Glomerular charge selectivity is impaired in hypertensive nephropathy

Rolf E. F. Christiansen1, Olav Tenstad2, Sabine Leh3 and Bjarne M. Iversen1

1Renal Research Group, Institute of Medicine, 2Department of Physiology, University of Bergen and 3Department of Pathology, Haukeland University Hospital, Bergen, Norway

Correspondence and offprint requests to: Rolf E. F. Christiansen, Institute of Medicine, N-5021 Haukeland University Hospital, Bergen, Norway. Email: rolf.christiansen{at}meda.uib.no



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. In hypertensive nephropathy the morphological changes and albuminuria seem to start in the inner cortex but the mechanism of proteinuria is unknown. We tested the hypothesis of reduced glomerular charge selectivity in the juxtamedullary cortex of old spontaneously hypertensive rats (SHR) as a cause of proteinuria in rats with hypertensive nephropathy.

Methods. The glomerular charge barrier was evaluated in 80-week-old SHR and age-matched normotensive Wistar–Kyoto rats (WKY) by measuring the glomerular clearance of radiolabelled cationic and anionic chymotrypsinogen (Chym and aChym, MW 25 000) accumulated by the proximal tubular cells in the outer, middle and inner cortex following intravenous injection. The glomerular filtration rates (GFR) in the cortical zones were obtained by aprotinin (MW 6500) and the sieving coefficient ({theta}) of the protein tracers calculated as their glomerular clearance/GFR.

Results. The {theta} aChym was similar in SHR and WKY except in the inner cortex where it was 35% higher in SHR (0.65±0.05) than in WKY (0.48±0.03) (P = 0.01). The ratio of {theta} aChym to {theta} Chym was increased from 0.55±0.06 to 0.77±0.05 (P<0.02) in the inner cortex of SHR, whereas {theta} Chym remained the same in all cortical zones. Finally, the percentage of glomeruli with adsorption droplets in podocytes quantified by light microscopy was higher in the inner than the outer cortex of SHR (P<0.05).

Conclusions. The study supports the theory of a functioning glomerular charge barrier. An increased relative clearance of aChym in the inner cortex of SHR indicates impairment of the charge barrier, which, at least in part, could explain the increased protein excretion in SHR with hypertensive nephropathy.

Keywords: glomerular charge selectivity; hypertensive nephropathy; proteinuria



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Hypertensive renal damage is a well known clinical entity and a common cause of end-stage renal failure in humans. Clinical features are persistent hypertension lasting for years without signs of renal involvement, eventually followed by proteinuria and declining glomerular filtration rate (GFR). Older people develop hypertensive renal disease more commonly than younger subjects.

The spontaneously hypertensive rat (SHR), which is recognized as a model of essential hypertension, develops hypertensive renal disease at high age and the morphological features are comparable with the findings in human kidneys exposed to long-lasting essential hypertension. The typical morphological findings are glomerulosclerosis, increased thickening of the vascular wall and interstitial fibrosis in the juxtamedullary cortex rendering the superficial parts of the renal cortex nearly normal [13].

During glomerular filtration, solutes are believed to be restricted in their passage into Bowman's space on basis of their size (size selectivity), shape (shape selectivity) and charge (charge selectivity). The property of glomerular selectivity to filtration of solutes is thought to be caused by functioning glomerular ‘pores’ and a net negative charge (negative charge density) in the glomerulus. The negative glomerular charge density is thought to repel anionic plasma proteins, and hence major serum proteins such as albumin are excluded from the tubular filtrate mainly by means of charge repulsion. The charge selective barrier has been documented in earlier work [4], where the filterability of anionic dextrans was studied demonstrating a lower fractional clearance of anionic compared with neutral or cationic dextrans of same molecular size. In recent years the validity of the classical model of glomerular charge selectivity has been questioned and the possibility of a physiological relatively high albumin clearance in conjunction with tubular retrieval has been proposed [5]. However, at the same time, experimental evidence supporting the concept of a glomerular charge barrier is newly presented [6].

