Skip Navigation


NDT Advance Access originally published online on January 12, 2006
Nephrology Dialysis Transplantation 2006 21(5):1223-1230; doi:10.1093/ndt/gfk050
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
21/5/1223    most recent
gfk050v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (2)
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Hjalmarsson, C.
Right arrow Articles by Haraldsson, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hjalmarsson, C.
Right arrow Articles by Haraldsson, B.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author [2006]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Original Articles: Experimental Nephrology

Beneficial effects of orosomucoid on the glomerular barrier in puromycin aminonucleoside-induced nephrosis

Clara Hjalmarsson1, Martin E. Lidell2 and Börje Haraldsson1

1 Department of Nephrology, Sahlgrenska Academy and 2 Department of Medical Biochemistry, Göteborg University, Gothenburg, Sweden

Correspondence and offprint requests to: Clara Hjalmarsson, MD, PhD, Department of Nephrology, Sahlgrenska University Hospital, SE-405 30 Gothenburg, Sweden. Email: Clara.Hjalmarsson{at}kidney.med.gu.se



   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Background. In a hitherto unconfirmed report, orosomucoid was reported to ameliorate the nephrotic syndrome induced by puromycin aminonucleoside (PAN) in rats.

Methods. We wanted to test this hypothesis and extend the analysis of the effects on the glomerular barrier. Glomerular filtration rate (GFR), and fractional clearance for albumin ({theta}albumin) and for neutral Ficolls were estimated in cooled isolated perfused kidneys. Modern transport equations were used to estimate glomerular size selectivity and charge selectivity. Also, podocyte morphology was studied. Four groups of rats (4x n = 8) were administered PAN intraperitoneally and treated daily for 5 days with orosomucoid in two different doses (groups A and B), albumin (group C) or saline (group D). Two additional groups of rats (2 x n = 8) were used as controls and these rats received either saline (group E) or orosomucoid (group F) but no PAN.

Results. Treatment with orosomucoid restored podocyte morphology and renal function from the damaging effects of PAN in a dose-dependent manner. GFR was significantly reduced by PAN (groups C and D) when compared with controls (groups E and F). This effect was partly (group A) or completely (group B) reversed by orosomucoid. The {theta}albumin was 0.002±0.001 (mean±SEM) in controls (group E) and was unaffected by orosomucoid per se (group F). PAN increased {theta}albumin to 0.020±0.001 in group C and to 0.021±0.001 in group D, while it was significantly less in group A, 0.014±0.001, P<0.05. The heterogeneous charged fibre model analysis revealed that PAN reduced the relative volume of negatively charged fibres, {phi}, from 7.1±0.08% (group E) to 48% of this value in groups C and D (P<0.001); {phi} was 4.5±0.04% in group A, 5.3±0.44% in group B, and 6.1±0.11% in group F.

Conclusion. High doses of orosomucoid completely normalized the glomerular barrier in six out of eight animals with puromycin-induced nephrotic syndrome. Thus, orosomucoid has a promising therapeutic potential for certain kidney disorders.

Keywords: {alpha}1-acid glycoprotein; charged fibre; glomerular; nephrotic; permeability; size selectivity



   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Various experimental disease models are extensively used for clarifying the intricate aspects of the structural and functional alterations encountered in proteinuric states. One commonly used model is the puromycin aminonucleoside (PAN)-induced nephrotic syndrome [1]. After a single intraperitoneal (i.p.) administration, PAN induces a disease similar to minimal-change nephrosis, characterized by heavy proteinuria, hypoalbuminaemia and dyslipidaemia. The exact mechanism underlying the PAN toxicity is still unclear. Many explanations have been proposed: damage to the glomerular epithelial cell structure [2], alteration of the main constituents of the glomerular basement membrane by means of cytokines and interleukin-1 [3], oxidative stress, and changes of the glomerular charge density [1] and size selectivity [4]. Several groups have studied the alterations of the glomerular barrier in PAN nephrosis. In a previous study, we found evidence for damage on both size selectivity and charge selectivity by PAN [5]. In that study, we evaluated glomerular permeability both in vivo and in cooled isolated perfused kidneys (cIPKs) where the low temperature was used to abolish tubular modification of the primary urine and protease activity. Moreover, glomerular size selectivity was similar in vivo and in the cIPK, which supports the validity of the cIPK model. One limitation is, however, that the charge selectivity cannot be quantitated in vivo, due to the potent tubular reabsorption and secretion processes. A gel membrane model was used to analyse the effects of PAN and it revealed, among other things, a dramatic increase in the number of large pores and a reduced charge density. Although most authors describe profound alterations in the glomerular barrier, there are reports suggesting that PAN acts solely by reducing tubular reuptake of proteins [6]. Thus, there is a research group that suggests nephrotic syndromes are due to tubular dysfunctions [7] rather than to alterations of the glomerular capillary wall, as assumed by most investigators. Albeit an attractive hypothesis, it must be remembered that there is no evidence for tubular reuptake of intact albumin [8]. On the contrary, recent developments in the tubular cell research area have revealed an obligatory incorporation of the albumin-binding megalin–cubilin complex into lysozomes with subsequent degradation [8].

