Department of Pediatrics, Karolinska Institutet, Huddinge University Hospital, Department of Pathology, Karolinska Hospital, Stockholm, Sweden
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Abstract |
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Methods. Sixteen MunichWistarFrömter male rats initially weighing median 247 g (range 171286) were used. Four rats served as controls. The other 12 rats were divided into three groups receiving daily subcutaneous injections of 1, 1.67, and 2.5 mg PAN/100 g body weight respectively, for 6 days. GFR was determined by clearance of inulin and the fractional urine albumin excretion was measured. Standard stereological methods were used to estimate the glomerular volume, the mean foot process width and the length density of slit pores.
Results. GFR decreased with increasing PAN doses. The glomerular volume was increased in the group receiving the lowest PAN dose, while it was decreased in the group with the highest PAN dose, compared with controls. The fractional albumin excretion and the foot process width increased and the total slit pore length decreased with increasing doses of PAN. GFR correlated directly with the glomerular volume as did the foot process width with the fractional albumin excretion. The foot process width correlated inversely with the glomerular volume as did the glomerular volume with the fractional albumin excretion, and GFR with foot process width.
Conclusions. The decreased GFR found in the nephrotic rats was inversely related to foot process width and directly related to glomerular volume, confirming our previous results in children in an early stage of the nephrotic syndrome.
Keywords: foot process width; glomerular filtration rate; glomerular volume; MunichWistarFrömter rats; nephrotic syndrome; puromycin aminonucleoside nephropathy
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Introduction |
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In a study of children during their first episode of minimal-change nephropathy, an inverse relation was found between mean foot process width and glomerular filtration rate (GFR) [8]. In the same group of MCNS patients, GFR and filtration fraction decreased significantly during the nephrotic stage, while renal plasma flow remained normal. This constellation of findings contradicts the idea that hypovolaemia is the main cause of the reduced GFR [9]. In another study including various types of the nephrotic syndrome, GFR was decreased in the nephrotic phase in all of the diagnostic groups and was directly correlated to the serum albumin concentration [10]. We also found increased glomerular volumes in patients with diffuse mesangial proliferation (DMP) and focal segmental glomerulosclerosis (FSGS), compared with MCNS patients [11].
The aim of the present study was to analyse further the renal haemodynamics and the glomerular volume, foot process width and total slit pore length in rats with nephrotic syndrome induced by PAN, to clarify whether similar relationships are found in rats and in children with nephrotic syndrome.
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Subjects and methods |
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The rats were housed with a 12-h lightdark cycle in controlled temperature and humidity. They were fed a standard rodent diet (B & K Universal, Sollentuna, Sweden) of pellets containing 20.5% protein and 0.3% sodium chloride, and tap water. The animal care and the protocols for experimental procedures were reviewed and approved by the Swedish National Ethics Board for Animal Research.
Induction of PAN nephropathy
PAN nephropathy was induced by subcutaneous (s.c.) injection of the drug (Sigma, St Louis, MO, USA). The animals were divided into three groups of four rats in each. They received a daily s.c. injection of 1 mg (group I), 1.67 mg (group II), and 2.5 mg/100 g body weight (BW) (group III) of PAN for six consecutive days [18]. A control group of four animals of the same age and weight were followed; they did not receive any treatment.
Urinary albumin excretion
The rats were weighed and placed in individual metabolic cages to determine albumin excretion in 24-h urine on the day before the first PAN injection. They were given water ad libitum and powder chow during the 24-h collection. Another 24-h urine collection for albumin excretion was performed on the day after the last PAN injection. Urine albumin was determined with a nephelometric method (Boehring Nephelometer Analyzer, Behringwerken AG, Marburg, Germany). The 24-h urine volumes collected before and after the PAN injections were incomplete in some rats. Therefore albumin excretion was calculated as the fractional log urine albumin concentration (log U-albumin/absolute GFR).