In SHR the glomerular capillary pressure in the deep cortex is higher than in the superficial cortex, an observation that is different from the normotensive animals where the glomerular capillary pressure is similar in the deep and superficial renal cortex [3]. Increased glomerular capillary pressure is found in the deep cortex of 10-week-old SHR and increases with age [3]. Proteinuria usually develops at 30–40 weeks of age, and the increase in glomerular capillary pressure consequently precedes development of proteinuria and morphological changes in the deep cortex of SHR.

The present study was designed to investigate the role of the glomerular charge barrier on protein filtration in hypertensive renal disease in SHR. To accomplish this, chymotrypsinogen A (Sigma C 4879, MW 25 000, Stokes radius 21 Å), a cationic polypeptide filtered with a sieving coefficient ({theta}) of 0.85±0.02, was anionized, resulting in an unchanged hydrodynamic radius but a reduction of {theta} to 0.53±0.02 confirming the existence of a glomerular charge barrier. Measuring the clearance ratio of anionic to cationic chymotrypsinogen (aChym to Chym) and GFR in different cortical zones using aprotinin (Sigma A 4529, MW 6500) [7,8], we found a reduction of GFR and an attenuated glomerular charge barrier in the juxtamedullary cortex of old SHR.



   Subjects and methods
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Experiments were performed in 80-week-old animals, 13 male SHR (Møllegaard, Denmark) and matched with seven male Wistar–Kyoto rats (WKY) as controls. Three rats were kept in each cage. They were fed ordinary rat chow (Special diets services) containing 0.25% sodium, 0.66% potassium, 0.71% calcium and 14.7% crude protein and had free access to water.

The experiments were performed with the approval of the Norwegian State Board for Biological Experiments with Living Animals.

The animals were anaesthetized with intraperitoneal pentobarbital (50 mg/kg body weight) and were placed on a servocontrolled heating pad to keep body temperature stable at 37–38°C. Infusion of 5% bovine serum albumin in a 0.9% sodium chloride solution was given at a rate of 1.0 ml/100 g body weight per hour through a polyethylene catheter, which was inserted into the right jugular vein. The same catheter was used for bolus injection of radioactive tracers.

Haemodynamic measurements
A polyethylene catheter was inserted into the right carotid artery for measurement of arterial blood pressure and blood sampling. The arterial blood pressure was measured with a Hewlett-Packard pressure transducer connected to a Gould-4000 recorder. Urine was collected from the left urether and the bladder.

After surgery, the rats were allowed to recover for 30 min before the experiment commenced.

Tracers
Chymotrypsinogen was acetylated and thereby anionized by blocking its free amino groups [9]. The net molecular charge of aChym was confirmed by isoelectric focusing (IEF) and ionic exchange chromatography (Figure 1). The hydrodynamic radius of aChym was 21.3 Å and of Chym 21.0 Å and was measured by size exclusion chromatography using low molecular weight calibration kit proteins as standards (Superdex 75-10/30 HR; Amersham Biosciences). Aprotinin, aChym and Chym were each dissolved (1 mg/ml) in a 0.05 M Na-phosphate buffer at pH 7.5 and labelled with 131I or 125I, respectively, by using an IODO-GEN technique (Pierce Chemical Company, Rockford, IL, USA). Iodination did not alter the hydrodynamic radius of the probes or their isoelectric points as measured by size exclusion chromatography and IEF.



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Fig. 1. (A) Scanned image of a dried, IEF gel (native conditions) showing Coomassie blue staining of the pI markers (Pharmacia broad kit, pI 3.5–9.3) in lanes 1, 3, 5 and 7 and autoradiographic representation of 125I-anionized chymotrypsinogen (125I-aChym) in lane 6 (pI 3.5). Human serum albumin (125I-Alb) and chicken hen ovalbumin (125I-ovAlb) labelled in our laboratory showed isoelectric points close to 4.9 (lane 2) and 4.6 (lane 4) in agreement with known values for the native proteins. The pH gradient indicated on the ordinate is based on point-to-point linear regression analysis of the pI markers (r2 = 0.98). (B) Cation exchange chromatography of native (Chym) and anionized chymotrypsinogen (aChym) on a RESOURCE S 1 ml (Amersham Pharmacia Biotech) column at 1 ml/min using 50 mM HEPES buffer, pH 6.9, and a linear NaCl (0–500 mM) gradient from 1 to 21 min using a gradient pump (P2000; Thermo Separation Products, USA).