Orosomucoid was first described >100 years ago, and since then it has been studied extensively. The glycoprotein has a molecular weight of 44 kDa, an isoelectric point of 2.8–3.8 and a pronounced microheterogeneity. While the structural and biochemical characteristics have been well described, the biological functions of orosomucoid are not yet fully understood. However, Haraldsson and Rippe showed that orosomucoid is essential for the maintenance of normal capillary permeability in skeletal muscle [9]. This was later confirmed for capillaries in the mesentery [10] and for glomerular capillaries [11]. Indeed, the glomerular barrier is highly dynamic, and serum components may alter its permselective properties [11].

A few years ago, it was suggested that orosomucoid may have therapeutic effects in certain nephrotic syndromes. Thus, intravenous injections of orosomucoid to rats with PAN nephrosis reduced the degree of proteinuria and improved podocyte morphology [12]. That study is the only report of an anti-proteinuric effect of orosomucoid and it mainly focused on the morphological alterations. Therefore, we wanted to test the hypothesis presented by Muchitsch et al. and to extend the analysis by focusing on the quantitative effects of orosomucoid on the size selectivity and charge selectivity of the glomerular barrier.



   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Experimental animals
All experiments were carried out on female Sprague–Dawley rats (strain Møllegaards Ltd, Stensved, Denmark) weighing 200–320 g. The animals had free access to food (standardized pellets) and tap water. The local ethics committee approved the experiments.

Kidney preparation
The rats were anaesthetized with 50 mg/kg of pentobarbitone® i.p. and then placed on a thermostatically controlled heating pad, preserving body temperature at 37°C. The trachea was isolated and a tracheostomy performed with a PE-240 catheter in order to assist respiration during anaesthesia. A tidal volume of 3 ml was provided by a respirator (UGO Basile type 7025, Biological Research Apparatus, Italy) at a frequency of 40/min. Arterial blood gas analyses were performed to ensure adequate ventilation.

The tail artery was cannulated by a PE-50 catheter, connected to a pressure transducer (PVB Medizintechnik GmbH, Kirchenseeon, Germany) for recording arterial pressure (PA). Laparotomy was performed and the kidneys isolated. The cannulation of the ureter (polyethylene catheter PE-25) was facilitated by prior injection of 2 mg/kg of furosemide® (Benzon Pharma A/S, Copenhagen, Denmark) and saline (0.3 ml), via the tail artery. The abdominal aorta was freed from surrounding tissues and prepared for cannulation.

The rats received heparin (2000 IU/kg, Lövens Läkemedel, Malmö, Sweden) via the tail artery, were carefully moved from the heating-pad to a Plexiglas working table, and were then prepared for the cIPK experiments. Two blood samples of 500–750 µl were collected from the tail artery, followed by administration of an equivalent amount of saline. The abdominal aorta was cannulated distal to the renal arteries in a retrograde direction using polyethylene tubing (PE-90). Just before the onset of the perfusion sequence, the right renal pedicle was isolated and ligated, and the kidney was removed and fixed in situ by subcapsular injection of Karnovsky fixative. The artificial perfusion of the left isolated kidney was started without interrupting blood flow, and a ligature was placed between the renal arteries. A peristaltic pump (Labinett type MS4, Ismatec SA, Laboratoriumstechnik, Switzerland) was used for pumping the perfusate through an isothermal chamber placed close to the kidney, maintaining the temperature of the perfusate at 8°C. Following a 15 min period of equilibration, four urine samples (200–450 µl) were taken from each animal. For each experiment, arterial pressure, perfusion pressure and urine flow were monitored using Labview® computer software.

Perfusate and tracers
The perfusate consisted of Tyrode solution containing human albumin (18 g/l, Baxter Medical AB, Vienna, Austria) and tracers. The final composition of the solution was as follows: NaCl, 113 mM; KCl, 4.3 mM; CaCl2, 2.5 mM; MgCl2, 0.8 mM; NaHCO3, 25.5 mM; NaH2PO4, 0.5 mM; glucose, 5.6 mM; nitroprusside (Merck, Darmstadt, Germany), 0.9 mM; furosemide® (Benzon Pharma A/S), 10 mg/l; [51Cr]EDTA (Amersham Pharmacia Biotech, Buckinghamshire, UK), 0.16 MBq/l; [125I]human serum albumin (HSA; Isopharma AS, Kettel, Norway), 0.6 MBq/l; and fluorescein isothiocyanate (FITC)-labelled Ficoll (Bioflor HB, Uppsala, Sweden), 15 mg/l. The sieving coefficients of albumin were analysed from the urinary output of the [125I]HSA. The free fraction of radioactive iodine was removed by filtration through an equilibrated desalting column (Sephadex® G-25 PD-10, Amersham Pharmacia Biotech) before [125I]HSA was added to the perfusate.

The perfusate (pH 7.4) was bubbled with 5% CO2 in O2, kept at 8°C, and protected from light.

