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-Nonalcoholic fatty liver disease (NAFLD) and albuminuria are associated with the aggravation of coexisting metabolic diseases.
-Of total 100 patients with both type 2 diabetes (T2DM) and NAFLD, but without chronic kidney disease were recruited.
-T2DM patients with significant hepatic steatosis had a higher prevalence of albuminuria.
-Albuminuria showed a correlation with hepatic fatty burden, and was independently associated with significant hepatic steatosis.
INTRODUCTION
Due to the increasing prevalence of obesity and type 2 diabetes mellitus (T2DM), nonalcoholic fatty liver disease (NAFLD) has emerged as one of the most common chronic liver diseases, with an estimated worldwide prevalence of 25.2% [1]. The prevalence of NAFLD in patients with T2DM is much greater than in the general population and is reported to be 55.5% [2]. As NAFLD shows a 3% rise in the annual incidence rate, the prevalence of NAFLD is expected to rise as individuals with obesity and T2DM age [3]. NAFLD is defined by the presence of fat accumulation in hepatocytes, after the exclusion of excessive alcohol use, hepatitis virus infection, iron overloading, autoimmune disease, or medications, which can cause chronic liver disease. NAFLD can advance to non-alcoholic steatohepatitis (NASH) that can progress to cirrhosis, end-stage liver disease, and finally, hepatocellular carcinoma (HCC) [4]. Moreover, NAFLD, per se, accounts for the most predominant cause of HCC, even without cirrhotic progression [5]. The risk of HCC rises at least two-fold in populations with T2DM and obesity [6].
Albuminuria is an independent risk factor for cardiovascular and renal diseases [7,8]. Additionally, it is a marker of insulin resistance and systemic inflammation. Consequently, an association between albuminuria and other metabolic diseases has been reported [9,10]. Although NAFLD and albuminuria are independently associated with the aggravation of coexisting metabolic diseases, the interactive influence of NALFD and albuminuria is not fully determined, especially in subjects with normal kidney function. Therefore, we aimed to investigate the association between the degree of steatosis and albuminuria, independent of other metabolic factors. Furthermore, we investigated whether albuminuria is associated with the hepatic burden in NAFLD.
METHODS
Study population
In this cross-sectional study, a total of 100 Korean subjects, aged 18 years or above, with T2DM were recruited at Keimyung University Dongsan Hospital, a tertiary university hospital in Korea. T2DM was defined according to the American Diabetes Association guidelines [11]. This study complied with the Declaration of Helsinki and Good Clinical Practice guidelines. The study protocol received ethical approval from the Institutional Review Board at the Keimyung University School of Medicine (2017-09-045). Informed consent was obtained from the patients prior to each examination.
All patients had NAFLD, as diagnosed using abdominal ultrasound or computed tomography and confirmed by a radiologist. Patients were excluded if they fulfilled any one of the following criteria: (1) diagnosis of type 1 diabetes mellitus, gestational diabetes mellitus, or any other form of diabetes other than T2DM; (2) history of addiction to alcohol, heavy alcohol consumption (≥210 g/week for men or ≥140 g/week for women); (3) other causes of liver disease (e.g., active viral or autoimmune hepatitis), liver cirrhosis, or HCC; (4) estimated glomerular filtration rate (eGFR) <45 mL/min/1.73 m2; (5) medications associated with fatty liver disease (e.g., amiodarone, methotrexate, tamoxifen, or valproate); and (6) pregnant or nursing women.
Measurement of steatosis burden and albuminuria
All patients underwent physical examination and clinical laboratory tests after an overnight (over 8 hours) fasting period for baseline parameters. Liver fat content was assessed via the controlled attenuation parameter (CAP), and liver fibrotic burden was assessed via the liver stiffness measurement (LSM). Both CAP and LSM measure ultrasonic attenuations at 3.5 MHz, using signals acquired by transient liver elastography (FibroScan; Echosens, Paris, France) with XL probe. To achieve an accurate CAP value, we calculated only ultrasonic attenuation when the matched LSM was valid while attempting to collect at least 10 valid LSMs. A success rate of ≥60% and a ratio for the interquartile range (IQR) to the median value of LSM of ≤30% were considered reliable and used for the final analysis [12]. The final CAP value was the median of each individual CAP value using the same valid measurements [13,14]. The steatosis grade (S) was determined using the following CAP-cutoff values that have been reported earlier: 236 dB/m for S ≥1, 270 dB/m for S ≥2, and 302 dB/m for S ≥3. We characterized significant steatosis when CAP was over 302 dB/m [15-17].