Renal function
On the 7th day, the rats were anaesthetized with Inactin (BYK-Gulden, 120 mg/kg BW, intraperitoneally). Tracheostomy was performed and indwelling polyethylene catheters were inserted into the right femoral artery for blood pressure monitoring and blood sampling and into the right femoral vein for infusion of radioactive inulin ([3H] inulin dissolved in 0.9% NaCl, 50 µCi/ml, Amersham, Buckinghamshire, UK). GFR was determined by the clearance of inulin. Inulin was given in a priming dose of 0.4 ml, followed by a continuous infusion of 1.2 ml/h. The urinary bladder was catheterized. After an equilibration period of 1 h, three 20-min urine samples were collected and blood samples were drawn in the middle of each urine sampling period. GFR was calculated as the mean of the three urine collection periods in absolute (ml/min), as well as in relative values (ml/min/100 g).
Blood pressure was recorded continuously using a Gould P23 pressure transducer connected to a Gould Recorder 2200S (Gould Electronics BV, Bilthoven, The Netherlands). Those rats that during the renal function test developed a severe fall in blood pressure that did not return to normal within 15 min were excluded from the study. Blood and urine samples were analysed for 3H activities with the liquid scintillation technique (LKB Wallac 1214 Betarack, Bromma, Sweden).
Tissue fixation
The left kidney was exposed and immobilized in a plastic micropuncture cup. After sealing the cup with agar, the kidney, still being reperfused, was continuously bathed in fixative (2% glutaraldehyde and 4% paraformaldehyde in a 0.05 mol/l sodium phosphate buffer, pH 7.4, temperature 37°C, osmolality 675 mOsmol/kg H2O) [19]. The fixation procedure was continued for 15 min during which time the fixative was renewed every 0.5 min. The kidney was then extirpated for morphometric studies and placed in a vial with the same fixative as above.
Light microscopy evaluation
The kidneys were cut into 1.5-mm thick coronal slices from which a set of two systematic random slices were collected and embedded in glycol methacrylate. The mean glomerular volume (GV) was estimated by the Cavalieri principle [20]. The tissue blocks were serially sectioned 2.5-µm thick, and the sections were stained with haematoxylin and eosin. The glomeruli were sampled in their order of appearance in the sections. The first profile of a glomerulus was sampled when its capillary loops became visible. Successive profile sections of the same glomerulus were then sampled at 7.5-µm intervals. The number of levels sampled per glomerulus ranged from 10 to 15. The area of the profiles was estimated by point-counting using an ocular grid with 19.9 µm between each point (d) at tissue level (410x). The sum of profile areas (A SUM) was estimated using the formula A SUM=Ptotxd2. For the final calculation of the GV, we estimated the mean of the actual section thickness (t) by a method based on confocal microscopy (CSLM) [21]. In brief, the xz profile of a section is scanned by CSLM, and the full-width half-maximum of the intensity profile obtained in the axial direction is used to estimate the actual section thickness. The precision of the method has been shown to be ±100 nm [21]. GV was then calculated as: A SUMx3t. In each rat, eight to 12 glomeruli were measured and the mean GV was calculated.
Electron microscopic quantification
For electron microscopy, systematic and random sampled tissue blocks of superficial cortex were post-fixed in 1% osmium tetroxide and embedded in Epon. Ultra-thin sections were stained with uranyl acetate and lead citrate, and studied with a Philips 420 electron microscope. Three to four superficial glomeruli were analysed from each rat. The same definition of the reference space of the glomerular tuft and the delineation of the mesangium to the peripheral capillary wall were applied as in the study by Østerby and Gundersen [22]. The average foot process width was estimated as the ratio of the surface density of the glomerular basement membrane, Sv (GBM) and the length density of filtration slits, Lv (slits) along the peripheral capillary walls. At approximately 1350x, sets of four to eight micrographs per glomerulus were taken in a systematic random manner by moving the specimen stage between predetermined points; the first point was chosen randomly. The final magnification was corrected using a grating grid with 2160 lines/mm (EF Fullan, Inc. UK). A superimposed square test grid was used with points defined by the intersections between the grid lines. The distance (d) between each point in the test grid was approximately 12 µm at tissue level. The intersections between the grid lines and the peripheral glomerular basement membrane (I), the points hitting the reference space (P), and the number of transected filtration slits along the peripheral basement membrane (Q) within the reference space were counted. The average number of filtration slits counted per animal in the control group was 1926 (13163219).
