Klinik für Nephrologie, Universitätsklinikum Magdeburg and Abteilung für Nephrologie, Medizinische Hochschule Hannover, Germany
Correspondence and offprint requests to: Klaus H. Neumann, MD, Klinik für Nephrologie, Universitätsklinikum, Leipziger Strasse 44, D-39120 Magdeburg, Germany. Email: Klaus.Neumann{at}medizin.uni-magdeburg.de
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Abstract |
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Methods. With the use of micropuncture techniques, ultrafiltration characteristics of cortical glomeruli were determined in isolated cell-free perfused kidneys. Because the filtration fraction in this preparation is low (3%) as a consequence of high perfusion rates at glomerular filtration rates comparable with in vivo conditions, uniform ultrafiltration conditions are provided over the whole filtering surface. After fixation at a defined perfusion pressure, the surface of glomerular capillaries (S) was obtained morphometrically on light microscopic sections of the glomeruli studied previously.
Results. The glomerular ultrafiltration coefficient (Kf) was 0.025 nl/s·mmHg in young rats and 0.038 nl/s·mmHg in old rats (P<0.0005) and S was 0.140 mm2 in young and 0.244 mm2 in old rats (P<0.0005). However, k was not significantly different (18.0 nl/s·mmHg·cm2 in young and 15.8 nl/s·mmHg·cm2 in old rats) despite a 2.4-fold increase of GBM thickness as estimated from electron microscopic sections.
Conclusions. These findings indicate that the age-dependent increase of GBM thickness in rat kidneys did not substantially increase hydraulic resistance of the glomerular capillary wall.
Keywords: ageing rat kidney; glomerular capillary basement membrane thickness; glomerular capillary surface area; glomerular effective hydraulic conductivity; isolated perfused kidney; micropuncture methods
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Introduction |
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Materials and methods |
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Isolated kidney
The operative procedure to set up the isolated kidney preparation is described elsewhere in detail [3]. In brief, after anaesthesia with 100 mg/kg body weight of Inactin® (Byk-Gulden, Konstanz, Germany), the animals were placed on a heated micropuncture table. After median laparotomy, the right ureter was cannulated and, after heparinization, the right kidney was placed into a temperature-controlled metal cup (37°C). The aorta was clamped distal to the right renal artery, a double-barrelled cannula was inserted distal to the clamp and perfusion was started after opening the distal aortic clamp and tying a proximal aortic ligature. The animal was then separated from the kidney and recirculation of renal venous effluent was established.
Perfusion apparatus
The perfusion apparatus was designed for recirculation. Perfusion medium circulated from a 200 ml reservoir through a capillary plate dialyser (Hoeltzenbein dialyser, 0.92 m2 M 1000, Travenol, Deerfield, IL), dialysing against a circulating 5 l volume of dialysate similar in composition to the perfusate except for albumin and calcium. It had been shown in a previous study that this arrangement is superior to a true recirculation system regarding functional stability of the preparation for time periods up to 150 min [3]. This arrangement allows for a sufficient supply of arginine as a source for intrarenal nitric oxide (NO) production [4]. The dialysate was equilibrated with a mixture containing 95% O2 and 5% CO2. The perfusate was pumped through the kidney by a feedback-regulated peristaltic pump via the double-barrelled cannula after passing an in-line filter. The perfusion pressure was monitored by a pressure transducer (P 23 dB, Statham Instruments, Oxnard, CA) attached to the inner cannula. The signal was used to regulate the perfusion pressure at a constant value of 100 mmHg. Oxygen tension in arterial and venous perfusate was measured (Clark-type electrode). Perfusion pressure, perfusion flow rate (generator signal of perfusion pump), oxygen tension and temperature of the kidney chamber were recorded continuously (KA 42H, Rikadenki Kogyo Co., Ltd, Tokyo, Japan).
