J. Lipid Res. Neurobiology of Lipids (ISSN1683-5506)
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Originally published In Press as doi:10.1194/jlr.M600178-JLR200 on June 20, 2006

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Journal of Lipid Research, Vol. 47, 2071-2079, September 2006
Copyright © 2006 by American Society for Biochemistry and Molecular Biology

Patient-Oriented Research

Role of the kidney in the metabolism of apolipoprotein A-IV: influence of the type of proteinuria

Arno Lingenhel*, Karl Lhotta{dagger}, Ulrich Neyer§, Iris M. Heid**,{dagger}{dagger}, Barbara Rantner*, Martina F. Kronenberg*, Paul König{dagger}, Arnold von Eckardstein§§, Maria Schober***, Hans Dieplinger* and Florian Kronenberg1,*

* Division of Genetic Epidemiology, Department of Medical Genetics, Molecular and Clinical Pharmacology, Innsbruck Medical University, Innsbruck, Austria
{dagger} Department of Clinical Nephrology, Innsbruck Medical University, Innsbruck, Austria
§ Department of Nephrology and Dialysis, Academic Teaching Hospital, Feldkirch, Austria
** Institute of Epidemiology, GSF-National Research Center for Environment and Health, Neuherberg, Germany
{dagger}{dagger} Institute of Biostatistics and Epidemiology, Ludwig-Maximilians-Universität München, Munich, Germany
§§ Institute of Clinical Chemistry, University and University Hospital of Zürich, Zürich, Switzerland
*** Central Laboratory of University Clinics, Innsbruck Medical University, Innsbruck, Austria

Published, JLR Papers in Press, June 20, 2006.

1 To whom correspondence should be addressed. e-mail: florian.kronenberg{at}i-med.ac.at


ABSTRACT

Increased plasma concentrations of apolipoprotein A-IV (apoA-IV) in chronic renal disease suggest a metabolic role of the kidney for this antiatherogenic protein. Therefore, we investigated patients with various forms of proteinuria and found increased serum concentrations of apoA-IV in 124 nephrotic patients compared with 274 controls (mean 21.9 ± 9.6 vs. 14.4 ± 4.0 mg/dl; P < 0.001). Decreasing creatinine clearance showed a strong association with increasing apoA-IV levels. However, serum albumin levels significantly modulated apoA-IV levels in patients with low creatinine clearance, resulting in lower levels of apoA-IV in patients with low compared with high albumin levels (21.4 ± 8.6 vs. 29.2 ± 8.4 mg/dl; P = 0.0007). Furthermore, we investigated urinary apoA-IV levels in an additional 66 patients with a wide variety of proteinuria and 30 controls. Especially patients with a tubular type of proteinuria had significantly higher amounts of apoA-IV in urine than those with a pure glomerular type of proteinuria and controls (median 45, 14, and 0.6 ng/mg creatinine, respectively). We confirmed these results in affected members of a family with Dent's disease, who are characterized by an inherited protein reabsorption defect of the proximal tubular system. In summary, our data demonstrate that the increase of apoA-IV caused by renal impairment is significantly modulated by low levels of serum albumin as a measure for the severity of the nephrotic syndrome. From this investigation of apoA-IV in urine as well as earlier immunohistochemical studies, we conclude that apoA-IV is filtered through the normal glomerulus and is subsequently reabsorbed mainly by proximal tubular cells.

Supplementary key words nephrotic syndrome • tubular proteinuria • atherosclerosis • Dent's disease

Human apolipoprotein A-IV (apoA-IV) is a 46 kDa glycoprotein (1, 2) synthesized in intestinal enterocytes during fat absorption and incorporated into the surface of nascent chylomicrons (3). In the fasting state, the majority of apoA-IV circulates in plasma as part of a lipid-poor, small HDL-like particle that does not contain apoA-I (4). Only small amounts are lipid-free (~4%) (5, 6). In vitro studies show strong evidence that apoA-IV plays an important role in reverse cholesterol transport (7–12). Because disturbances in the reverse cholesterol transport are obvious in patients with chronic kidney disease (13, 14), apoA-IV was investigated and found to be markedly increased in hemodialysis and peritoneal dialysis patients (15–19). It was identified as a marker of kidney impairment that starts increasing in the earliest stages of kidney disease (20), and high apoA-IV concentrations predict the progression of primary nondiabetic kidney disease (21).