 
The principle of the method
The technique, based on cortical accumulation of iodinated polypeptides filtered in the glomeruli following intravenous injection, has been described in detail and validated by Tenstad et al. [7,8]. When a tracer protein is injected intravenously it is filtered in the glomeruli and taken up by the brush border of the proximal tubules. Endosomes carry the polypeptide to the lysosomes where it is broken down to amino acids, which are then returned to peritubular plasma with the free radiolabel. This process takes time and for aprotinin [7] and chymotrypsinogen the first free radiolabel (125I or 131I) is observed in the plasma ~20 and ~15 min after intravenous injection, respectively. In the meantime, all filtered protein is accumulated together with its radiolabel in the kidney cortex, only a minor part that is not reabsorbed being excreted in the urine. The total renal plasma clearance of the protein tracer, calculated as the total radioactivity of urine and the kidneys excised 5–10 min after tracer injection divided by the time-integrated plasma activity is therefore a direct measurement of tracer protein filtration in the glomeruli. Using protein probes like aprotinin and chymotrypsinogen that are nearly completely reabsorbed in the proximal tubules, measurements of local protein filtration in tissue samples from the outer, middle and inner cortex is possible. Since the filtration of the small polypeptide aprotinin (MW 6500) is not restricted by the glomerular barrier its accumulation clearance can be used to calculate GFR in the different cortical zones [7]. Furthermore, the ratio between the accumulation clearance of aChym and Chym provides a direct assessment of the charge barrier in the different cortical layers.

Clearance of aChym, Chym and measurement of zonal and total GFR
At zero time, 131I-labelled aprotinin (131I-Ap) and 125I-labelled anionic chymotrypsinogen (125I-aChym) or chymotrypsinogen (125I-Chym) in a volume of ~100 µl of saline was injected as a bolus into the right jugular vein. Arterial blood samples (0.1 ml) were taken at time 15 s, 1, 3, 5 and 7 min after the start of the experiment. A 0.1 ml volume of 5% bovine serum albumin was injected after each blood sample to compensate for the blood loss. Total and zonal GFR was obtained by measuring tubular uptake of 131I-Ap as described by Tenstad et al. [7,8]. Similarly, total and zonal clearance of 125I-aChym and 125I-Chym was obtained by dividing the total 125I activity in the kidney or tissue samples from the outer, middle and inner cortex by the time-integrated plasma concentration during the 7 min clearance period.

The blood samples were centrifuged in a Hettich Rotanta Centrifuge at 2700 r.p.m. for 5 min, and 20 µl of plasma was taken from each sample and radioactivity was counted in a gamma counter (Cobra 2; Auto Gamma) for 5 min. Ten microlitres of plasma was also taken from each sample for measurement of total protein concentration with a refractometer (American Optical) together with 15 µl of plasma from each sample for HPLC (Superdex 75-10/30 HR; Amersham Biosciences), performed to measure protein binding of the tracers.

At the end of the experiment, 1 ml of 2% Alcian blue was injected intra-arterially and the renal arteries were ligated. The kidneys were removed and frozen in isopenthane prechilled to –80°C.

The left kidney was counted in a gamma counter and thereafter placed in a constantly cooled dish containing isopenthane at ~–10°C. The kidney was first divided in three 3–4 mm transverse slices, sectors of these slices were cut using a scalpel, and then five tissue samples of similar thickness from the outer, middle and inner cortex were prepared from each of these sectors under the microscope at 16x magnification. The corticomedullary border was determined by the use of Alcian blue and by the position of the arcuate vessels.

Each piece of cortical tissue was put into pre-weighed counting tubes, reweighed and radioactivity was counted for 5 min. The urine was collected in a test tube and radioactivity counted.

Clearance of aChym and Chym was corrected for their protein binding of ~20%, as measured by HPLC in each experiment. Aprotinin clearance was corrected for its protein binding of ~5% and GFR was calculated by correcting for a glomerular Gibbs–Donnan distribution according to an empirical formula developed by Tenstad et al. [8]:

where P denotes the plasma protein concentration. Zonal GFR is the term given to the average GFR of five tissue samples in one zone.

