Association Between Levels of Depression Symptoms and Moderately Increased Levels of the Inammation Marker Albuminuria Is Explained by Age and Comorbidity.

Background: The study aimed to examine whether there are associations between levels of depression symptoms and levels of the inammation marker albuminuria. Materials and methods: The 8303 participants in this cross-sectional study were subjects from the second survey of the Trøndelag Health Study (HUNT, Norway). Depression symptoms were assessed by the Hospital Anxiety and Depression Scale (HADS). Logistic regression analysis was performed to estimate the odds ratio (OR) for moderately increased albuminuria (ACR > 3.0 mg/mmol) according to different HADS subgroups and -scores. Results: Unadjusted ORs for moderately increased albuminuria were signicantly increased in those with HADS > 8 (OR 1.27, 95% CI 1.05-1.54, p=0.013) and HADS > 11 (OR 1.59, 95% CI 1.19-2.14, p=0.002). However, after adjusting for age and sex, only HADS > 11 was signicantly associated with ACR > 3.0 mg/mmol (OR 1.46, 95% CI 1.08-1.98, p=0.014), and after multivariable adjustments for cardiovascular risk factors and comorbidity, there were no signicant associations. Conclusion: The positive and signicant association between moderately increased albuminuria and symptoms of depression found in unadjusted analyses weakened and disappeared after adjustments. Although individuals with depressive symptoms had albuminuria more often than individuals without such symptoms, albuminuria may reect other comorbidity and inammation conditions than depression.


Introduction
The immune system may play a part in the pathophysiology of mood disorders 1,2 . Major depression has now for several years been a studied area regarding involvement of immune function and in ammation, like oxidative stress-mediated brain damage 3 . Studies have also shown that use of anti-in ammatory agents in combination with conventional therapy may improve outcomes in mood disorders 4,5 .
The excretion of moderately increased levels of albumin in the urine, former called microalbuminuria, has been well documented to predict cardiovascular (CV) morbidity and mortality in both diabetic and nondiabetic persons 6-8 . Severely increased albuminuria is probably a direct cause of in ammation in the kidney interstitium, leading to increased progression towards kidney failure, whereas moderately increased levels are more likely a biomarker of subclinical vascular pathology and atherosclerosis. The link between moderately elevated urine albumin excretion (UAE) and atherosclerosis seems to be in ammation with endothelial dysfunction, leading to increased permeability and leakage of albumin through the vessel wall 9,10 . Today, moderately increased albuminuria seems to be a crucial renal marker for a generalized damage and systemic in ammation in the vascular system. Studies support this, showing that in ammatory parameters are signi cantly and independently associated with UAE in prehypertensive, otherwise healthy individuals 11 .
There have not been many previous studies examining the relationship between depression and albuminuria [12][13][14] , and to our knowledge, there have not been carried out a study of this kind in a large population, aged 20-90 years, as this health survey. We hypothesised a positive association between moderately increased albuminuria, re ecting in ammation, and levels of the depression dimensions.

Study subjects
During 1995-1997, the second survey of the Trøndelag Health Study (HUNT, Norway), invited all residents >20 years (n=93 898), and a total of 70% participated. The survey comprised questionnaires, which included questions on CVD (angina pectoris, myocardial infarction and stroke), smoking habits, other lifestyle factors, and a clinical examination. Details of the HUNT study design and albuminuria screening have been published previously 32,43 . All those with self-reported diabetes mellitus and/or treated hypertension and a 5% randomly selected sample of the total population were included in the albuminuria screening study. A total of 9598 participants delivered three morning urine samples for albuminuria analysis (overall response rate 84%).Of these, 8801 had answered questions with depression and/or anxiety dimensions. Those who answered con rmatory to one of the questions about UTI in the previous week, persistent haematuria in the previous year, menstruation at time of urine collection or pregnancy were excluded from the analysis (n=338). So were also 160 individuals with severe albuminuria (ACR > 30 mg/mmol), leaving a total of 8303 subjects included in the main analysis.

Clinical examination
The clinical examination included standardized measurement of height, weight, blood pressure and pulse rate. Height was measured without shoes to the nearest centimetre, and weight was measured to the nearest half-kilogram while wearing light clothing without shoes. Three consecutive standardized blood pressure measurements were recorded with one-minute interval. After a minimum of two minutes rest, the measurements were performed in the sitting position, using an automatic oscillometric method (Dinamap 845XT; Criticon, Tampa, FL).

Measurement of signs and symptoms of clinical depression and anxiety
Symptoms of anxiety and depression were assessed by the HADS 15 . HADS consists of seven depression related items (HADS-D) and seven anxiety related items (HADS-A), each with a four-point ordinal scale to describe symptom severity from 0 to 3. Valid HADS response was de ned as completed answer on ve or more items on the subscale. Missing responses among those who lled in 5 or 6 items were replaced based on the sum of completed items multiplied by 7/5 or 6/5, respectively.