Patients with kidney disease have an extremely high cardiovascular risk (22). Several other metabolic disturbances have been described that might contribute to this high risk (19). These include high triglyceride and lipoprotein[a] levels, low HDL cholesterol concentrations, and disturbed lipolysis.

Severe changes in lipoprotein metabolism have also been described in patients with proteinuria and nephrotic syndrome (23–25). The nephrotic syndrome is the consequence of increased permeability of the glomerular basement membrane, resulting in urinary loss of plasma proteins, including albumin, transferrin, IgG, hormone binding proteins, inhibitors of the clotting cascade such as antithrombin, or protein S. Therefore, nephrotic syndrome is characterized by severe proteinuria and hypoalbuminemia. In response to the subsequently decreased plasma oncotic pressure, mainly the synthesis of large hepatic proteins, including apolipoproteins and lipoproteins, is increased and causes hyperlipidemia (23, 24). Although increased triglyceride concentrations in nephrotic patients result from decreased VLDL catabolism, the increase in LDL and lipoprotein [a] derives from an increased synthesis (26, 27). Severe proteinuria may also result from disturbed tubular reabsorption of small proteins that are physiologically secreted through the glomerula.

In recent immunohistochemical studies, we observed immunoreactivity of apoA-IV in the kidney tubular cells, which suggests a direct role of the human kidney in the metabolism of apoA-IV (28). To elucidate this role in more detail and by a different approach, we investigated apoA-IV serum concentrations in patients with various degrees and types of proteinuria. ApoA-IV serum concentrations were measured in 124 patients with a wide range of nephrotic proteinuria. In addition, serum and urinary apoA-IV were analyzed in 66 patients with glomerular and tubular types of proteinuria and a large family with Dent's disease. This disease is an X-chromosome-linked tubular syndrome (29, 30) caused by mutations in the renal chloride channel gene CLCN5. It is characterized by pronounced tubular proteinuria attributable to a failure in the reabsorption of low molecular weight proteins by the proximal tubular system (31) and the urinary loss of these proteins.


METHODS

Patients and controls
Nephrotic syndrome One-hundred twenty-four patients with nephrotic syndrome were recruited at the Department of Nephrology at the University of Innsbruck as part of a recently described study (25). Patients were included in the study when they underwent kidney biopsy and when they had at least one exact measurement of 24 h proteinuria of >3.5 g/24 h, serum creatinine, height and weight, and a fasting blood withdrawal with collection of serum. Patients with diabetic nephropathy were excluded from the study. All patients were Caucasians and not in need of renal replacement therapy. Patients were recommended a balanced diet with daily 0.8–1 g protein/kg body weight with neither protein restriction nor protein overconsumption. Table 1 shows the clinical characteristics of patients, including the histological diagnosis of the primary cause of renal disease. Patients were compared with 274 controls frequency-matched for age and sex and of the same ethnic origin without renal impairment or liver disease, who were recruited in 1997 from one of the Prospective Cardiovascular Münster (PROCAM) study centers.


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  		TABLE 1. Anthropometric and biochemical data of patients with nephrotic syndrome and age- and gender-matched controls

 
Proteinuric patients For serum and urine analysis, 66 patients with different forms of proteinuria were recruited at the Department of Nephrology at the University of Innsbruck and at the Feldkirch Hospital during 1999–2002. Thirty randomly selected healthy controls with comparable age and gender distribution consisted of individuals from the staff of our department.

Dent's disease One family with 5 affected male patients with Dent's disease, 5 female carriers, and 42 unaffected members was recruited at the Department of Nephrology and Dialysis at the Feldkirch Hospital. Three of the five affected patients had normal serum creatinine levels, and the other two patients had already undergone kidney transplantation. The diagnosis of Dent's disease was based on the following clinical criteria: 1) low molecular weight proteinuria; 2) hypercalciuria; and 3) the X-chromosome mode of inheritance. All family members were analyzed for CLCN5 mutations. The affected patients carried a novel CLCN5 mutation (K. Lhotta et al., unpublished results). All female carriers were shown to be heterozygous for this CLCN5 mutation.