Sv (GBM), was calculated according to the following:
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Lv (slits) was estimated as:
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The total slit pore length was calculated as the product of GV and Lv (slits).
Statistical analyses
Mean values±standard error of mean (SEM) or range with median values are given. Linear regression and correlation coefficients were calculated with the least-squares method. The paired t-test was used. Variance analysis was done using ANOVA with the TukeyKramer and Dunnet post-tests.
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Results |
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Renal morphology
Light microscopy showed no obvious interstitial fibrosis, tubular atrophy, global or focal segmental glomerulosclerosis. A direct correlation was found between GV and BW in the control rat group (r=0.98, P=0.019), but not in the other three groups. After 6 days, the mean GV in the control rats was 0.77±0.05 Mµm3 and the mean albumin excretion 71±10 mg/24 h. Figure 2a, b
, c
shows the GVs, the foot process width and the total slit pore length in the various groups. The mean GV in group I was significantly larger than that in the control group (P<0.035) and group III (P<0.0008), and the GV in group III was almost significantly smaller than the controls (P<0.065). The PAN-treated rats showed significantly broader foot processes than the controls (P<0.001) while group III (P<0.01) showed broader foot processes than group I (Figure 2b
). The total slit pore length of the glomerulus was significantly higher in the controls than in the PAN-treated rats (P<0.0001) (Figure 2c
). There was a strong inverse correlation between foot process width and GV (r=0.849, P=0.0005) (Figure 3
).
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Renal function in relation to morphology
A direct correlation was found between absolute GFR and GV in the PAN-treated rats (r=0.813, P=0.0013) (Figure 4a). An inverse correlation was seen between foot process width and absolute GFR (r=0.607, P=0.036) in the PAN-treated rats (Figure 4b
). With inclusion of controls, the correlation coefficient was higher (r=0.883, P=0.0001). A nearly significant correlation was found between absolute GFR (ml/min) and total slit pore length (r=0.568, P=0.0542). There was a significant inverse correlation between log U-albumin/absolute GFR and GV (r=0.857, P=0.0004) (Figure 5a
) and total slit pore/glomerulus (r=0.613, P=0.0342) and a significant direct correlation between log U-albumin/absolute GFR and foot process width (r=0.65, P=0.022) (Figure 5b
).
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The major findings in this study were that as the doses of PAN increased, GFR and total slit pore length decreased, and foot process width and fractional albumin concentration increased. The rats receiving the lowest dose of PAN had significantly (17%) larger glomeruli than the controls, while GVs were smaller in the groups of rats receiving higher PAN doses. In fact, there was a direct correlation between GFR and GV, and between foot process width and fractional albumin concentration. Furthermore, we found an inverse correlation between foot process width and GV as well as GFR, and between GV and fractional albumin concentration.
The decrease in relative GFR and simultaneous increase in foot process width and fractional albumin concentration with increasing PAN doses are in agreement with the findings of Inokuchi et al., who reported decreasing creatinine clearance with increasing proteinuria and increased foot process width [23]. Trachtman et al. also found that the creatinine clearance decreased with increasing proteinuria in PAN-treated SpragueDawley rats [24]. These results accord with ours in a group of MCNS children during their first episode of nephrosis. We then found an inverse correlation between mean foot process width and GFR and the filtration fraction [8]. A reduction in epithelial slit pore length occurred concomitantly with the broadening of the foot processes. It led to the hypothesis that the reduction in the total length of glomerular epithelial slit pores, due to the fusion of foot processes, resulted in a reduced glomerular capillary permeability to water and small solutes [8]. This hypothesis is supported by findings in adult patients by Guasch and Myers and by Drumond et al. [25,26]. Drumond et al. [25] showed that a decrease in filtration slit frequency due to foot process fusion may explain the decreased hydraulic permeability of the filtration barrier and the decreased GFR. They also found a significant increase in GV, which, however, they did not discuss. Guasch and Myers [26] studied two groups of MCNS patients with normal or depressed inulin clearance. They found that the capillary surface area available for filtration was preserved, if not slightly enhanced, in both groups, but the filtration slit frequency was significantly reduced by epithelial podocyte broadening, which correlated with the computed ultrafiltration coefficient. They suggested that the reduction in plasma oncotic pressure leading to an increase in ultrafiltration pressure counteracted a decrease in the ultrafiltration coefficient. Thus the ultrafiltration coefficient must be substantially reduced to reduce GFR [26].
