marker, endothelin-1, and indicators of low-intensity inflammation markers in patients with the early stages of CKD against the background of obesity.

2. Materials and methods

A total of 158 patients (67 women and 91 men) with chronic kidney disease, who were hospitalized in the Department of Arterial Hypertension of Communal noncommercial enterprise “Ivano-Frankivsk Regional Clinical Cardiological Centre of Ivano-Frankivsk Regional Council” and Urological and Cardiological Departments of Communal noncommercial enterprise “Central City Clinical Hospital of Ivano-Frankivsk City Council” in Ivano-Frankivsk City, Ukraine, were examined. The average age of the examined was 55.36 ± 2.02 years in women and 47.45 ± 2.66 years in men.

Body mass index was calculated by the Kettle formula (kg/m²): BMI = body weight (kg)/height (m²). Stage 1 of CKD was diagnosed in 70 patients (31 women and 39 men) (the average age was 46.43 ± 3.77 years), and stage 2 was diagnosed in 88 patients (36 women and 52 men) (the average age was 53.07 ± 2.61 years). Patients were divided into two groups: 1-a (70)—patients with stage 1 of CKD and obesity, and 2-a (88)—patients with stage 2 CKD and obesity. The control group consisted of 10 healthy individuals (three women and seven men), whose average age was 36.7 ± 8.6 years.

The reasons for the development of CKD are as follows: infections of the upper urinary system were in 11.8%, urolithiasis was in 19.89%, glomerulonephritis with symptomatic renoparenchymal hypertension was in 15.76%, abnormalities in the development of urogenital system were in 6.9%, essential arterial hypertension (AH) was in 29.1%, and coronary heart disease with heart failure was in 18.89%. The duration of CKD was on average 7.1 years. Exclusionary criteria were diabetes, hypothalamic and endocrine obesity, acute myocardial infarction, stages 3 and 4 of New York Heart Association congestive heart failure, hepatic failure, and stages 3–5 of CKD.

General clinical examinations were conducted for all patients. Albumin in daily urine was examined by the turbometric method, using the diagnostic kit “Microalbumin” (Germany) and evaluated in mg/day. The glomerular filtration rate was calculated according to the chronic kidney disease epidemiology collaboration (CKD-EPI) formula based on creatinine and cystatin C levels and their combination (CKD-EPIcysC/cr) (mL/min/1.73 m²) using a calculator of the U.S. National Renal Fund (http://www.kidney.org/professionals/kdoqi/gfr_calculator.cfm). Serum cystatin C levels (0.79–2.15 mg/L in healthy individuals) were examined by enzyme-linked immunosorbent assay using Human Cystatin C ELISA kit (Czech Republic) on the STAT FAX analyzer (No. 7898). The level of neutrophil-gelatinase-associated lipocalin (NGAL) (ng/mL) was examined by sandwich enzyme-linked immunosorbent assay in urine (0.16–10 ng/mL in healthy individuals) using the HUMAN NGAL ELISA Kit (USA). The concentration of tumor necrosis factor-alpha (TNF-α) in serum (0.02–2.3 pg./mL) was determined by solid-phase enzyme-linked immunosorbent assay using Vector BEST kit (Belarus). The quantitative determination of C-reactive protein (CRP) (0.07–2.5 μg/mL in healthy individuals) in serum was determined by enzyme-linked immunosorbent assay using the Accu-Bind ELISA Microwells kit (USA). The level of endothelin-1 (reference values within 2.5–7.8 pg./mL) in serum was examined by enzyme-linked immunosorbent assay using the Human ET-1 (Endothelin-1) ELISA Kit (USA). All enzyme-linked immunosorbent assays were performed on STAT FAX analyzers (No. 7898).

The research protocol was approved by the Ethics Commission of Ivano-Frankivsk National Medical University, protocol No. 97/17 of October 19, 2017. All patients gave informed consent to participate in it. The research was conducted in accordance with the principles of Helsinki Declaration of the World Medical Association “Ethical principles of medical research with human participation as an object of study” No. 900_005 of October 1, 2008.

The statistical analysis of the results was performed using the statistical software package Statistica 6.0 using Student’s t-test. Correlation was assessed by Spearman’s rank correlation coefficient. The discrepancy of the results at p < 0.05 was considered statistically significant.