Urine-and blood sampling
The participants included in the albuminuria screening received a unit with three plastic receptacles for three rst morning urine samples and three transport tubes, and one envelope for return by mail back to the laboratory. Additionally, the participants received instructions for urine collection, information about the albuminuria-screening and a questionnaire about UTI, haematuria and menstruation.
Blood sampling was carried out whenever subjects attended (i.e. in non-fasting state). Fresh serum and urine samples were analyzed at the Central Laboratory at Levanger Hospital, on Hitachi 91 Autoanalyzer (Hitachi, Mito, Japan). Details of the laboratory methods have been published 44 . Urine albumin and creatinine were measured by using an immunoturbidimetric method (anti-human serum albumin, Dako Norway, Oslo) and Jaffé method respectively. ACR was used as an expression for urine albumin excretion.

Statistical analysis
Body mass index (BMI) was calculated in kilograms per meter squared. Systolic (SBP) and diastolic blood pressure (DBP) were included as the mean of the second and third of three measurements. ACR was calculated as [urine albumin (mg)]/[urine creatinine (mmol)], and was de ned as the mean of three ACRs. Moderately increased albuminuria was de ned as ACR > 3.0 mg/mmol and < 30 mg/mmol. We used the t-test for independent samples, Mann-Whitney U statistics and chi-square to examine baseline differences between two means or proportions.
Logistic regression analysis was performed to estimate the odds ratio (OR) for moderately increased albuminuria (ACR > 3.0 mg/mmol) according to different HADS subgroups and -scores. A priori selected potential confounding factors were used in both bivariate and multivariate adjusted analyses, i.e age, sex (in the total population), SBP, waist circumference, cholesterol, creatinine, history of CVD (yes/no), daily smoking (yes/no), treated hypertension (yes/no), diabetes (yes/no), education (primary and secondary school [< 12 years] and college or university [> 12 years]) and strenuous physical activity (< 1 hour/week, > 1 hour/week). All statistical analyses were conducted with the Statistical Package for the Social Sciences, version 25.0 (SPSS Inc., Chicago, USA), and signi cant alpha criterion was set to 0.05 or above.

Results
The baseline characteristics of the population showed that those with Hospital Anxiety and Depression Scale (HADS)-depression (D) > 8 had signi cant higher mean albumin/creatinine ratio (ACR) than those with HADS-D < 8 (1.91 mg/mmol v.s. 1.68 mg/mmol, p=0.030). They had higher proportion of moderately increased albuminuria (ACR > 3.0 mg/mmo; 13.3% v.s. 10.8%, p=0.012), corresponding result was found in those with HADS-D > 11. However, in addition to increased albuminuria, those with HADS-D > 8 or >11 were signi cant older, had higher BMI, creatinine and lipid levels compared to those with lower HADS-D score. They also had more frequently diabetes, treated hypertension and cardiovascular disease (CVD), had lower education, and were more often smokers and less physical active than those with HADS-D score < 8 (Table 1).
The signi cant association disappeared especially by variables as SBP, smoking, diabetes, CVD, creatinine, and waist circumference.