Our studies of patients with kidney impairment were approved by the institutional ethics committees, and participants gave informed consent.

Laboratory procedures
Serum and EDTA plasma were taken after a 12 h overnight fast. After low-speed centrifugation, samples were frozen and kept at –80°C before analysis. We calculated the creatinine clearance (CrCl) using the formula of Cockcroft and Gault (32) corrected to 1.73 m2 body surface. Measurement of serum albumin, lipids, and apoA-IV was performed in batches. Serum albumin concentrations (brom-cresol green method) and total and HDL cholesterol were measured using kits from Roche Diagnostics (Basel, Switzerland). Serum apoA-IV quantification was performed with a double-antibody ELISA using an affinity-purified polyclonal rabbit anti-human apoA-IV antibody for coating and the same antibody coupled to horseradish peroxidase for detection. Plasma with a known content of apoA-IV (standardized with purified apoA-IV after phenylalanine quantification by HPLC) served as the calibration standard. Each sample was analyzed in duplicate, and intra-assay and interassay coefficients of variation were 2.7% and 6%, respectively. The lower detection limit of this assay was 0.002 mg/dl.

Urinary apoA-IV from patients and controls was analyzed with the ELISA described above. Immunoblot analysis of urinary apoA-IV was performed using a Novex gel system (BisTris 4–12%, 1x MOPS, 200 V for 1 h) under reducing conditions with 1:4 diluted urine, applying 10 µl per sample. Control plasma and purified apoA-IV were diluted 1:40, and 2 µl was applied to the gel. Gels were blotted for 45 min at 120 V in a cooled Hofer transphor unit. Incubation with a HRP-labeled affinity-purified polyclonal rabbit anti-human apoA-IV antibody was followed by detection with ECL substrate (Amersham Bioscience).

{alpha}1-Microglobulin in urine was measured by nephelometry using the N {alpha}1-Microglobulin Kit on the Behring Nephelometry II System (Dade Behring Marburg GmbH, Marburg, Germany). Renal proteinuria was classified as being of the glomerular type if {alpha}1-microglobulin/creatinine was <=14 mg/g and of the tubular type if >14 mg/g.

Statistical procedures
Statistical analysis was performed with SPSS for Windows 12.0 and SAS 9.1.3. Unadjusted comparisons of continuous variables between controls and nephrotic patients were done by unpaired t-test or by the nonparametric Wilcoxon rank sum test for not normally distributed variables such as serum triglycerides, CrCl, proteinuria, urinary protein/creatinine, or apoA-IV/creatinine. Spearman correlation coefficients were computed to investigate the relationship between apoA-IV and parameters of renal dysfunction or lipid levels. The Mantel-Haenszel test of one degree of freedom was used to compare the frequency distribution of apoA-IV serum levels in categories between patients and controls. A linear model (SAS GLM procedure) was used to investigate the association of different variables with apoA-IV serum concentrations, adjusting for gender with and without inclusion of the interaction parameter.


RESULTS

The anthropometric, clinical and biochemical parameters of nephrotic patients and the age- and gender-matched healthy controls are summarized in Table 1. As expected, plasma levels of albumin, total protein, creatinine, and CrCl differed significantly between patients and controls. Patients showed a 24 h proteinuria of on average 7 g.

Hyperlipidemia and nephrotic syndrome
Hyperlipidemia is one striking characteristic of the nephrotic syndrome. As shown in Table 2 , all plasma lipids but HDL were increased significantly in the nephrotic patients. Mean total and LDL cholesterol and triglyceride levels were at least 50% above the values of the control group.


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  		TABLE 2. Lipids and apoA-IV serum concentrations in patients with nephrotic syndrome and age- and gender-matched controls

 
ApoA-IV and nephrotic syndrome
ApoA-IV serum concentrations were markedly increased in nephrotic patients compared with controls (21.9 ± 9.6 vs. 14.4 ± 4.0 mg/dl; P < 0.001), and the frequency distribution of apoA-IV concentrations by categories showed major differences between patients and controls (Mantel-Haenszel test with one degree of freedom: P < 0.001) (Fig. 1 ).