Urinary albumin and creatinine levels were measured in a fasting, morning spot urine sample obtained from each participant. Urinary albumin levels were expressed as the urinary albumin-to-creatinine ratio (ACR) to minimize the influence of variations in kidney function. The urine albumin level was measured by an immunoturbidimetric method using an AU680 automated chemistry analyzer (Beckman Coulter Inc., Brea, CA, USA). The urine creatinine level was also measured using the AU680 analyzer (Beckman Coulter Inc.) by the kinetic Jaffe method. Albuminuria was defined as urinary ACR ≥30 mg/g.
Clinical parameters and biochemical analysis
Body weight, waist circumference, blood pressure, glycemic parameters (i.e., fasting plasma glucose and glycosylated hemoglobin [HbA1c]), lipids (i.e., total cholesterol, high density lipoprotein cholesterol [HDL-C], low density lipoprotein cholesterol [LDL-C], and triglycerides), and liver enzymes (i.e., aspartate aminotransferase [AST], alanine transaminase [ALT], and gamma-glutamyl transferase [γ-GT]) were measured. An eGFR was calculated using the Modification of Diet in Renal Disease equation [18]. The homeostasis model assessment of insulin resistance (HOMA-IR) was calculated as follows: fasting glucose (mg/dL)×fasting insulin (μIU/L)/405. Subjects were considered as obese when their body mass index (BMI) was ≥25 kg/m2. Central obesity was defined on the basis of the waist circumference criteria from the Korean Society for the Study of Obesity (≥90 cm for men and ≥85 cm for women) [19]. Regular exercise was defined as more than 20 minutes per session and at least three times per week. Hypertension was defined as systolic pressure over 140 mm Hg and diastolic pressure over 90 mm Hg, or if the subject was currently prescribed antihypertensive medications. Cardiovascular disease was defined as any prior history of acute coronary syndromes, cerebrovascular accidents, or peripheral arterial diseases. The presence of diabetic neuropathy and diabetic retinopathy was defined by the patient’s medical record diagnosed by a neurologist/ophthalmologist, or if the patient was currently prescribed medication for neuropathy/retinopathy.
Statistical analysis
Data are presented as mean±standard deviation for parametric variables and as median (IQR) for non-parametric variables or percent for categorical variables. We analyzed differences in the patient characteristics between the two groups using paired t-tests for parametric continuous variables and chi-square tests for categorical variables. The urinary ACR, total cholesterol, triglyceride, HDL-C, LDL-C, AST, ALT, γ-GT, insulin, and HOMA-IR values were not normally distributed; analyses, therefore, were performed using the Mann-Whitney U test. The association between NAFLD and albuminuria was evaluated using the chi-square test after transforming the variables into tertiles. Correlations between urinary ACR, CAP, and other parameters were analyzed using Spearman’s correlation coefficients. Backward multiple linear regression analysis was performed on CAP to analyze the relationship between CAP and demographic and laboratory parameters. The relative risk factors for severe NAFLD were obtained using multiple logistic regression, and the risk was reported as odds ratios (ORs) with 95% confidence intervals (CIs). All statistical analyses were performed using SPSS version 25.0 (IBM Corp., Armonk, NY, USA). For all tests, P<0.05 was considered as statistically significant.
RESULTS
Baseline characteristics of the study population
Baseline characteristics according to the status of NAFLD are shown in Table 1. The prevalence of significant steatosis and albuminuria was 56.0% and 21.0%, respectively. Subjects with significant steatosis were significantly younger and had a significantly shorter duration of T2DM. They had greater waist circumferences, higher BMI, total cholesterol, triglycerides, and LDL-C, than subjects without severe NAFLD (all P<0.05). Furthermore, the proportion of central obesity and obesity was significantly higher in subjects with significant steatosis, whereas the proportion of hypertension and cardiovascular diseases was similar between the two groups. Liver function tests, including ALT, AST, γ-GT levels, were mostly within the normal range for both groups, with no significant difference between the values. The prevalence of albuminuria was higher in patients with significant steatosis than in subjects without significant steatosis (32.1% vs. 6.8%, P=0.002). Ninety-seven patients were prescribed metformin, and 35 patients were using angiotensin-converting enzyme (ACE) inhibitors or angiotensin II receptor blockers (ARB), and the proportion of anti-diabetic medications and ACE inhibitors/ARB was similar between the severe and non-severe NAFLD groups. In addition, the fibrotic burden, LSM was comparable between the two groups.