Measurements of urinary protein excretion
Urine was collected for 24 h while the rats were in metabolic cages with free access to food and water during the collection period. Urinary protein and albumin concentration was measured by agarose gel electrophoresis on a Hydrasys (Sebia) semi-automated analyser. The normal rats excreted <20 mg of protein/24 h.

Light and electron microscopy
Kidneys from both WKY and SHR were fixed in 10% formalin and embedded in paraffin by standard procedures. Frontal paraffin sections were cut (4 µm) and stained with periodic acid-Schiff (PAS). All analyses were performed in a blinded manner. 100 glomeruli from the outer cortex and at least 70 glomeruli from the inner cortex were analysed regarding podocytes with adsorption droplets (magnification x400) and development of synechiae/adherences. The degree of tubulointerstitial damage was assessed using a semi-quantitative scoring system (0–4). In PAS-stained sections, at least 20 fields from the inner or outer cortex were randomly sampled. At a magnification of x200 they were graded as follows: grade 0, no changes; grade 1, lesions involving <25% of the area; grade 2, lesions involving 25–50% of the area; grade 3, lesions involving >50% of the area; grade 4 involving (almost) the entire area [10]. Tubulointerstitial damage was defined as tubular dilatation, atrophy, cast formation or interstitial expansion with inflammation or fibrosis.

For ultrastructural investigation pieces of superficial and deep cortical tissue from both WKY and SHR were fixed in McDowells solution. The pieces were post-fixed in 1% osmium tetroxide, dehydrated and embedded in Epon 812. Semi-thin sections were stained with toluidine blue. Ultra-thin sections were stained with uranylacetate and lead citrate and studied in a Philips 400 electron microscope.

Statistical analysis
The results are presented as means±SE. Differences between groups were assessed by non-parametric ANOVA on Ranks followed by one-way ANOVA in groups to be compared. Where significant differences were found, the groups were compared by unpaired t-test. Differences between cortical layers within the same strain in the histological analysis were assessed by paired comparisons. A value of P<0.05 was accepted as statistically significant.



   Results
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
As shown in Table 1, the body weight of the SHR was lower than in WKY (P<0.01), and similarly the kidney weights were lower in SHR than in WKY (P<0.05). Mean arterial blood pressure (MAP) was higher in SHR (167±4 mmHg) than in WKY (101±5 mmHg, P<0.01). Urinary protein excretion was increased in SHR (54.3±9.0 mg/24 h) vs WKY (17.2±0.7 mg/24 h, P<0.01) consisting mainly of albumin (65.4±1.0 vs 21.9±1.9%, P<0.01). In spite of the difference in protein excretion, total serum proteins were not different between the two strains (P = 0.9).


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Table 1. MAP, proteinuria, albuminuria, plasma protein concentration, body and left kidney weight, and aChym, Chym and aprotinin excretion

 
The urinary excretion of the protein probes was similar in the two strains of rats. As shown in Table 1, urinary excretion of 131I-Ap was negligible in SHR and WKY (P = 0.9), excretion of 125I-aChym was 9–10% in both SHR and WKY (P = 0.9) while excretion of 125I-Chym was <1% in SHR. The protein binding of the probes was not significantly different in SHR and WKY: 131I-Ap SHR = 5.3±0.2% vs 131I-Ap WKY = 4.7±0.4% (P = 0.2), 125I-aChym SHR = 21.5±2.4% vs 125I-aChym WKY = 22.1±2.0% (P = 0.9) and 125I-Chym SHR = 19.3±0.8%. The high molecular weight protein-bound fraction of the tracers in plasma, found with HPLC, was not detectable in urine.