Figure 1
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  		Fig. 1. Frequency distribution of apolipoprotein A-IV (apoA-IV) serum concentrations by categories in 124 nephrotic patients and 227 controls. Mantel-Haenszel test: Chi-square = 89, degrees of freedom = 1, P < 0.00001.

 
In patients, serum apoA-IV levels showed the strongest correlations with serum albumin levels (r = 0.510, P < 0.001) (Fig. 2A ) and weaker correlations with proteinuria (r = –0.304, P < 0.001) (Fig. 2B). In addition, apoA-IV increased with decreasing renal function (r = –0.367, P < 0.001) (Fig. 2C). Generally, the correlations of apoA-IV levels with other variables, notably creatinine and CrCl, were weaker in controls than in patients (Table 3 ). We did not find any correlation between apoA-IV and triglyceride concentrations even when we performed the correlation analysis stratified for patients in the tertiles of triglycerides or serum albumin levels.


Figure 2
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  		Fig. 2. Correlation of serum apoA-IV concentrations with serum albumin (A), proteinuria (B), and creatinine clearance (C). Correlation coefficients (r) were calculated according to Spearman.

 

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  		TABLE 3. Bivariate Spearman correlation coefficients of plasma apoA-IV with anthropometric, biochemical, and lipid parameters

 
To analyze the influence of the severity of the nephrotic syndrome, as measured by serum albumin and CrCl, on apoA-IV levels, we stratified the patients according to the sex-specific medians of serum albumin (2.68 and 3.05 for women and men, respectively) and CrCl (54 and 63 ml/min/1.73 m for women and men, respectively). Figure 3A shows 5 mg/dl lower serum apoA-IV levels in serum albumin below compared with above median (P = 0.0014). Nephrotic patients with CrCl below median showed ~7 mg/dl higher serum apoA-IV levels than those with CrCl above median (P < 0.0001). In a next step, we included an interaction between CrCl groups and albumin groups, which showed borderline significance (P = 0.099) using the significance level of 0.10 usually applied for interaction analysis. Figure 3B shows the apoA-IV levels from this analysis for the four groups (high albumin and high CrCl, low albumin and high CrCl, high albumin and low CrCl, low albumin and low CrCl). It can be seen that the differences between subjects with low or high albumin in apoA-IV levels were small in cases of high CrCl (mean ± SD: 16.9 ± 7.4 vs. 19.4 ± 9.3 mg/dl; P = 0.25). However, patients with low CrCl had significantly higher apoA-IV levels in cases of concomitant high albumin compared with low albumin (29.2 ± 8.4 vs. 21.4 ± 8.6 mg/dl; P = 0.0007). Therefore, it seems that the known apoA-IV-increasing effect of a decreased creatinine clearance (20) is strongly modified by low albumin levels and, therefore, by the severity of the nephrotic syndrome. When we performed the analysis stratified by tertiles of CrCl and tertiles of serum albumin levels, we found a consistent pattern even indicating a trend per tertile (Fig. 3C). Although women have generally lower apoA-IV levels, the observed association was the same in men and women (data not shown).


Figure 3
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  		Fig. 3. A, B: Influence of albumin status and creatinine clearance (CrCl) status on apoA-IV serum levels in nephrotic patients (n = 124). Values shown are mean ± SEM apoA-IV levels adjusted for sex, albumin status, and CrCl status. Albumin status and CrCl status were defined using the sex-specific medians of albumin (2.68 and 3.05 for women and men, respectively) and CrCl (54 and 63 ml/min/1.73 m for women and men, respectively) as cutoff points. C: Analysis similar to B but stratified by tertiles of CrCl and tertiles of serum albumin levels, indicating a trend per tertile.