Correlation between CAP and urinary ACR
After stratifying into CAP and urinary ACR tertiles (<2.39, 2.39 to 11.41, >11.41 mg/g), individuals with a higher CAP had a higher urinary ACR than those with a lower CAP (Fig. 1A). When stratified by severity of NAFLD (S0, S1, S2, and S3 stages assigned to 12, 14, 18, and 56 subjects, respectively), the proportion of the highest urinary ACR tertile and significant albuminuria gradually increased and was the highest in subjects with S3 stage NAFLD (Fig. 1B and C). To assess the association between CAP and other metabolic components, we performed a linear correlation analysis. As shown in Table 2, BMI, waist circumference, ALT, γ-GT, and urinary ACR positively correlated with CAP, displaying statistical significance, whereas age and duration of T2DM showed a negative correlation with CAP. Furthermore, to determine which factors were significantly associated with CAP, we performed multiple linear analyses (Table 3). After adjusting for age and sex in Model 1, BMI, waist circumference, and urinary ACR were independently associated with CAP. In Model 2, adjusting further for the geodemographic parameters, including duration of T2DM, diabetic retinopathy, and ACE inhibitors/ARB use, led to a significant association of triglyceride level, BMI, waist circumference, and urinary ACR with CAP. On further adjusting Model 2 for laboratory findings (HbA1c, ALT, γ-GT, and triglyceride)(Model 3), BMI, waist circumference, and urinary ACR remained independently associated factors. Finally, Model 3 adjusted for BMI and waist circumference (Model 4) showed a significant association between urinary ACR and CAP.
Association between significant steatosis and urinary ACR
We demonstrated a relationship between CAP and urinary ACR; likewise, we analyzed the clinical and demographical factors related to significant steatosis. Table 4 shows that age, BMI, waist circumference, duration of T2DM, triglycerides, LDL-C, and urinary ACR demonstrated a significant association with significant steatosis. Similar to the linear association, younger age and shorter duration of T2DM was associated with a higher risk of significant steatosis ([OR, 0.97; 95% CI, 0.94 to 0.99; P=0.030 for age], [OR, 0.94; 95% CI, 0.89 to 0.99; P=0.034 for the duration of T2DM]), along with higher BMI, waist circumference, triglycerides, LDL-C, and urinary ACR (OR ranges 1.02 to 3.79; all P<0.05). To determine the independence of these risk factors, we performed multivariable logistic regression analysis. After adjusting covariate factors including age, duration of T2DM, BMI, use of ACE inhibitors or ARB, LDL-C, and BMI, LDL-C, urinary ACR still remained statistically significant ([OR, 1.34; 95% CI, 1.15 to 1.57; P< 0.001 for BMI], [OR, 4.33; 95% CI, 1.20 to 15.63; P=0.025 for LDL-C], [OR, 1.88; 95% CI, 1.31 to 2.71; P=0.001 for urinary ACR]).
DISCUSSION
This prospective and cross-sectional study demonstrates a significant association between CAP and urinary ACR, and an increased risk of significant steatosis in subjects with albuminuria and T2DM. Urinary ACR showed a significant positive correlation with CAP and independently predicted fatty burden of NAFLD after adjustment for clinical variables, including age, sex, waist circumference, BMI, duration of T2DM, presence of diabetic retinopathy and the use of ACE inhibitor/ARB, along with laboratory test results including HbA1c, ALT, γ-GT, triglycerides, and LDL-C. After further adjustments, we observed an approximately 2-fold rise in the adjusted risk of significant steatosis in patients with albuminuria and T2DM (OR, 1.88; 95% CI, 1.31 to 2.71; P=0.001).
There have been several attempts made to elucidate the association between NAFLD and albuminuria. Although previous studies consistently indicated a strong association between NAFLD and chronic kidney disease (CKD) [20,21], the results from investigations regarding NAFLD and albuminuria are conflicting; especially in populations with T2DM. A meta-analysis study showed a 2.25-fold elevated risk of albuminuria among patients with NAFLD without diabetes; however, they could not find any significant association between albuminuria and NAFLD among subjects with diabetes [22]. A Chinese cross-sectional study of 1,763 patients with T2DM and NAFLD (ascertained using transient elastography), showed only a significant association between fibrotic burden and albuminuria, but not between steatosis burden and albuminuria, in subjects with T2DM [23]. Compared to the Chinese study, our study population was relatively younger (mean age 53.0±15.5 years vs. 60.7±11.5 years in the Chinese study), and had a shorter duration of T2DM (7.1±7.3 years vs. 10.8±8.5 years in the Chinese study). Moreover, we excluded patients with liver cirrhosis, splenomegaly, or chronic liver diseases other than NAFLD, based on imaging studies, along with subjects with CKD to clarify the association between the degree of steatosis and albuminuria. These factors could account for the discrepancies between the two studies. In concordance with our results, studies have confirmed the association between NAFLD and albuminuria in prediabetic patients and those diagnosed recently or with a shorter duration of T2DM [24,25]. As albuminuria is regarded as an early clinical manifestation of diabetic kidney disease (DKD), and fatty burden accumulation precedes fibrotic change, the association between the degree of steatosis and albuminuria might emerge in the early course of T2DM. This finding is clinically relevant to identify high-risk patients of NAFLD for further screening and evaluation.