GFR was calculated from the aprotinin clearance. As shown in Figure 2, total GFR was numerically 17% lower in SHR (0.69±0.07) than in WKY (0.83± 0.08 ml min–1 g–1), although not a statistically significant finding (P = 0.2). GFR in different layers of the renal cortex were, however, different in WKY and SHR. Outer cortex GFR was numerically 11% lower in SHR (1.09±0.11) than in WKY (1.22±0.11 ml min–1 g–1), but failed to reach significance (P = 0.4). GFR in the middle part of the renal cortex was 31% lower in SHR (0.74±0.07) than in WKY (1.07±0.09 ml min–1 g–1) (P<0.05), while GFR of the inner part of the renal cortex was 55% lower in SHR (0.33±0.04) than in WKY (0.73±0.09 ml min–1 g–1) (P<0.01).



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Fig. 2. Total and outer (Oc), middle (Mc) and inner (Ic) cortical glomerular filtration rate (GFR) and clearance of aChym (CaChym) in WKY and SHR. GFR values are represented by filled circles and CaChym values are represented by open circles.*P<0.05, difference in GFR in WKY vs SHR. **P<0.01, difference in GFR in WKY vs SHR. +P<0.01, difference in clearance of aChym in WKY vs SHR.

 
Clearance of aChym (CaChym) for the whole kidney and outer cortex was not significantly different between the strains (P = 0.1), in contrast, CaChym in middle cortex was 30% lower in SHR (0.32±0.02) than in WKY (0.46±0.03 ml min–1 g–1) (P<0.01) and CaChym in inner cortex was 40% lower in SHR (0.21±0.02) than in WKY (0.35±0.03 ml min–1 g–1) (P<0.01).

The sieving coefficient of aChym ({theta} aChym) and Chym ({theta} Chym) was calculated as their clearances divided by GFR as measured by the aprotinin method. As shown in Figure 3A, {theta} aChym was similar in SHR and WKY for the whole kidney and in the outer and middle cortical zones, while {theta} aChym in the inner cortex was 35% higher in SHR (0.65±0.05) than in WKY (0.48±0.03) (P = 0.01). The {theta} aChym of ~0.85 was similar in all cortical zones in SHR (P = 0.36), and the ratio of {theta} aChym to {theta} aChym was increased from 0.55±0.06 to 0.77±0.05 (P<0.02) in the inner cortex of SHR as shown in Figure 3B.



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Fig. 3. (A) Total and outer (Oc), middle (Mc) and inner (Ic) cortical sieving coefficient of aChym ({theta} aChym) in WKY and SHR. SHR values represented by filled circles and WKY values represented by filled triangles. *P = 0.01, difference WKY vs SHR. (B) Total and outer (Oc), middle (Mc) and inner (Ic) cortical ratio of sieving coefficient of aChym to Chym ({theta} aChym/{theta} Chym) in SHR. *P<0.02, difference between groups.

 
The most striking change in light microscopic examination was segmental sclerosis of the inner cortex glomeruli in SHR (Figure 4). Histomorphometric quantitation showed more pronounced morphological changes in the inner than in the outer cortex in SHR, whereas there were scant morphological changes in WKY and the difference between the outer and inner cortex disappeared (Table 2). The percentage of glomeruli with adsorption droplets in podocytes was higher in inner cortex than in outer cortex in SHR (22.0±1.2 vs 8.5±2.5%, P<0.05). WKY showed only few glomeruli with adsorption droplets in podocytes in both the inner and outer cortex (5.2±4.5 and 0.5±0.3, P>0.05). The same pattern applied to percentage glomeruli with adherences. Like the glomerular changes, tubulointerstitial damage was marked in the inner cortex in SHR (tubulointerstitial damage index 0.7±0.2 inner cortex vs 0.3±0.1 outer cortex, P<0.05). There was no significant difference between the outer and inner cortex in WKY. We did not perform systematic morphometric investigations of the renal vasculature. However, the arteries in SHR showed marked hypertrophy of the muscular wall compared with WKY, and in one midcortical interlobular artery fibrinoid necrosis was seen.



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Fig. 4. SHR inner cortex: glomerulus with segmental sclerosis. There is an area with obliterated capillaries, endothelial cells with vacuolated cytoplasm, hyalinous lesions and deposition of extracellular matrix in Bowman's space (semi-thin section, toluidine blue, x500).