 
When we offered 24 h proteinuria dichotomized by the sex-specific median (6.90 and 6.16 for men and women, respectively) instead of the dichotomized albumin to the model, the differences in the four groups (low proteinuria and high CrCl, high proteinuria and high CrCl, low proteinuria and low CrCl, high proteinuria and low CrCl) were similar but less pronounced (19.0 ± 8.5, 16.8 ± 7.8, 28.6 ± 9.2, and 22.8 ± 8.7 mg/dl, respectively). A smaller percentage of the variance of apoA-IV was explained by proteinuria versus albumin: with sex, albumin, and CrCl in the model, 37.4% of the variance of apoA-IV was explained, to which the three variables contributed 3.3, 22.4, and 11.7%, respectively. The analogous model exchanging 24 h proteinuria for albumin explained only 20.0%, to which the three variables contributed 3.6, 5.1, and 11.3%, respectively.

Urinary ApoA-IV and the type of renal proteinuria
To analyze whether apoA-IV is lost in urine and whether the type of proteinuria (glomerular or tubular) influences the amount of apoA-IV in urine, we investigated a further sample of 66 patients with isolated glomerular (n = 17) or tubular (n = 49) proteinuria (Table 4 ) compared with 30 healthy controls. We found significantly higher apoA-IV levels in urine of proteinuric patients compared with controls (median 26 vs. 0.6 ng/mg; P < 0.001). This difference was caused mainly by significantly higher urinary apoA-IV concentrations in patients with the tubular type of proteinuria compared with those with an isolated glomerular proteinuria (median 45 vs. 14 ng/mg; P < 0.001). The comparison of the three groups is presented in Fig. 4A . Figure 4B shows a significant correlation of the logarithmically transformed urinary apoA-IV/creatinine with {alpha}1-microglobulin/creatinine concentrations (Spearman correlation coefficient = 0.44; P < 0.001), demonstrating a significant association of urinary apoA-IV excretion with the intensity of tubular damage. Western blot analysis confirmed the ELISA data and the presence of intact apoA-IV and apoA-IV fragments only in urine samples from patients with a tubular component of proteinuria (Fig. 4C). To find significant amounts of apoA-IV in urine, patients had to have a tubular defect as well as a sufficient amount of apoA-IV glomerulus-filtered that was no longer able to be reabsorbed by the tubular cells.


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  		TABLE 4. Comparison of serum and urinary parameters between patients with only glomerular and the tubular type of proteinuria

 

Figure 4
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  		Fig. 4. A: Urinary concentrations of apoA-IV/creatinine in patients with isolated glomerular (n = 17) or a glomerular-tubular or tubular type (n = 49) of proteinuria and 30 healthy controls. The horizontal lines represent the median concentration of each group. B: Correlation of logarithmically transformed urinary apoA-IV/creatinine with {alpha}1-microglobulin/creatinine concentrations in patients with the two forms of proteinuria (r = correlation coefficient according to Spearman). C: Western blot of urinary apoA-IV. Lane Pl. shows the apoA-IV pattern from plasma (10 ng applied to the gel), lane ST shows an affinity-purified plasma apoA-IV standard (20 ng applied to the gel), lanes C1 and C2 represent samples from controls, and lanes P1–P8 represent samples from patients with various amounts of proteinuria (up to 0.3 ng applied to the gel). The amount of apoA-IV/creatinine, {alpha}1-microglobulin/creatinine ({alpha}1-MG/Cr), and proteinuria/creatinine is provided for each sample. The amount of applied apoA-IV was calculated from ELISA measurements. The apoA-IV antibody could detect only one protein band in the plasma sample (lane 1) but a double band in the apoA-IV preparation (lane 2). Peptide sequencing in an earlier project confirmed that both protein bands of the apoA-IV preparation represent apoA-IV. The second, smaller apoA-IV isoform might result from deglycosylation or proteolytic degradation.