Perfusion medium
Perfusate contained 5 g of fraction V bovine albumin/100 ml (Armour Reheis Chemical Co., USA) dissolved in a modified KrebsHenseleit bicarbonate solution and dialysed against the same buffer. The perfusate contained the following substances (mmol/l): sodium (140), potassium (5), calcium (2.5), magnesium (1.2), chloride (104), bicarbonate (25), phosphate (0.72), urea (6), glucose (8.3), oxaloacetate (1), pyruvate (1), lactate (2), glutamate (2), methionine (0.5), alanine (2), serine (2), glycine (2), arginine (1), proline (2), isoleucine (1) and aspartic acid (3). Antidiuretic hormone (ADH) was added (10 mU/l Pitressin®, Parke Davis).
Micropuncture techniques
Proximal convolutions of surface nephrons were punctured with sharpened glass pipettes (tip size 810 µm) filled with isotonic sodium chloride solutions containing 0.1% FD&C green dye no. 3 (Allied Chemical Corp., USA). The pipettes were mounted on the lucite pipette holder of a microperfusion pump (W. Hampel, Frankfurt, Germany) with a side arm connection to a micropressure transducer. After recording the tubular free flow pressure (PT), the tubule was perfused for a short period of time with the coloured pipette solution. If an early loop was punctured as judged from the number of coloured proximal loops downstream (at least four, range 47, mean 5.3), the pressure pipette was left in place. Since the number of proximal surface loops in Wistar rats has been reported to be 5.2 on average, it can be assumed that the pressure pipette was located in the earliest accessible part of the proximal tubule. The last proximal surface loop was then punctured with a second pipette filled with coloured paraffin oil, and a timed collection (58 min) of tubular fluid was begun after insertion of a small droplet of oil into the tubular lumen. The collection rate was adjusted so that the continuously recorded tubular pressure was kept at the original PT and the oil droplet was kept distal to the collection pipette. After collection was completed, the pipette was withdrawn and the tubule distal to the pressure pipette was blocked via another pipette with paraffin oil. The stop flow pressure was then recorded and the tubule was charted for later identification. Since the glomerulo-tubular feedback mechanism is not demonstrable in this isolated perfused kidney preparation (unpublished observation), a possible influence of proximal collection of tubular fluid on glomerular capillary hydraulic pressure could be disregarded. At the end of the experiment, the kidney was perfused with a 1.25% glutaraldehyde solution with phosphate buffer and 6% hydroxyethyl starch as a colloid at the original perfusion pressure. The tubules previously studied were then gently injected in the retrograde direction with microfil (Canton Biomedical Products, Boulder, CO) so that the marked glomeruli could be identified later on histological sections by small particles of microfil in Bowman's space.
Macroanalytical techniques
The GFR was estimated as inulin (polyfructosan) clearance. The inulin concentration in urine and perfusate was measured after acid hydrolysis by adding a phosphohexose isomerase reaction to the hexokinase/glucose-6-phosphate dehydrogenase method for determination of glucose [5]. The albumin concentration in the perfusate was measured with the biuret technique.
Microanalytical techniques
The volume of collected tubular fluid was estimated from the length of the fluid column in a calibrated constant bore capillary glass tube with an eye-piece micrometer. The inulin concentration in microsamples of tubular fluid and plasma was measured with a microadaptation of the macroanalytical technique [5].
Morphometry
The GBM surface area (S) was measured according to the technique of Aeikens [6]. After fixation of the kidney, wedges of tissue each containing one glomerulus marked with microfil were dehydrated and embedded in Araldite (Serva, Heidelberg, Germany). Beginning at the kidney surface, 400600 serial sections, of which 150 pertained to the marked glomerulus, were obtained (0.9 µm thickness) on an ultramicrotome (MT-22-B Servall Dupont Instruments). The thickness of the sections was measured interferometrically [6]. The sections were stained with methylene blue and covered. The inner circumferences of the glomerular capillary sections were then estimated interactively with an ASM Leitz digitizer (Leitz, Wetzlar, Germany) in connection with a computer (Digital Equipment, Maynard, MA). S was calculated from the number and the thickness of the microscopic sections and the total inner circumference of the capillaries. S thus represents the total (mesangial plus peripheral) inner surface area of the glomerular capillaries.