 

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Table 2. Glomerular and tubulointerstitial measurements

 
Ultrastructural investigation of the inner cortex glomeruli from SHR, normal by light microscopy, showed podocytes with larger nuclei compared with WKY. There was patchy fusion of foot processes but this change was also observed in WKY. Some podocytes contained partly membrane-bound electron dense material corresponding to adsorption droplets. Glomeruli of the outer cortex showed no abnormalities compared with glomeruli from WKY. Ultrastructural investigation of glomeruli with segmental sclerosis showed collapsed capillary loops with a thickened and wrinkled basement membrane. There were endothelial cells with vacuolated cytoplasm. Podocytes showed diffuse fusion of foot processes associated with condensation of cytoplasm near the basement membrane. The obliterated capillary loops were embedded in a newly formed extracellular matrix and there was a large adherence between the area with segmental sclerosis and the Bowman's capsule. Podocytes in the neighbourhood of the lesion showed attenuation of the cell body with formation of pseudocysts (Figure 5). Diffuse fusion of foot processes was also seen in some capillary loops in unaffected parts of glomeruli with segmental sclerosis (Figure 6). No quantitative estimation of foot process fusion was performed in SHR and WKY.



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Fig. 5. SHR inner cortex: a podocyte next to an area with segmental sclerosis with pseudocysts (p); open capillary (arrow) (TEM, x17 000).

 


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Fig. 6. SHR inner cortex: capillary with diffuse fusion of foot processes and condensation of cytoplasm near the basement membrane (TEM, x8600).

 


   Discussion
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
The main observation that emerges from the present study is that the increased urinary protein excretion in hypertensive nephropathy in SHR is due, at least partially, to impairment of the glomerular charge barrier in glomeruli from the juxtamedullary cortex. This is shown by an increased sieving coefficient of aChym coexistent with an unchanged sieving coefficient of Chym in this part of the cortex in SHR. In the normotensive animals, the sieving coefficient for aChym was similar in all cortical zones.

SHR is a commonly accepted experimental model for essential hypertension in man and the morphological picture of human hypertensive renal disease is comparable with renal histological changes observed in old SHR. Focal and segmental glomerulosclerosis associated with tubular atrophy and interstitial fibrosis fits well with the morphological picture in man, described as decompensated benign nephrosclerosis by Ratschek et al. [11]. As in SHR, the glomerular and tubulointerstitial changes begin and are most severe in the juxtamedullary cortex.

In SHR proteinuria usually develops at the age of 30–40 weeks and GFR starts to decline at higher age [3], although in the present study reduced GFR was only found in the middle and inner renal cortex. Other investigators have demonstrated that the urinary protein excretion in old SHR originated from juxtamedullary glomeruli [1,2], protein casts in tubuli were most abundant in juxtamedullary nephrons and no protein excretion was found in superficial glomeruli of male SHR using a micropuncture technique. The observations correlate with the well known morphological changes in SHR where glomerulosclerosis, increased interstitial cell infiltration and vascular wall thickness were found in deep cortex, while the more superficial part of the cortex showed minor changes [13].

The glomerular capillary filtration barrier consists of the glomerular basement membrane (GBM) with the fenestrated endothelium of the capillaries lining the inside while the visceral epithelium covers the outside. The epithelial cells are in contact with the GBM through the numerous epithelial podocyte foot processes. Proteoglycans, especially heparan sulphate proteoglycan (HSPG), in the form of perlecan and agrin are molecular constituents of the GBM which constitute parts of the negative glomerular charge density and are produced mainly in the epithelial cells [12]. There are also other negative charges in the GBM and important negatively charged glycoproteins on the endothelium, podocytes, podocyte foot processes and slit diaphragms. The precise localization of the anionic sites which are most important in conferring charge selectivity to the glomerular barrier is disputable [13]. Evidence suggest that anionic sites in the GBM are not crucial for the charge selectivity since knockout mice without glomerular basal membrane HSPG do not have proteinuria [14] and clearance values from studies of isolated basal membranes do not indicate a functioning charge barrier [15].