 
Dent's disease
Dent's disease is an X-linked renal tubular disorder that is characterized by low molecular weight proteinuria (29). Its primary causes are loss-of-function mutations in the renal chloride channel gene CLCN5, which are responsible for the defective endocytic uptake of low molecular weight proteins in proximal tubular cells. To prove the proteinuric loss of apoA-IV as a tubular malfunction, we measured urinary apoA-IV concentrations in a family with Dent's disease. Four of five affected males showed increased urinary apoA-IV concentrations compared with the five female carriers and the noncarriers (Fig. 5 ). Two of the patients had already undergone kidney transplantation. One of these two patients had normal transplant function and no apoA-IV excretion in urine as well as a normal {alpha}1-microglobulin/creatinine level in urine. The other patient had a chronic transplant nephropathy, with serum creatinine of 2 mg/dl, {alpha}1-microglobulin/creatinine of 157 mg/g, and increased urinary apoA-IV/creatinine of 50 ng/mg. Female carriers and normal individuals showed no significantly increased urinary apoA-IV levels.


Figure 5
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  		Fig. 5. Urinary concentration of apoA-IV per creatinine in a family with Dent's disease. Comparison of affected males, female carriers, and healthy family members. Two affected males had already undergone renal transplantation. One of these patients had normal transplant function and no apoA-IV excretion in urine as well as a normal {alpha}1-microglobulin/creatinine level in urine (patient RTX). The other patient had a chronic transplant nephropathy with a serum creatinine of 2 mg/dl, {alpha}1-microglobulin/creatinine of 157 mg/g, and increased urinary apoA-IV/creatinine of 50 ng/mg (patient RTX+CTN).

 

DISCUSSION

Increased serum apoA-IV concentrations are a characteristic feature of renal disease (15–19) and are even an early marker of renal impairment (20) as well as progression of kidney disease (21). No study in humans until now has investigated apoA-IV in nephrotic syndrome. Our results show major differences between nephrotic patients and nonnephrotic patients with impaired kidney function. In a previous study, we had demonstrated that apoA-IV levels were explained mainly by the loss of glomerular clearance function in patients with nonnephrotic kidney disease (20). This study shows that apoA-IV levels in patients with nephrotic syndrome are strongly influenced by the severity of nephrotic syndrome. We used serum albumin levels as a surrogate marker for proteinuria, because this parameter is measured with greater reliability and reproducibility than is 24 h proteinuria. It is well known that any clearance technique that relies on urine excretion measurement is markedly influenced by substantial errors associated with 24 h urine collection, which is inconvenient and difficult for most patients (33). Serum albumin also provides a better insight into the metabolic situation of the disease, as low albumin levels are the result not only of urinary but also of extrarenal losses, insufficient hepatic protein production, and disturbances in the distribution of albumin between the intravascular and extravascular albumin pools (33). Our data clearly demonstrate that the increase of apoA-IV caused by renal impairment is significantly modulated by a low level of serum albumin. Nephrotic patients with CrCl below the sex-specific median had ~7 mg/dl higher apoA-IV levels than those with CrCl above the median (Fig. 3A).

In our previous study (20) in nonnephrotic renal disease, we observed with each 11 ml/min decrease of glomerular filtration rate an increase of 1 mg/dl apoA-IV, which is similar to the increase in this study. In nephrotic patients, low serum albumin levels had a significantly apoA-IV-decreasing effect (–5 mg/dl) (Fig. 3A). This was most pronounced in patients with CrCl below the sex-specific median (–7.8 mg/dl) (Fig. 3B). If we repeated the regression analysis in the data from our former study (20) of nonnephrotic patients entering serum albumin into the model, we did not observe any effect of serum albumin on apoA-IV serum levels (Fig. 6 provides a scatterplot of serum albumin and apoA-IV levels in these nonnephrotic patients for comparison with the nephrotic patient data in Fig. 3A). This argues for a strong independent effect of the severity of nephrotic syndrome on apoA-IV serum levels and therefore for major differences in the metabolism of apoA-IV in nephrotic and nonnephrotic patients. These and our previous data suggest that decreasing kidney function determined by CrCl has a strong apoA-IV-increasing effect as long as a patient has only nonnephrotic proteinuria. As soon as a pronounced nephrotic syndrome, with its major influence on metabolic pathways, develops, the disturbances in these pathways mask the effects of renal impairment to a large extent. This is most pronounced in patients with albumin levels and CrCl below the median of the patients. This is in agreement with calculations using a linear model in nephrotic patients, which revealed that, together with sex, ~22.4% and 11.7% of the apoA-IV variance is explained by serum albumin levels and CrCl, respectively.