Experimental protocol
Four groups of rats, A, B, C and D (4x n = 8), were administered a single i.p. injection of PAN (150 mg/kg body weight, Sigma-Aldrich, Steinheim, Germany) dissolved in 1 ml of saline. Two control groups of rats, E (n = 8) and F (n = 8), were given a single injection of saline (1 ml). Experiments were performed 6 days after the administration of PAN/saline as described under ‘Kidney preparation’. Under the 6 day period, the rats in all six groups were given the following daily i.p. injections: group A, human orosomucoid (kindly provided by Dr H. P. Schwartz, Baxter Inc., Austria) 20 mg in 1 ml/100 g body weight; group B, human orosomucoid 60 mg in 1 ml/100 g body weight; group C, Albumin Immuno (Baxter Medical AB) 20 mg in 1 ml/100 g body weight (the orosomucoid contamination of the HSA was 0.1 mg/ml); group D, saline 1 ml/100 g body weight; group E, saline 1 ml/100 g body weight; and group F, human orosomucoid 20 mg in 1 ml/100 g body weight.

Thus, each rat got the same amount of fluid (orosomucoid solution, albumin solution or saline) in all six groups of rats, and groups A, C and F received equal amounts of protein.

Data analysis
To determine the sieving coefficients ({theta}) for albumin and the glomerular filtration rate (GFR), the plasma and urine concentrations of [125I]HSA and [51Cr]EDTA were measured in all samples (CobraTM, AutoGamma® Counting Systems, Packard Instrument Company, Meridan, CT). Background radioactivity and the spillover of [51Cr]EDTA radiation to the iodine channels were corrected for. Additionally, the albumin concentrations of all urine samples were determined by radioimmunoassay (Amersham Biosciences Inc., USA).

All perfusate and urine samples were subjected to both gel filtration (BioSepTM-SEC-S3000, Phenomenex, Torrance, CA) and detection of fluorescence (RF 1002 Fluorescence HPLC Monitor, Gynkotek, Germering, Germany) using ChromeleonTM (Gynkotek, Germering, Germany) software for determining the sieving coefficients of FITC–Ficoll. A 0.05 M phosphate buffer with 0.15 M NaCl at pH 7.0 was used as eluent. The emission and excitation wavelengths were 520 and 492 nm, respectively, and 5–10 µl from each sample were analysed. The sampling frequency (1/s); the flow-rate (1 ml/min); the temperature (8°C); and the pressure (4 MPa) were all maintained constant during the analysis. For most molecular sizes, the errors in the CU/CP ratios for Ficoll were estimated to be <1%.

Calculations
Glomerular filtration rate
The GFR was determined using the formula

Formula 1(1)
where CU and CP represent the urine and plasma concentrations of [51Cr]EDTA, and Qu is the urinary flow.

Fractional clearances of albumin and Ficoll
The fractional clearance {theta} of a solute X was calculated as:

Formula 2(2)
where CP represents the average concentration of three samples collected from the perfusate, and CU is the concentration of the tracer in each ‘primary urine’ sample collected during the perfusion period.

The charged fibre model with discontinuities of low fibre density
One way of estimating the size- and the charge-selective properties of the glomerular barrier is to use a charged fibre model with small discontinuities (large pores) with considerably fewer fibres [13]. This model emerged from the classic partitioning theory of Ogston [14]. It was developed by Johnson and Deen [15] and extended by us for the case of heterogeneous fibre density and for clearance across capillary barriers [13].

The parameters of the model are: the fibre radius (rf), the relative concentration of fibres in the gel ({phi}), the surface charge densities of the solute (qs) and the fibre (qf), the unrestricted exchange area over diffusion distance (A0/{Delta}x), the large pore fraction of the hydraulic conductance (fL) and the dilution factor for the fibre density in the large pore (XQL). A non-linear regression analysis was employed. Some of the parameters mentioned above were constant, namely rf and qs for albumin and Ficoll, while others ({phi}, qf, A0/{Delta}x, fL and XQL) were varied in order to obtain a good fit between the experimental and the modelled data. The gel/plasma concentration ratio at equilibrium in a fibre matrix is described by the partition coefficient ({Phi}):

Formula 3(3)
where g(h) is the probability of finding the closest fibre at a distance, h, from a spherical solute in a dilute solution:

Formula 4(4)
where {phi} is the volume fraction of fibres, rs the solute radius and rf the fibre radius. By integrating Equation 4, Ogston [14] reached the following expression for {Phi}:

Formula 5(5)

Johnson and Deen [15] introduced a Boltzmann factor to describe the relative probability at different energy states in charged gels. Multiplying g(h) by this factor gives:

Formula 6(6)
where E(h) is the electrostatic free energy of the interactions between the solute and the nearest fibre divided by kT (k is Boltzmann's constant and T is the absolute temperature). E is dependent on one position variable, h, only. Please note that the model assumes interactions between the solute and the nearest fibre, while interactions with multiple fibres (and other solutes) are expected in vivo.