3. Results

Currently, the clinic is considering new biomarkers for the early diagnosis of CKD of various etiologies. According to Melnikov et al., NGAL is expressed by tubular epithelial cells in response to inflammation, trauma, and tubulointerstitial damage [19]. According to the results of our research, the excretion of lipocalin-2 in the urine in the patients of groups 1 and 2 exceeded that in healthy individuals by 3.6 and 6.3 times, respectively (p_1,2 < 0.001). The increase of the NGAL excretion in urine was greater in the patients of group 2, who had a higher body mass index. Thus, comparing u-NGAL in the patients of both the groups, it was found that in patients with stage 2 CKD against the background of obesity, it was 1.8 times higher (p < 0.05) than in patients with stage 1 of CKD against the background of obesity (Table 1). The level of daily microalbuminuria in the patients of groups 1 and 2 also exceeded that in healthy individuals by 7.4 and 8.9 times, respectively (p_1,2 < 0.001). Comparing the indexes of daily microalbuminuria in the patients of both the groups, it was found that MAU in patients with stage 2 CKD against the background of obesity was 1.2 times higher (p > 0.05) than in patients with stage 1 of CKD against the background of obesity. Increasing NGAL level in urine has been shown to be a significant predictor of albuminuria, especially in the patients of group 2. The level of cystatin C also exceeded the indexes of healthy individuals in both the groups by 1.8 and 2.29 times, respectively (p_1,2 < 0.001). The level of cystatin C in the patients of group 2 was higher by 1.25 times than in the patients of group 1 (p > 0.05). The increase of cystatin C in serum reflects the state of glomerular filtration and the degree of renal function decrease [20]. GFR calculated according to the formula CKD-EPIcysC/cr indicates the decrease of this index by 1.5 times (p < 0.001) in the patients of group 2 compared with group 1, which confirms decreased kidney function, despite normal creatinine values.

Indices	Health, n = 10	Group 1, n = 70	Group 2, n = 88
Age, years	36.7 ± 8.6	46.43 ± 3.77	53.07 ± 2.61
BMI, kg/m^2	21.44 ± 0.39	34.15 ± 1.1	37.59 ± 0.82
Albuminuria, mg/day	19.0 ± 0.96	141.1 ± 12.66*	170.05 ± 17.6*
Cystatin C, mg/L	0.78 ± 0.02	1.43 ± 0.13*	1.79 ± 0.13*
Creatinine, μmol/L	77.71 ± 1.48	94.26 ± 2.32*	105.03 ± 1.56*°
GFR, mL/min/1.73 m^2; CRD-EPIcysC/cr	103.3 ± 1.27	95.09 ± 2.76*	64.5 ± 2.07*
CRP, μg/mL	1.31 ± 0.42	7.73 ± 2.19*	12.85 ± 4.14*
TNF-α, pg/mL	0.73 ± 0.15	2.66 ± 0.29*	4.27 ± 0.57*°
Endothelin, pg/mL	3.4 ± 0.29	5.87 ± 0.48*	9.04 ± 0.38*°
NGAL, ng/mL	3.83 ± 0.56	13.6 ± 0.62*	24.13 ± 0.91*°

Table 1.

Clinical and laboratory characteristics of the examined patients.

*

Reliable when compared to the healthy.

°

Reliable when comparing groups 1 and 2.

A positive average correlation was found between NGAL and BMI in both the groups (r₁ = 0.45 and r₂ = 0.58; p_1,2 < 0.05), between NGAL and microalbuminuria (r₁ = 0.45 and r₂ = 0.48; p_1,2 < 0.05), and between NGAL and cystatin C (r₁ = 0.46 and r₂ = 0.68; p_1,2 < 0.05). In the patients of group 2, a direct average correlation was found between daily albuminuria and BMI (r = 0.56; p < 0.05), which indicates a deterioration in the functional state of the kidneys against the background of obesity. In both the groups, there was a negative mean correlation between NGAL and GFR (CKD-EPIcysC/cr) (r₁ = −0.56 and r₂ = −0.58; p_1,2 < 0.05), indicating a violation of tubular functions even in the early stages of CKD.