Figure 6
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  		Fig. 6. Correlation of serum apoA-IV concentrations with serum albumin in 227 patients with nonnephrotic primary kidney disease from our previous study (20). The correlation coefficient (r) was calculated according to Spearman. To describe the patient population briefly, we included Caucasian patients aged 19–65 years who had visited the outpatient department at least once during the preceding year. Exclusion criteria were serum creatinine > 6 mg/dl, diabetes mellitus, malignancy, liver, thyroid, or infectious disease at the time of recruitment, nephrotic syndrome defined as daily proteinuria > 3.5 g/1.73 m2, organ transplantation, allergy against ionic contrast medium, and pregnancy.

 
We hypothesize that the apoA-IV-decreasing effect of low serum albumin levels in severe nephrotic syndrome could be caused either by a decreased production or increased loss in the intestinum or by an increased loss of apoA-IV by glomerular filtration. A decreased production in enterocytes is conceivable considering the pronounced mucosal edemas that can be observed in severe nephrotic syndrome. Furthermore, the production of apoA-IV is stimulated by leptin (34). Large amounts of leptin are lost in urine of nephrotic children, although serum leptin levels remain stable (35). Because an infusion of leptin in patients with lipodystrophy with proteinuric nephropathy results in a reduction of proteinuria and hyperfiltration (36), it is conceivable that disturbances in leptin metabolism have some influence on the production of apoA-IV in the intestinum. On the other hand, experiments in experimental nephrotic rats revealed a compensatory increase in intestinal apoA-IV mRNA levels in response to the urinary loss of apoA-IV (37). ApoA-IV could also be lost into the intestinum by an intestinal hyperpermeability, resulting in a leakage of enterocyte-derived apoA-IV into the intestinal lumen (38). Similar intestinal losses in patients with nephrotic syndrome have been described for albumin (39, 40).

A renal loss of apoA-IV is supported by an almost 6-fold relative increase of apoA-IV in urine of proteinuric patients compared with controls. These investigations in urine present strong evidence that apoA-IV is filtered through the glomerulus and reabsorbed by the proximal tubular systems. In general, many plasma proteins are handled in this way. For example, the most abundant plasma protein, albumin, with a molecular mass of ~65 kDa, shows a wide range of glomerular filtration. Various techniques used to measure glomerular filtration revealed an amount filtered to the ultrafiltrate of 0.2 to almost 10 g/24 h. Therefore, this large amount of filtered albumin has to be reabsorbed subsequently in the proximal tubular cells, mostly by receptor-mediated endocytosis. The multiligand receptors megalin and cubulin are responsible for the uptake of the vast majority of filtered plasma proteins, including albumin, in the renal tubular system (for review, see 41).

A glomerular filtration of apoA-IV with subsequent reabsorption in the proximal tubular cells is supported by differentiating between patients with an isolated glomerular proteinuria and those with an additional tubular component of proteinuria in our study. Those with a tubular component of proteinuria show an almost 6-fold higher excretion of apoA-IV into urine compared with those with pure glomerular proteinuria, and the amount of urinary apoA-IV correlates very well with the urinary excretion of {alpha}1-microglobulin as a marker of tubular damage. Furthermore, it is strongly supported by data from a family with Dent's disease, an X-chromosome-linked syndrome characterized by tubular proteinuria attributable to a failure of protein reabsorption by the proximal tubules. Affected male family members showed a pronounced increase in urinary apoA-IV that is in agreement with a recent proteomic approach that identified apoA-IV in higher amounts in these patients compared with controls (42). The involvement of the proximal tubular system is in agreement with our recent data on apoA-IV immunoreactivity observed in healthy human renal tubular cells, indicating a direct role of the human kidney for apoA-IV metabolism. ApoA-IV was found predominantly in the brush border of proximal tubules and in intracellular granules and various plasma membrane domains of both proximal and distal tubules (28). Finally, previous data in rats demonstrated that apoA-IV is catabolized by kidney and liver, and histological analysis found apoA-IV to be localized within proximal tubular cells (43).