Although most studies show that the glomerulus has a net negative charge density, the hypothesis of a functioning glomerular charge barrier as postulated in earlier studies is debated [16] and a model of a negligible glomerular charge barrier with a normally relatively high albumin clearance together with tubular albumin reabsorption and degradation is presented [5]. There are, however, several problems in accepting a crucial role of tubular reabsorption and cellular processing in the control of albuminuria. Dysfunctions of the receptors for tubular albumin uptake (cubulin and megalin receptors) for example, are not followed by heavy proteinuria and acute tubular necrosis is not associated with marked albuminuria [5]. In addition, evidence supporting the classical theory of a glomerular charge barrier normally restricting the passage of albumin has been recently published [6]. Sorensson et al. [6] studying different proteins and Ficoll changed the ionic strength of a perfusate to alter glomerular charge interactions concluding that glomerular negative charges are important determinants of the glomerular permselectivity.

Methodological considerations
In the present study we have examined the glomerular charge barrier in different cortical zones of SHR and WKY by a new approach. The use of aprotinin, aChym and Chym is methodologically a technique where glomerular clearance can be measured in the outer, middle and inner cortex. It should be noted that even though the single glomerular filtration rate (SNGFR) is highest in the larger glomeruli of the deep cortex, the GFR per gram wet weight as estimated by the aprotinin method is lower due to the fewer number of glomeruli in the deep cortex than in the superficial cortex.

Chym is filtered with a {theta} of 0.85, whereas aChym with a practically identical hydrodynamic radius has a restricted clearance ({theta} = 0.53), which is evidence of an existing glomerular charge barrier. Since the fractional Chym clearance is normally high ({theta} = 0.85), this marker has limited capability to detect a potential charge- independent increase in filterability. Nevertheless, the clearance ratio between aChym and Chym provides a direct assessment of the charge barrier in the different cortical layers and the finding of a clear increase in the aChym/Chym clearance ratio only in the inner cortex of SHR without any change in Chym sieving suggests that the charge barrier in the juxtamedullary cortex of SHR is impaired.

The circulatory pattern in the juxtamedullary cortex may interfere with the measurements of CaChym and CChym. Increasing the time of exposure of the tracers to the glomerular filter would probably increase the clearance values due to increased diffusive transport through the glomerular membrane. However, in this part of the renal cortex in SHR, the glomeruli are enlarged [3], SNGFR are probably increased and the glomeruli hyperperfused. Taking this into account, the values for CaChym and CChym may be underestimated rather than overestimated in the juxtamedullary cortex, however, a disproportionate change in the clearance of the tracers would not be expected.

Using the aprotinin method to measure zonal GFR, we found unchanged GFR in outer cortex (numerically 11% lower) and a significantly lower GFR in the middle (31%) and inner cortex (55%) of SHR compared with WKY, whereas the total GFR was not significantly reduced (numerically 17% lower). Due to the spherical shape of the kidney and the method of dividing the kidney cortex into three different zones, the GFR in the outer, middle and inner cortex may represent ~60, ~30 and ~10% of the total GFR. This intracortical distribution of GFR implies a 22% reduction of total GFR in SHR compared with WKY, in fairly good agreement with the observed value of a 17% reduction.

It is well known [17] that anionic peptides are less readily reabsorbed in proximal tubular cells than cationic peptides. This could explain the observed urinary excretion of aChym compared with Chym and aprotinin (~10 vs <1%). The handling of aChym is, however, similar in SHR and WKY and should therefore not interfere with our overall conclusion. However, one fundamental reservation should be stated. In spite of detailed studies in our group on the proximal tubular reabsorption of peptides, we cannot yet totally exclude the possibility of a different outer, middle and inner proximal tubular reabsorption of aChym in old WKY and SHR. Increased inner cortical reabsorption of aChym in SHR could theoretically be a pitfall that could explain the increased inner cortical sieving coefficient in these animals. However, the finding of unaltered urinary excretion of aChym and unaltered reabsorption of native chymotrypsinogen in the outer, middle and inner cortical zones makes this explanation unlikely.