According to recent studies, endothelin-1 is considered a marker for the diagnosis of endothelial dysfunction in a variety of vascular pathology, including kidney disease [21]. In both the groups, we found an increase in endothelin-1 by 1.7 and 2.7 times, respectively (p_1,2 < 0.05) in contrast to the control group. As GFR decreased and body weight increased, there was a 1.6-fold increase in endothelin-1 levels in group 2 patients in contrast to group 1 patients (p < 0.05). In the patients of both the groups, there was a positive correlation between the level of lipocalin-2 and endothelin-1 (r₁ = 0.49 and r₂ = 0.44; p_1,2 < 0.05). In both the groups, there was also a negative average correlation between glomerular filtration rate and ET-1 (r₁ = −0.44 and r₂ = −0.63 (p_1,2 < 0.05) and a positive strong correlation between albuminuria and ET-1 in patients with stage 2 CKD against the background of obesity (r₂ = 0.74; p < 0.001). Our data are consistent with the studies of Davenport et al., who found that increased levels of ET-1 can damage renal podocytes, cause structural changes, and reduce their function, and an increase in NGAL indicates the early signs of tubulointerstitial sclerosis [14].

We found an increased CRP level in both the groups by 5.9 and 9.8 times, respectively, in contrast to the healthy (p_1,2 < 0.05). There was a weak reverse correlation between GFR and CRP levels in the patients of group 1 (r₁ = −0.34; p > 0.05) and an average reverse correlation between these indicators in the patients of group 2 (r₂ = −0.45; p < 0.05). A positive correlation was also found between the levels of CRP and BMI (r₁ = 0.66 and r₂ = 0.78; p_1,2 < 0.05) and between CRP and NGAL (r₁ = 0.46 and r₂ = 0.58; p_1,2 < 0.05). According to Rysz et al., an increase in the level of C-reactive protein by more than 3 mg/L contributes to a high risk of cardiovascular events, and when the GFR falls below 89 mL/min, the risk of cardiac events increases markedly [22]. According to Lumlertgul et al., uNGAL is produced in the kidney epithelium and leukocytes, and its level increases in response to prolonged tubular damage and systemic inflammation [23]. Tumor necrosis factor-α (TNF-α), a proinflammatory cytokine required for the regulation of inflammation, apoptosis, and renal oxidative stress, also increases in CKD and obesity [24]. We found an elevated level of TNF-α by 3.6 times in the patients of group 1 and by 5.8 times in the patients of group 2 in contrast to the healthy (p_1.2 < 0.001). In the patients of group 2, the level of TNF-α was 1.6 times higher than in the patients of group 1 (p₂ < 0.05). We also found the average positive correlation between TNF-α and BMI in the patients of group 1 (r₁ = 0.60; p₁ < 0.01) and group 2 (r₂ = 0.69; p₂ < 0.05) and the average reverse correlation between GFR and TNF-α levels in the patients of group 2 (r₂ = 0.67; p₂ < 0.05). At the same time, a strong correlation was found between the levels of NGAL and TNF-α in the patients of group 2 (r₂ = 0.72; p₂ < 0.05) and between microalbuminuria and TNF-α in the patients of groups 1 and 2 (r₁ = 0.46; p₁ < 0.05 and r₂ = 0.72; p₂ < 0.05). Our results showed that TNF-α and u-NGAL levels were elevated and independently correlated with albuminuria status, suggesting that they may be used as the markers of CKD progression against the background of obesity, which is consistent with the research of Darde et al. [25].