The direct correlation between foot process fusion and fractional albumin concentration shown in the present study is in agreement with the observations of Mahan et al. [27] in rats, who noted increases in foot process width and in urinary albumin excretion after the intraperitoneal injection of PAN. Our previous finding [8] of an inverse correlation between foot process fusion and serum albumin concentration agrees with our present results. In rats with unilateral nephrosis induced by an intra-arterial injection of PAN, Baboolal and Meyer [28] reported decreased GFR and increased proteinuria in the nephrotic kidney, compared to the contralateral kidney. We also found a decreased GFR in the nephrotic stage in a group of MCNS children [9] and in a larger group of children with various histological entities of nephrotic syndrome [10]. Thus the decreased GFR seen in our PAN-treated rats seems to be due to a severely reduced ultrafiltration coefficient, caused by decreased total slit pore length secondary to foot process fusion.
In the group of rats receiving the lowest dose of PAN (1 mg/100 g), GV was larger than that in the controls (17%), which might indicate glomerular hypertrophy in this group. Both Trachtman et al. [24] and Cahill et al. [29] induced nephrosis in SpragueDawley rats by giving seven PAN injections (2 mg/100 g) every week or every other week and found that the rats had developed glomerular hypertrophy. Cahill reported biphasic glomerular hypertrophy, the first phase of which, after 7 weeks, was explained by an increase in total capillary length per glomerulus, while the second phase, after 13 weeks, was more related to an increase in capillary diameter and/or mesangial matrix expansion. They suggested that the glomerular hypertrophy was an attempt by the glomerulus to increase the filtration area and compensate for the loss of filtration slits. Large glomerular volumes have been reported by Fogo et al. in patients with focal segmental glomerulosclerosis (FSGS) as well as MCNS, who later progressed to FSGS. Thus a large GV in MCNS may be a predictor of FSGS [30].
In a previous study from our unit, we found increased GV in nephrotic children with DMP and FSGS, as well as in children with steroid-dependent and steroid-resistant nephrotic syndrome [11]. A prerequisite of the development of FSGS seems to be glomerular hypertrophy with subsequent podocyte detachment. We also found a strong inverse correlation between foot process width and GV in this study of acute PAN nephrosis. The much more severe foot process effacement that was found in group III suggests a more severe podocyte injury as well as an increased risk of podocyte detachment and progression to FSGS [31]. However, in the same group of animals we found neither manifest FSGS nor podocyte detachment. We suggest that the decreased glomerular volume in group III (18% less than the controls) had a protective effect on the podocytes, preventing them from detaching, and thereby hindering the development of FSGS. This is supported by Trachtman et al. [24], who reported that growth hormone treatment of PAN-nephrotic rats resulted in significant glomerular hypertrophy and pronounced FSGS, compared with nephrotic rats not treated with growth hormone. Reduced extracellular volume has been reported to cause a substantial decrease in GV in MunichWistar rats [32]. One explanation of the small glomeruli found in rats given the highest PAN dose might be volume contraction secondary to severe oedema and high urine volumes. This group of rats showed almost three times higher urine volumes than the other groups, which might have been caused by severe tubular changes secondary to increased albumin reabsorption.
In conclusion, we confirm our previous results in children with nephrotic syndrome: reduced GFR in the nephrotic stage [10], an inverse correlation between GFR and foot process fusion [8], and a direct correlation between GFR and GV [11]. In the present study, we also found an inverse correlation between foot process width and GV as well as a direct correlation between foot process width and fractional albumin concentration.
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Acknowledgments |
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Notes |
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References |
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