Our finding of an increased sieving coefficient for the anionic peptide in the inner cortex of old SHR with a selective proteinuria (65% albumin) is strongly supported by the observations of Feld et al. [2]. In their study using female SHR with proteinuria and hypertensive nephropathy, the glomeruli were examined by the use of polyanionic staining. Marked decreased staining affinity for polyanions was found in the juxtamedullary cortex compared with the superficial glomeruli in the rats with hypertensive nephropathy. However, to the best of our knowledge, there are little available data on the pathophysiological importance of the glomerular charge barrier in relation to increased urinary protein excretion in human hypertensive renal disease. Heintz et al. [18] demonstrated a decreased content of HSPG in GBMs of hypertensive subjects. In addition, a diminished post exercise urinary excretion of HSPG was observed in hypertensive subjects. This could suggest that the HSPG production is reduced or the turnover is changed in hypertensive individuals. Abnormal proteoglycan metabolism is involved in the onset of the functional derangement in diabetic nephropathy and this determines the loss of glomerular membrane anionic charges, which may lead to proteinuria. Hertzan-Levy et al. [19] observed reduced membrane polyanions such as HSPG in salt-sensitive hypertension in rats. Taken together, these data support our observation of a functional effect of reduced negative glomerular charge density in the kidney damaged by hypertension.

The function of the podocytes and their slit diaphragms in the barrier function of the filter has gained increased interest [20]. Lack of podocyte expression of a slit diaphragm peptide named nephrin is associated with congenital nephrotic syndrome of the Finnish type and heavy proteinuria [21], hence emphasizing the importance of the slit diaphragms. The slit pores were not studied in the present paper. We observed podocyte hypertrophy and loss of the normal cell shape with fusion of the foot processes and prominent cytoskeleton in the inner cortex. These changes are commonly seen in renal diseases with proteinuria [22]. They are considered adaptive to different injuries and thought to be reversible. Podocytes neighbouring areas with damage of the filtration barrier reabsorb filtered proteins and store them in lysosomes [20]. These adsorption droplets are easily detected as cytoplasmatic PAS positive droplets using light microscopy under high magnification and serve as a parameter of a distorted glomerular filter. Increased numbers of glomeruli in the inner cortex of SHR showed adsorption droplets compared with glomeruli in the outer cortex or compared with WKY. This observation fits nicely with the results of our functional studies. Podocyte pseudocysts, a sign of more severe podocyte damage, were also seen in the inner cortex. It is thought that damaged podocytes detach from the GBM and that bare basement membranes get attached to the parietal layer of Bowman's capsule, thus forming a synechia. Synechiae are considered the earliest lesions in the development of segmental sclerosis. They have a tendency to grow, and established segmental sclerosis usually shows broad adherences with the Bowman's capsule [20]. Both synechiae and adherences represent different stages in the progress of irreversible sclerotic lesions in glomeruli and were found significantly more often in the inner than in the outer cortex of SHR in the present study. The possible interplay between foot process fusion and effacement, detachment from the basement membrane, decreased negative glomerular charge density and proteinuria is not clear, but most observations indicate that the glomerular anionic sites play an important role.

A common feature in hypertensive as well as diabetic nephropathy is the increased glomerular capillary pressure. We have shown earlier that the increase in capillary pressure is mainly localized to the juxtamedullary cortex and the more superficial glomeruli have a normal capillary pressure [3]. How increased glomerular capillary pressure increases protein excretion and possibly interferes with glomerular anionic charge is not known. One may speculate that stretching of the glomerular capillary loops due to increased glomerular capillary pressure may interfere with podocyte attachment to the basal membrane [20] and synthesis of HSPG and thus reduce the negative glomerular charge density.

Our data support the notion of a functioning glomerular charge barrier, and the finding of an increased {theta} aChym to {theta} Chym ratio in the inner cortex of old SHR suggests an impairment of the glomerular charge barrier, which could be of pathogenetic importance for the proteinuria observed in SHR hypertensive nephropathy.



   Acknowledgments
 
The authors want to thank Odd Kolmannskog, Terje Ertkjern, Anne-Marie Austrheim, Turid Guldbrandsen and Nina Holmelid for excellent technical assistance. Financial support was provided by a grant from the Norwegian Council on Cardiovascular Disease.

Conflict of interest statement. None declared.



   References
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 17. 6.03
Accepted in revised form: 10.12.03