To calculate the reflection coefficient of a solute from the partition coefficient, the following equation was used [16,17]:

Formula 7(7)
where, {sigma} is the reflection coefficient and {Phi} is the partitioning coefficient for a solute in a gel.

The fractional clearance of a solute was calculated by use of a non-linear flux equation [18]:

Formula 8(8)
where PS is the diffusion capacity for the solute estimated as:

Formula 9(9)
To describe the transport accurately, the fibre network was considered heterogeneous, with small areas of low fibre concentration, i.e. large pores. The fractional clearance of a solute will then be the sum of the {theta} through the main gel and the {theta} through the large pores; for details, see Jeansson and Haraldsson [13].

Electron microscopy
After fixation in situ by subcapsular injection of Karnovsky fixative, 1 mm cubes of renal cortex were excised and further fixed in 2.5% glutaraldehyde in pH 5.7 acetate buffer containing 0.3 M MgCl2 and 0.05% cupromeronic blue (Seikagaku Chemical Company). After en bloc staining with phosphotungstate, the specimens were dehydrated and embedded in plastic resin. Sections 50–60 nm thick were obtained with an ultramicrotome (Reichert-Jung, Austria) fitted with a diamond knife. A Zeiss 902 electron microscope was used to examine the sections. We compared podocyte morphology and quantified the alterations induced by PAN in the albumin-treated group (C) and the orosomucoid-treated group (A).

SDS–PAGE and western blot
The presence of orosomucoid in the rat plasma was checked by SDS–PAGE followed by immunoblotting. Samples from five animals in each group (except groups B and F) were diluted 1 : 100 in water; 10 µl of each diluted sample was supplemented with Laemmli sample buffer [62.5 mM Tris–HCl, 10% (v/v) glycerol, 2.5% (w/v) SDS, pH 6.8] with 100 mM dithiothreitol for 5 min at 95°C. The samples were separated on a 10% polyacrylamide gel, and then electrotransferred to a polyvinylidene fluoride membrane (Immobilon-PSQ, 0.20 µm, Millipore) in a Transfer-Blot SD-Dry Transfer Cell (Bio-Rad) at 1.6 mA/cm2 for 1 h. The transfer buffer used contained 48 mM Tris, 39 mM glycine, 1.3 mM SDS and 15% (v/v) methanol. After blotting, the membrane was incubated in blocking solution [phosphate-buffered saline (PBS) containing 5% (w/v) milk powder, 0.1% (v/v) Tween-20] for 2 h at room temperature, followed by incubation with an anti-human orosomucoid monoclonal antibody (mouse; Sigma-Aldrich Chemie Gmbh, Steinheim, Germany) diluted 1 : 10 000 in blocking solution. The membrane was washed three times for 5 min with PBS-T [PBS containing 0.1% (v/v) Tween-20] and incubated with secondary antibody [goat anti-mouse coupled to horseradish peroxidase, 10 ng/ml in blocking solution] for 2 h at room temperature. Immune complexes were detected by enhanced chemiluminescence according to the recommendations of the manufacturer (SuperSignal®, West Pico, Pierce, Rockford, IL).

Morphological data and statistical analysis
We analysed glomerular capillaries from all rats of groups A and C by electron microscopy. A total of 587 capillaries in group A and 523 capillaries in group C were screened for podocyte morphology. The capillary profile was virtually divided into four sectors, each representing ~25% of the capillary area. Thus, for each capillary, we could estimate the percentage of podocytes with normal morphology and preserved slit diaphragms covering the basement membrane. The percentages of normal podocytes in capillaries from the two groups (A and C) were then compared.

Statistics
Results are presented as mean±SEM. For all parameters except podocyte morphology, differences among groups were assessed by one-way analysis of variance (ANOVA), with post-test pair-wise comparisons between groups according to the Tukey HSD test. The {chi}2 test was used to test differences in podocyte morphology. For all statistical tests, P ≤ 0.05 was considered significant.



   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Perfusion pressure and glomerular filtration rate
Perfusion pressure and GFR (mean values±SEM) during cIPK for the six groups of rats are shown in Table 1. The average GFR of the control rats (group E) was 0.076±0.008 ml/min/g body weight. PAN decreased the GFR to 30% of this value in groups C and D. However, in the group treated with low dose orosomucoid (group A), the GFR was twice as high as in groups C and D (P<0.05). The GFR of the rats of group B (high dose orosomucoid) was not statistically different from that of the control group (E). There was no significant difference between GFR of groups E (0.076±0.008 ml/min/g body weight) and F (0.101±0.008 ml/min/g body weight).