Because of its molecular mass of ~46 kDa, at least the lipoprotein-unbound (free) form of apoA-IV can be filtered through the glomeruli (5, 7). An uptake by proximal tubular cells may then be followed by degradation, intracellular usage, or even return to circulation. An at least partial intracellular usage or return to circulation by transcytosis is supported by an intact apoA-IV protein band in kidney tissue (28). A similar rescue transport in proximal tubular cells that is mediated by the receptor megalin has been described for other serum molecules such as vitamin B12 and retinol (44) as well as apoA-I (45, 46). In nephrotic syndrome, apoA-IV can be largely reabsorbed as long as the nephrotic syndrome is not too pronounced and as long as no major tubular involvement of proteinuria occurs. This is in agreement with the results shown in Fig. 3, which suggest that nephrotic patients with CrCl below the median and low serum albumin levels, and therefore a high likelihood of already having tubular damage, show a pronounced decrease in serum apoA-IV levels compared with patients with high serum albumin levels. Because of the tubular damage, they are no longer able to reabsorb the large amounts of glomerulus-filtered apoA-IV and therefore show relatively low apoA-IV serum levels, despite pronounced impairment of kidney function.

Our results are in agreement with findings in rats with puromycin-induced nephrotic syndrome (37). Those animals showed, in addition to a pronounced decrease in serum albumin levels, a strong decrease in serum apoA-IV levels despite a significant increase in mRNA levels in the jejunum and ileum. The authors suggested that the decrease of serum apoA-IV levels was caused by urinary loss of apoA-IV. Because of the severity of the disease, it is conceivable that the tubular reabsorption system for apoA-IV was no longer intact, resulting in decreased apoA-IV serum levels.

Limitations of the study
When we take into account the small absolute amount of measured apoA-IV in urine, this cannot explain the large apoA-IV-decreasing effect in patients with severe nephrotic syndrome observed in the linear model. We suspect that our ELISA systematically underestimates the absolute amount of urinary apoA-IV, for example, by missing apoA-IV fragments, which we observed in immunoblot analysis of urine apoA-IV. Furthermore, a semiquantitative comparison of an apoA-IV standard and urinary apoA-IV in immunoblots reveals that a much higher amount of apoA-IV is excreted with the urine than is measured with our apoA-IV ELISA. Although we applied only up to 0.3 ng of urinary apoA-IV measured by ELISA to the gel, we found bands of similar intensity as serum samples or apoA-IV preparations from which we applied up to 20 ng according to ELISA measurements (Fig. 4C).

In summary, our data demonstrate that the increase of apoA-IV caused by renal impairment is significantly modulated by a low level of serum albumin as a measure of the severity of the nephrotic syndrome. From the investigation of apoA-IV in urine of patients with various forms of proteinuria, as well as from our previous immunohistochemical studies, we conclude that glomerulus-filtered apoA-IV is reabsorbed mainly via the renal tubular system.


ACKNOWLEDGMENTS

The authors thank Anna Schlögl and Sonja Wintersteiger (deceased) for excellent cooperation and technical assistance. This study was supported by grants from the Austrian Nationalbank (Project 9331), the University of Innsbruck (Project M30), and the Genomics of Lipid-Associated Disorders of the Austrian Genome Research Programme to F.K.; from the Austrian Science Fund to H.D. (P-12358); from the Deutsche Forschungsgemeinschaft (Wi621/12-1) to GSF-Institute of Epidemiology; and from Hans Drexel and Herwig Wallmann to the Vorarlberg Institute of Vascular Investigation and Treatment. B.R. was supported by a doctoral fellowship from the Austrian Academy of Sciences.

Manuscript received April 20, 2006 and in revised form June 2, 2006.


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B. C.H. Kwan, F. Kronenberg, S. Beddhu, and A. K. Cheung
Lipoprotein Metabolism and Lipid Management in Chronic Kidney Disease
J. Am. Soc. Nephrol., April 1, 2007; 18(4): 1246 - 1261.
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