Discussion
This study demonstrated a signi cant positive association between the measured depression symptoms and moderately increased albuminuria. However, the association weakened after adjusting for age and sex, and after multivariable adjustment the signi cant association disappeared. Therefore, the unadjusted correlation between depression symptoms and albuminuria in the study population seemed to be explained by other factors causing possible in ammation, like increased CV risk and other comorbidities.
A positive association between albuminuria and depressive symptoms/depressive episodes has been published by Martens et al 12 . This study has also a cross sectional design, however with fewer participants than ours. As mentioned by the authors, almost 1/3 had diabetes type 2, compared to 16% in our study, and although adjustment of this comorbidity in both studies, the population from Netherlands might have a higher cardiovascular (CV) risk pro le. Additionally, the studies differed in methods regarding classi cation of depressive symptoms (PHQ-9 versus HADS), and Martens et al. also included a clinical interview (MINI) to assess the presence of minor or major depressive episodes, which gives a more valid classi cation of depressive episodes. HADS is not a diagnostic instrument for clinical diagnoses, but it identi es seven depressive symptoms. A literature review, which includes 31 studies, concluded that the HADS holds good properties for depression in patient populations in primary care and hospital settings 15 . Even high scores of HADS-D do not give a depression diagnosis, but HADS has been used in several studies, including HUNT studies 16,17 . Various cut-off levels have been reported from various study populations, and studies comparing HADS with «gold standard» or reference standard diagnostic interviews for depression disorder have disclosed reasonable validity 18, 19 . The Norwegian version of HADS is a relatively well validated screening instrument for anhedonia and psychological distress 20,21 .
Depression and stress related symptoms may be associated with a chronic, low-grade in ammation response, and studies have shown association between depression and activation of cell-mediated immunity as well as increased oxidative and nitrosative stress 22 . Several factors increase the risk for development of depression whilst they also are associated with systemic in ammation; psychosocial stressors, poor diet, physical inactivity, obesity, smoking, altered gut permeability, atopy, dental cares, sleep and vitamin D de ciency 22 . Some of these factors are also associated with albuminuria, like physical inactivity 23 , obesity 24,25 and smoking 26,27 , and were included as confounders in our analyses, and might have been stronger associated with albuminuria than depression symptoms.
The cerebral small vessel disease, de ned as a disease of the small blood vessels in the brain, and a frequent cause of stroke and dementia, is also hypothesized to lead to depression (the vascular depression hypothesis) through disruption of neuronal circuits involved in mood regulation 28, 29 . Although we recently published the results of a positive association between vascular dementia and lowgrade albuminuria 30 and have found signi cant association between lacunar stroke and albuminuria 31 in the same HUNT-material, we could not support the vascular depression hypothesis by our results. However, the results may re ect the limitation of HADS in measurement of depression more than the true association between depression and vascular endothelial dysfunction.
There are several strengths of this study. The population-based approach and the high attendance rate make selection bias less likely. The response rate in the albuminuria screening was especially high in the elderly and those with diabetes and treated hypertension 32 . Another strength was that albuminuria analyses were performed in fresh urine samples without long-term storing, in contrast to other studies 33 .
Several studies conclude that measuring ACR is a speci c and sensitive alternative to twenty-four-hour urine collection in population-based albuminuria screenings 34,35 .
One of the most important limitations is the self-reported symptoms of depression, which always may lead to misclassi cation. As the HADS questionnaire does not mirror somatic depressive symptoms, cases with predominantly somatic depressive, i.e. biologically based symptoms, as weight loss, fatigue and insomnia, might be undetected. However, these symptoms overlap with several other morbidities, which might be associated with albuminuria, thus somewhat underestimating than overestimating our results. There is probably considerable variability of in ammation within the depressed population, and there are studies that nd subtypes of depression which are more in ammation-related 36 . On the other hand, HADS has shown in other studies association with in ammation-variables, as CRP 37 . Further, we had no information about medical treatment of depression, where treatment by tricyclic antidepressant could increase CV risk 38 in contrast to selective serotonin reuptake inhibitors which are associated with reduced CV risk 39 . Another limitation is the lack of other in ammatory markers. The HUNT study includes a large population and questions about several diseases, and because of limited resources speci c in ammation-or biological parameters for most diseases were not included. Neither we have information about all comorbidities in this epidemiological study since comorbidities are very heterogeneous. We therefore adjust for risk factors and comorbidities most correlated with albuminuria and depression.
Further, the information about use of other medication, which could have in uenced albuminuria levels, was lacking, i.e. use of among others angiotensin converting enzyme (ACE) inhibitors and angiotensin II blockers (ATB). However, at the time of HUNT 2, the use of ACE inhibitors and ATB in Norway were still low. Another limitation includes the diagnosis of urinary tract infection (UTI). Those who reported UTI in the previous week were excluded from the analyses, but we were not able to adjust for asymptomatic UTI. This could contribute to a non-differential misclassi cation that might further weaken the associations found. The cross-sectional design limits the possibility for discovering cause-effect relationships.
Although the association between symptoms of mood disorders and albuminuria was non-signi cant after multi adjustment in this study, there was a signi cant increased prevalence of albuminuria in the depressive symptoms sample compared to the non-depressive. Intervention in albuminuria positive individuals with diabetes or hypertensive are recommended and include more aggressive treatment of CV risk factors like blood pressure, smoking, hyperlipidemia and overweight 40,41 . So far, there are no recommendations of albuminuria screening in otherwise healthy people, although small studies have found reduction of CVD by intervention with ACE inhibition 42 . Individuals with depression and elevated peripheral in ammatory markers, included albuminuria might have bene t of anti-in ammatory agents, such as acetyl-salicylic acid (ASA), statins, ACE inhibitors and ATB to improve the treatment of depression and other mood disorders, leading to reduction of disability, even though there is much more research to be carried out here.
In conclusion, the positive and signi cant association between moderately increased albuminuria and depressive symptoms found in unadjusted analyses weakened and disappeared after adjustments, indicating that albuminuria is stronger associated with age, other comorbidities, and CV risk factors.
Another screening or diagnostic tool for depressive symptoms should also be considered in this case.