View this table:
[in this window]
[in a new window]
  		Table 1. Arterial pressure (AP) and glomerular filtration rate (GFR) measured as [51Cr]EDTA plasma clearance in control and PAN-treated rats

 
The fractional clearance for albumin, {theta}albumin
The {theta}albumin in the control group E was 0.0025±0.0007. In rats given saline and orosomucoid (group F), {theta}albumin was 0.0013±0.0003. A 10-fold increase in {theta}albumin was induced by the administration of PAN in the rats treated with albumin (group C) or saline (group D). Compared with rats in groups C and D, the group treated with 20 mg/100 g/day of orosomucoid (group A) had a significantly lower {theta}albumin, 0.0137±0.0012, P<0.01. Moreover, treating the nephrotic rats of group B with a high dose of orosomucoid resulted in an albumin filtration that was not different from the controls (group E and F), {theta}albumin 0.0015±0.0003 (see Figure 1). The difference between {theta}albumin of groups E and F was not significant.


Figure 1
View larger version (15K):
[in this window]
[in a new window]
  		Fig. 1. Fractional clearance ({theta}) ±SEM for albumin in PAN + low dose orosomucoid-treated rats (group A, 20 mg/100 g body weight), PAN + high dose orosomucoid-treated rats (group B, 60 mg/100 g body weight), PAN + albumin-treated rats (C), PAN + saline-treated rats (D), saline + saline-treated rats (E) and saline + orosomucoid-treated rats (group F, 20 mg/100 g body weight). There was a difference between the control and all the nephrotic groups (groups E and F vs groups A, C and D, P<0.001) except for that with high dose orosomucoid (group B vs group E or F, NS). Group A had lower {theta}albumin than groups C and D, P<0.01.

 
The fractional clearance for Ficoll35.5 Å
Ficoll with the same size as albumin had a fractional clearance of 0.082±0.023 in control rats (group E), and similar values were found for all experimental groups (A–F). The nephrotic animals showed higher U/P ratios for the larger Ficolls, as previously described [5].

The heterogeneous charged fibre model
In this model, charged fibres account for both size and charge interactions. The unrestricted exchange area over diffusion distance, A0/{Delta}x, was 213 331 cm [95% confidence interval (CI) 171 626–255 036 cm] in the control group (E), while in the groups treated with PAN + albumin (group C) and PAN + saline (group D) it decreased to 880 cm (95% CI 810–950 cm) and 720 cm (95% CI 640–800 cm), respectively. For the nephrotic rats treated with low dose orosomucoid (group A), A0/{Delta}x was 4270 cm (95% CI 3800–4700 cm), significantly different from that of groups C and D. In the group treated with high dose orosomucoid (B), A0/{Delta}x was 52 332 cm (95% CI 5500–100 000 cm) (see Figure 2).


Figure 2
View larger version (18K):
[in this window]
[in a new window]
  		Fig. 2. The unrestricted exchange area over diffusion distance (A0/{Delta}x) ±SEM in PAN + low dose orosomucoid-treated rats (group A), PAN + high dose orosomucoid-treated rats (group B), PAN + albumin-treated rats (C), PAN + saline-treated rats (D), saline + saline-treated rats (E) and saline + orosomucoid-treated rats (F). This parameter decreased dramatically in the PAN-treated rats, P<0.01, but it was improved by orosomucoid, P<0.01.

 
The relative fibre volume, {Phi}, was 7.0% (95% CI 6.6–7.6%) in the control group (E) and 6.1% (95% CI 5.9–6.4%) in group F. The PAN administration decreased {Phi} to 3.5% (95% CI 3.4–3.5%) in group C and to 3.4% (95% CI 3.4–3.5%) in group D. Treatment with low dose orosomucoid (group A) resulted in some preservation of the fibre volume, as {Phi} was 4.5% (95% CI 4.4–4.6%) in this group. For group B, {Phi} was 5.3% (95% CI 4.2–6.4%), i.e. similar to that of the controls (group E) (see Figure 3).


Figure 3
View larger version (12K):
[in this window]
[in a new window]
  		Fig. 3. The charged fibre volume fraction, {phi}, in PAN + low dose orosomucoid-treated rats (group A), PAN + high dose orosomucoid-treated rats (group B), PAN + albumin-treated rats (C), PAN + saline-treated rats (D), saline + saline-treated rats (E) and saline + orosomucoid-treated rats (F). This parameter decreased in the PAN-treated rats, groups C and D, P<0.001, but it was improved by orosomucoid, groups A and B P<0.01.

 
The fibre radius was set to 5.0 Å for all groups. The fibre surface charge density was –0.2 C/m and albumin had a surface charge density of –0.022 C/m. With these values, we obtained a good fitting of the modelled and the 228 measured data pairs (Ficoll U/P ratios vs Stokes–Einstein radius). The statistical analysis was performed using the average values for each of the four groups.

SDS–PAGE, western blotting and immunodetection
The presence of human orosomucoid in plasma samples from rats of groups A, C, D and E was assessed by SDS–PAGE followed by detection with an anti-human orosomucoid monoclonal antibody. Bands close to 42 kDa were detected with the mouse anti-human orosomucoid antibody. Figure 4 shows a representative gel for three animals from each group. The primary antibodies cross-reacted with the intrinsic rat orosomucoid existent in the plasma samples. For this reason, we were unable to quantify exactly the human fraction of orosomucoid. Using the cross-reactivity with the rat orosomucoid, it was possible to determine that the plasma levels of rat orosomucoid in the PAN-treated groups C and D were lower than in the control group E. Moreover, the total orosomucoid concentration was estimated to be between 0.1 and 0.3 g/l in group A.