4. Discussion

The problem of obesity in recent years has attracted the attention of a wide range of professionals. The prevalence of this multifactorial disease, connected with the excessive accumulation of adipose tissue in the body, in all the age groups is steadily increasing, according to the National Health and Nutrition Examination Survey (NHANES) [26]. There is growing evidence that the kidneys, along with the cardiovascular system, are vulnerable target organs in obesity, and the irreversible process of changes in them occurs even in the absence of arterial hypertension (AH) and diabetes mellitus (DM) or in compensation for these conditions [27]. Thus, an increase in body mass index (BMI) by 10% increases the probability of a steady decrease in glomerular filtration rate (GFR) by 1.27 times [28]. In routine clinical practice, kidney damage is usually detected by changes in serum creatinine and creatinine-based GFR, as well as by protein excretion. However, recently, much attention has been paid to the study of the tubulointerstitial apparatus, which is involved in the pathological process of inflammatory or ischemic lesions before the glomerulus and is a marker of tubular kidney damage in preclinical stages [29]. In particular, NGAL is considered an early marker of kidney damage, the expression of which is enhanced in response to inflammation or damage to the endothelium in the renal tubules and leads to the proliferation of new epithelial cells [30]. According to Lim et al., NGAL is also a marker of disease severity and clinical outcomes in patients with CKD, in particular, in 45 patients with CKD stages 2–4, systemic NGAL concentrations were significantly related to GFR [31]. And according to research by Malyszko et al., in 92 patients without diabetes mellitus with CKD stages 2–4, both the systemic and urinary NGAL correlated with GFR and the systemic NGAL/NGAL ratio in urine showed the best correlation [32]. Current data also treat microalbuminuria as a marker of early renal dysfunction. We found that a decrease in GFR was accompanied by an increase in microalbuminuria, and an increasing level of NGAL in the urine was a significant predictor of albuminuria, especially in the patients of group 2. An increase in serum cystatin C levels is also associated with the initial manifestations of tubulointestinal lesions [33]. Cystatin C in the blood and NGAL in the urine may confirm chronic renal impairment earlier than serum creatinine monitoring. We found a positive average correlation between NGAL and cystatin C and between NGAL and BMI in the patients of both the groups and, at the same time, a negative average correlation between NGAL and GFR (CKD-EPIcysC/cr) in both the groups, indicating impaired tubular function even in the initial stages of CKD. According to Gharishvandi et al., serum and urine NGAL levels were significantly higher in obese people, and compared to creatinine and GFR, increased cystatin C and u-NGAL provides information on a worse prognosis in patients with renal disease and increasing body weight [34].

Endothelial dysfunction is considered the first stage in the development of systemic atherosclerosis, and endothelin-1 is a marker of its dysfunction in chronic kidney disease [35]. It is thought to be an early, noninvasive marker of kidney damage that can be detected before clear clinical manifestations. With the increasing concentration of ET-1, vasospasm is formed, which causes systemic changes in the microcirculatory tract, promotes ischemia, and increases the permeability of glomerular vessels, which subsequently causes proteinuria [36]. According to our data, the patients of both the groups had a positive correlation between the levels of lipocalin-2 and endothelin-1 (p_1,2 < 0.05), a negative average correlation between the glomerular filtration rate and ET-1 (p_1,2 < 0.05), and a positive strong correlation between the levels of albuminuria and ET-1 in patients with stage 2 CKD against the background of obesity. According to Karol et al., chronic renal tissue hypoxia and decreased GFR against the background of increasing body weight is one of the causes of damage to the tubulointerstitial apparatus, leading to the progression of CKD, and the main markers of this process are albuminuria, as well as levels of cystatin C and interleukin-18 (IL-18), including lipocalin-2 [37].

CRP is a recognized risk factor in CVD and mortality in the general population, as well as in patients with CKD. CRP plays a multifaceted role, as it is a highly sensitive marker of inflammation and tissue destruction, and is considered a “new” proatherogenic, proinflammatory adipokine, whose source is adipocytes, and its level in the blood may correlate with BMI and visceral adipose tissue [38]. According to Shankar et al., TNF-α and IL-6, more than CRP, are associated with a prevalence and severity of CKD, regardless of other identified risk factors, a history of CVD [39]. We found a strong positive correlation between the levels of NGAL and TNF-α in the patients of group 2 (p₂ < 0.05) and between microalbuminuria and TNF-α in the patients of both the groups (p₂ < 0.05), the average positive correlation between TNF-α and BMI in the patients of groups 1 and 2, and the average inverse correlation between GFR and TNF-α levels in the patients of group 2. According to Flannery et al. [40], TNF-α is a proinflammatory cytokine required for the regulation of inflammation, apoptosis, and oxidative stress in the kidneys, and the increase in serum and NGAL levels in urine correlates with kidney damage and glomerular permeability barrier in patients with chronic kidney disease, and the correlation between NGAL levels and albuminuria indicates that glomeruli and tubules may be affected at the same time.

Thus, lipocalin-2, as a new marker of immunity and acute phase response to infectious lesions, is actively involved in fatty acid transport, inflammation modeling, and metabolic homeostasis. NGAL is also considered an early marker of tubulointerstitial damage. Research has shown an increase in the secretion of lipocalin-2 in the urine, especially with a higher body mass index, in patients with CKD. It can be a significant predictor of albuminuria and positively correlates with cystatin C levels. There is a close correlation between NGAL levels and markers of systemic inflammation, including C-reactive protein and TNF-α, and an increase in microalbuminuria indicates a progressive decline in renal function.

Conflict of interest

The author declare that there is no conflict of interest.