With respect to risk factors for NAFLD in populations with T2DM, traditional metabolic risk factors such as BMI, waist circumference, and dyslipidemia were independent variables associated with CAP and the aggravation of NAFLD in our study. In addition to these traditional markers, albuminuria showed at least a comparable potential to predict NAFLD severity. In other words, patients with T2DM and additional metabolic diseases are at a higher risk of developing advanced NAFLD and may benefit from a liver assessment. Moreover, it is important to note that among patients with T2DM, urinary ACR test may be useful for screening NAFLD as well as DKD. Among current valid liver assessments, transient elastography has the advantage of providing quantitative measurements, with validations done in different populations, showing high reproducibility [17,26]. In contrast to the scoring system, confounding factors such as age and liver enzymes do not influence the results of transient elastography [27-29].
The possible mechanisms for the association between albuminuria and NAFLD might be explained by insulin resistance and systemic inflammation. Albuminuria reflects insulin resistance, even in a healthy population, and the reduced insulin sensitivity promotes hepatic steatosis [30,31]. An increase in systemic inflammatory markers (interleukin 6, tumor necrosis factor α, fibrinogen, monocyte chemoattractant protein-1) is observed in subjects with albuminuria, attributing to NAFLD, and its progression [32-34]. Recently, the concept of crosstalk between the liver and kidney has emerged, involving the renin-angiotensin system. Adipocytes (fat cells) express components of the renin-angiotensin system, impacting the ACE (angiotensin II) angiotensin II type 1 receptor axis, which is also expressed in the liver and kidney, leading to organ damage [35]. In addition, the altered activation of nutrient/energy sensors sirtuin-1 and adenosine monophosphate-activated kinase, and impaired antioxidant defense mediated by nuclear factor erythroid 2-related factor-2 (NRF2), support the crosstalk between liver and kidney [36]. Patatin-like phospholipase domain-containing protein 3 (PNPLA3) gene, which is expressed in the liver, adipose tissue, and kidney, is also linked with the crosstalk. PNPLA3 is a lipid droplet-associated protein that has hydrolase activity toward triglycerides and retinyl esters, leading to hepatic steatosis. Recent studies have also reported the association of PNPLA3 polymorphism with albuminuria [37,38].
Previous studies reported the increased prevalence of NAFLD with age [39,40]. However, recent studies have also demonstrated an increased presence of NAFLD in young adults with T2DM [41,42]. A Danish cross-sectional study showed that the risk of NAFLD nearly doubled in patients diagnosed with T2DM before the age of 40 [41]. In addition, NAFLD and NASH have shown an increased prevalence in young adults, and, if unchecked, could lead to liver transplantation [43]. In agreement with recent studies, we observed that patients with significant steatosis were younger and more obese than those without significant steatosis. However, after adjusting for age and BMI, this association was dismissed. A selection bias might have affected the results achieved in our study since we actively performed imaging studies in young and recently diagnosed T2DM patients. However, such patients usually do not show any symptoms of NAFLD, and are, therefore, left treated. Screening and early identification of NAFLD should, therefore, not be neglected in this population, and screening should be emphasized as for older patients with T2DM.
We acknowledge that our study has some limitations. Firstly, despite the acceptance of transient elastography for detecting NAFLD, this diagnostic tool does not replace liver biopsy, which is the gold standard for detecting NAFLD. Liver biopsy is regarded as a precise diagnostic tool for evaluating NAFLD and NASH. However, owing to its complications, non-invasive diagnostic devices, including transient elastography, have been widely utilized. Additionally, although we applied XL probe in the current study to enhance its accuracy, CAP could be overestimated in patients with morbid obesity. Secondly, since this was a cross-sectional study, we could not elucidate a causal relationship between the observed findings. Thirdly, since this study was based on a population visiting a single tertiary university hospital, our results may not be reflective of the entire Korean population. In addition, selection bias could be present and might have affected the results. Further multi-center, large-scale, longitudinal studies are required to show a clear association between albuminuria and hepatic burden in T2DM.
In conclusion, our results show that patients with T2DM and NAFLD frequently have albuminuria, which is associated with a higher risk of significant steatosis. Additionally, we showed that the level of urinary ACR was associated with an increased hepatic steatosis burden in NAFLD patients. Hepatic complication should be kept in mind in T2DM patients with albuminuria. Further studies with a larger number of patients and longer periods of observation, along with liver biopsy, are needed to elucidate the association more clearly between albuminuria occurrence and NAFLD progression.
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