Figure 4
View larger version (34K):
[in this window]
[in a new window]
  		Fig. 4. Identification of orosomucoid in rat plasma samples in a representative gel from 12 animals (A1–A3, C2–C4, D1–D3 and E1–E3). After SDS–PAGE on a 10% gel, the glycoprotein was blotted and detected by an anti-orosomucoid monoclonal antibody. The positions of molecular mass standards (Precision Plus Protein Standards, Bio-Rad Laboratories) are indicated to the left.

 
Podocyte morphology
Normal morphology of the glomerular capillary wall was observed in the control group E (see Figure 5A) while the other three groups showed architectural alterations of the glomerular epithelial cells. Flattening and detachment of the podocytes were present in groups A, C and D (see Figure 5C and D). However, in group A, areas with preserved slit diaphragms and normal podocyte morphology were also present (see Figure 5B). Sections from the groups treated with orosomucoid (group A) and albumin (group C) were compared in detail. In group A, 587 glomerular capillaries from 52 glomeruli (6–7 glomeruli from each of the eight rats) were examined and 523 from 39 glomeruli in group C (6–7 glomeruli from each of the eight rats). In kidney sections from rats treated with orosomucoid (group A), we found 25–50% morphologically normal podocytes in 78 capillaries (i.e. 13.3% of the capillaries), and <25% normal podocytes in the remaining 509 capillaries. In kidneys from the albumin-treated rats (group C), 96.4% of the glomerular capillaries had <25% preserved podocytes, i.e. more severe damage than in group A (P<0.001) (see Figure 6).


Figure 5
View larger version (159K):
[in this window]
[in a new window]
  		Fig. 5. (A) Electron micrograph from a control rat (group E) showing the normal architecture of the glomerular capillary wall. CL = capillary lumen; EC = endothelial cells; BM = basement membrane; P = podocyte; US = urinary space. In vitro fixation with Karnovsky fixative, magnification x85 000. (B) Electron micrograph of a glomerular capillary from a nephrotic rat treated with 20 mg/kg of orosomucoid, (group A) morphologically normal podocytes with preserved slit diaphragms are seen. In vitro fixation with Karnovsky fixative, magnification x31 500. (C) Electron micrograph of a glomerular capillary from a nephrotic rat treated with 20 mg/kg of albumin (group C); flattened giant podocytes and the absence of slit diaphragms are observed. In vitro fixation with Karnovsky fixative, magnification x21 500. (D) Electron micrograph of a glomerular capillary from a nephrotic rat treated with saline (group D); unorganized podocyte morphology, flattened podocytes and the absence of slit diaphragms are observed. In vitro fixation with Karnovsky fixative, magnification x15 000.

 

Figure 6
View larger version (9K):
[in this window]
[in a new window]
  		Fig. 6. The podocyte morphology was improved in the orosomucoid-treated group compared with the albumin-treated group. The bars represent the percentage of glomerular capillaries with 25–50% morphologically normal podocytes in PAN + orosomucoid-treated rats (group A) and in PAN + albumin-treated rats (group C).

 


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
PAN caused extensive damage to the glomerular barrier as demonstrated by morphological and functional measurements. The purpose of this study was to test the hypothesis that the acute phase protein orosomucoid could ameliorate the nephrotic effect of PAN. Indeed, giving orosomucoid to rats with PAN nephrosis improved the GFR and the clearance of albumin in a dose-dependent manner. In fact, six out of eight rats receiving the high orosomucoid dose were completely cured of their nephrotic syndrome. In comparison, treatment with albumin did not seem to have any beneficial effect. The low dose of orosomucoid partially restored normal glomerular function and improved the morphological parameters. Muchitsch et al. [12] used the dose of 60 mg/100 g, which is the same as we used for group B, but the beneficial effects were greater in the present study. This is probably due to differences in administration; they started treatment at day 5, while daily i.p. injections were initiated at day 1 in the present study. In functional terms, PAN markedly reduced the charged fibre volume fraction of the glomerular barrier, an effect that was prevented by orosomucoid, but not by administration of saline or albumin. Western blot analysis showed that the plasma levels of orosomucoid were markedly reduced in the nephrotic rats of groups C and D. i.p. injections of orosomucoid normalized these levels.

By which mechanism does orosomucoid ameliorate the effect of PAN? Orosomucoid is a classical acute phase protein showing a 3- to 4-fold increase during inflammation or tissue damage. Its anti-inflammatory and immunomodulatory roles are connected to the strong antineutrophil and anticomplement activity. It inhibits proliferation, invasion and metastasis of cancer cells and interleukin-2 secretion by lymphocytes. Orosomucoid seems to protect against reactive oxygen species by binding free radicals rather than by inhibition of neutrophil activation. Several studies suggest that the induction of PAN nephrosis is correlated with the appearance of highly reactive oxygen species [19]. Thus, orosomucoid might act as a scavenger for reactive oxygen species formation induced by PAN and thereby diminish the structural damage of podocytes. In previous work, we found that orosomucoid is required for maintaining normal capillary permselectivity in skeletal muscle and in glomerular capillaries as well.

All of the mechanisms cited above could play a role in reducing the renal damage induced by PAN. Moreover, orosomucoid may interact with the endothelial surface layer (ESL) of the capillaries, which per se may increase the charge density of the luminal surface of the vessel wall. The endothelial cells themselves are able to synthesize orosomucoid [20] and they respond to stimulation by the protein [21].

It has been reported that removal of the sialic acid-rich surface coat of the podocytes induces changes of the cell architecture: loss of foot processes and filtration slits, and formation of junctional complexes between many of the podocytes, followed by the retraction of the processes towards the cell body. This seems to occur both in differentiating glomeruli and in certain glomerular diseases. Indeed, PAN has been reported to decrease the total glomerular sialic acid content and the sialic acid content of podocalyxin [22]. The filtration slit loss that appears in PAN nephrosis coincides with a reduction of the GFR. Indeed, filtration slits are considered to be the main resistance components for hydraulic flux across the glomerular capillary wall. Previous studies have shown that altered podocyte morphology is related to a decrease in hydraulic conductance and GFR [23]. In the present study, orosomucoid partially preserved the podocyte morphology, thus assuring a somewhat better glomerular hydraulic conductance and GFR. Possible interactions between orosomucoid and the podocytes have not yet been studied, but such experiments seem to be worthwhile.

The ESL of the glomerular capillaries can be viewed as a three-dimensional fibrous network of glycoproteins reinforced by plasma proteins. However, the relative importance of the ESL for the normal glomerular permselectivity has hitherto been neglected. Apart from the podocytes and the basement membrane, ESL may be important for size and charge selectivity. Thus, for analysis of this complex barrier, the heterogeneous charged fibre model is highly appropriate since it takes both solute size and charge into account. A Ficoll over an albumin clearance ratio of 40–50 during control is obvious evidence for charge selectivity, irrespective of theoretical modelling. PAN reduced this ratio one order of magnitude, indicating a decrease in charge selectivity. Theoretical analysis, however, did confirm and extend these observations, and orosomucoid was found to improve the density of charged fibres of the capillary wall, and to some extent increase the exchange area parameter, A0/{triangleup}x. Note that the increased fraction of charged fibres reflects improvement in both size and charge selectivity.

This study reveals important benefits of the heterogeneous charged fibre model compared with less advanced theories where size and charge interactions are treated as separate entities. Thus, orosomucoid was found to increase the relative charged fibre density, which will affect both size and charge selectivity, compared with the treatment with albumin or saline. Please note that simply looking at a clearance ratio between a neutral and a charged solute (e.g. Ficoll35.5 Å and albumin) may be misleading. In situations with important alterations of A0/{Delta}x, the volume of charged fibres may alter the U/P vs aSE curve. Indeed, such a pronounced reduction of A0/{Delta}x was found in three of the PAN-treated groups (A, C and D), resulting in low U/P ratios for low molecular weight Ficolls. Reduced size selectivity increased the U/P for large Ficolls, while these effects cancelled each other out for Ficoll35.5 Å. Thus, based on the {theta} for Ficoll35.5 Å, it might incorrectly be concluded that the size barrier was not affected by PAN. Hence, the more advanced charged fibre model gives us new and useful insights into the functional details of the glomerular barrier.

We conclude that orosomucoid has the ability to restore the glomerular damage induced by PAN completely in terms of both functional and morphological parameters. The exact molecular mechanism for this action remains to be elucidated, but we propose several potential sites of action including direct effects on the podocytes and/or the endothelial cells. Clinically, there are few therapies for patients with nephrotic syndrome, and those that exist have severe side effects. Therefore, this study opens up new avenues for treatment with orosomucoid or with drugs having similar actions on glomerular cells.



   Acknowledgments
 
The authors are grateful for the advice and support of Professor Bengt R. Johansson at the EM Unit of the Institute of Anatomy and Cell Biology, Göteborg University. The technical assistance of Mrs IngaBritt Persson and Mrs Martina Bassen is gratefully acknowledged. This study was supported by the Swedish Medical Research Council, Grant 9898; the Knut and Alice Wallenberg Research Foundation; the IngaBritt and Arne Lundberg Research Foundation; the Göteborg Medical Society; the National Association for Kidney Diseases; and Sahlgrenska University Hospital, Grant LUA-S11733.

Conflict of interest statement. None declared.



   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Kanwar YS, Rosenzweig LJ. Altered glomerular permeability as a result of focal detachment of the visceral epithelium. Kidney Int 1982; 21: 565–574[Medline]
  2. Suzuki T, Takemura H, Noiri E et al. Puromycin aminonucleoside induces apoptosis and increases HNE in cultured glomerular epithelial cells. Free Radicals Biol Med 2001; 31: 615–623[CrossRef][Medline]
  3. Diamond JR, Pesek I. Glomerular tumor necrosis factor and interleukin 1 during acute aminonulceoside nephrosis. An immunohistochemical study. Lab Invest 1991; 64: 21–28[Medline]
  4. Olson JL, Rennke HG, Venkatachalam MA. Alterations in the charge and size selectivity barrier of the glomerular filter in aminonucleoside nephrosis in rats. Lab Invest 1981; 44: 271–279[Web of Science][Medline]
  5. Hjalmarsson C, Ohlson M, Haraldsson B. Puromycin aminonucleoside damages the glomerular size barrier with minimal effects on charge density. Am J Physiol 2001; 281: F503–F512
  6. Osicka TM, Hankin AR, Comper WD. Puromycin aminonucleoside nephrosis results in a marked increase in fractional clearance of albumin. Am J Physiol 1999; 277: F139–F145[Medline]
  7. Greive KA, Nikolic-Paterson DJ, Guimaraes MA et al. Glomerular permselectivity factors are not responsible for the increase in fractional clearance of albumin in rat glomerulonephritis. Am J Pathol 2001; 159: 1159–1170[Abstract/Free Full Text]
  8. Zhai XY, Nielsen R, Birn H et al. Cubilin- and megalin-mediated uptake of albumin in cultured proximal tubule cells of opossum kidney. Kidney Int 2000; 58: 1523–1533[CrossRef][Web of Science][Medline]
  9. Haraldsson B, Rippe B. Orosomucoid as one of the serum components contributing to normal capillary permselectivity in rat skeletal muscle. Acta Physiol Scand 1987; 129: 127–135[Web of Science][Medline]
  10. Curry FE, Rutledge JC, Lenz JF. Modulation of microvessel wall charge by plasma glycoprotein orosomucoid. Am J Physiol 1989; 257: H1354–H1359[Medline]
  11. Johnsson E, Haraldsson B. Addition of purified orosomucoid preserves the glomerular permeability for albumin in isolated perfused rat kidneys. Acta Physiol Scand 1993; 147: 1–8[Web of Science][Medline]
  12. Muchitsch E, Pichler L, Schwarz HP, Ulrich W. Effects of human alpha-1-acid glycoprotein on aminonucleoside-induced minimal change nephrosis in rats. Nephron 1999; 81: 194–199[Medline]
  13. Jeansson M, Haraldsson B. Glomerular size and charge selectivity in the mouse after exposure to glucosaminoglycan-degrading enzymes. J Am Soc Nephrol 2003; 14: 1756–1765[Abstract/Free Full Text]
  14. Ogston AG. The spaces in a uniform random suspension of fibers. Trans Faraday Soc 1958; 54: 1754–1757[CrossRef]
  15. Johnson EM, Deen WM. Electrostatic effects on the equilibrium partitioning of spherical colloids in random fibrous media. J Colloid Interface Sci 1996; 178: 749–756
  16. Anderson JL, Malone DM. Mechanism of osmotic flow in porous membranes. Biophys J 1974; 14: 957–982[Medline]
  17. Curry FE, Michel CC. A fiber matrix model of capillary permeability. Microvasc Res 1980; 20: 96–99[CrossRef][Web of Science][Medline]
  18. Rippe B, Haraldsson B. Transport of macromolecules across microvascular walls: the two-pore theory. Physiol Rev 1994; 74: 163–219[Abstract/Free Full Text]
  19. Nakakura H, Ashida A, Hirano K, Tamai H. Oxidative stress in a rat model of nephrosis can be quantified by electron spin resonance. Pediatr Nephrol 2004; 19: 266–70[Medline]
  20. Sörensson J, Matejka GL, Ohlson M, Haraldsson B. Human endothelial cells produce orosomucoid, an important component of the capillary barrier. Am J Physiol 1999; 276: H530–H534[Medline]
  21. Sörensson J, Ohlson M, Björnson A, Haraldsson B. Orosomucoid has a cAMP-dependent effect on human endothelial cells and inhibits the action of histamine. Am J Physiol 2000; 278: H1725–H1731
  22. Kerjaschki D, Vernillo AT, Farquhar MG. Reduced sialylation of podocalyxin—the major sialoprotein of the rat kidney glomerulus—in aminonucleoside nephrosis. Am J Pathol 1985; 118: 343–349[Abstract]
  23. Daniels BS. The role of the glomerular epithelial cell in the maintenance of the glomerular filtration barrier. Am J Nephrol 1993; 13: 318–323[Web of Science][Medline]
Received for publication: 12.10.04
Accepted in revised form: 13.12.05


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Physiol. Rev.Home page
B. Haraldsson, J. Nystrom, and W. M. Deen
Properties of the Glomerular Barrier and Mechanisms of Proteinuria
Physiol Rev, April 1, 2008; 88(2): 451 - 487.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
21/5/1223    most recent
gfk050v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (2)
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Hjalmarsson, C.
Right arrow Articles by Haraldsson, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hjalmarsson, C.
Right arrow Articles by Haraldsson, B.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?