Measurement of GBM thickness
The thickness of the GBM was estimated from the width of the GBM on photographs of transmission electron microscopic sections from the kidneys investigated. The perpendicular distance from the endothelial cell boundary to the epithelial cell boundary of the peripheral basement membrane was measured with an ASM Leitz digitizer. About 100 measurements on at least four peripheral capillary walls of two glomeruli of each kidney were performed (criterion for perpendicular sections: depiction of slit membrane as a narrow line).
Calculations
The single nephron glomerular filtration rate (SNGFR) was calculated as
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The effective ultrafiltration pressure was calculated as
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The glomerular ultrafiltration coefficient was obtained by dividing the SNGFR by 60 and by the effective ultrafiltration pressure:
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All values are means±SEM unless indicated otherwise. Statistical analysis was performed by the unpaired t-test. Statistical significance is defined as P<0.05.
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Results |
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GBM thickness
The width of the GBM on electron microscopic sections as a measure of the GBM thickness was 129±2 nm (n = 420) in group I and 305±2 nm (n = 542) in group II (P<0.0005).
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Discussion |
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To calculate k, the inner surface of the glomerular capillaries and the driving forces for ultrafiltration were measured. Since the actual anatomical surface area available for filtration has not yet been clearly defined, the effective hydraulic conductivity k was calculated from the total inner surface of the glomerular capillaries. To our knowledge, the present study is the first investigation in which both physical forces and capillary surface area (S) were measured in identical glomeruli. The value of 0.140 mm2 of S in 2-month-old rats (250 g body weight) is similar to the estimation of 0.132 mm2 Olivetti and co-workers obtained for Wistar rats of comparable size (calculated from their value of 0.088 mm2 for the two-thirds fraction of peripheral membrane [8]. Variance between strains of rats seems to be quite large, as was demonstrated by the results of Aeikens who found S values of 0.08 and 0.06 mm2 for two different colonies of Munich Wistar rats endowed with surface glomeruli but 0.17 mm2 in a standard Wistar strain of comparable size and age [6]. These findings emphasize the importance of measuring physical forces of filtration and capillary surface area in comparable glomeruli in order to calculate k.
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Since our investigations were performed on a strain of rats without surface glomeruli, PGC had to be estimated by the indirect stop flow technique. Whereas most investigators found identical values for the glomerular capillary hydrostatic pressure, both by direct puncture and by the stop flow technique [9,10], some authors reported discrepant findings between these two techniques. According to Ichikawa [12], the directly measured PGC rises when the proximal tubule is blocked and the flow through the loop of Henle is zero. The rise in PGC was explained by the diversion to the efferent vasculature of fluid which without tubular blockade would have been filtered. This possible rise of PGC should be negligible in the isolated kidney, since the increment of efferent flow rate after tubular blockade is small due to the low filtration fraction of 3%.
In addition to the constant filtration forces, an advantage in studying the passive properties of the glomerular capillary filtration barrier in the isolated perfused kidney is derived from the finding that the protein concentration itself influences Kf in such a way that Kf is reduced when the plasma protein concentration is lowered [13]. This implies that the local k could also change with a rising protein concentration along the glomerular capillary. This effect would be abolished in our model. Also, of the numerous hormonal substances which have been demonstrated to reduce Kf, only ADH was present in the perfusion solution at a physiological non-pressor concentration, thus ruling out possible age-dependent ADH effects.
While we think that the isolated cell-free perfused kidney is a suitable model to study the physical properties of the glomerular capillary wall, it should be kept in mind that the Kf and k values obtained may not be directly comparable with in vivo values. Factors such as the absence of molecules larger than albumin, e.g. globulins, fibrinogen and others such as orosomucoid, might alter the filtration characteristics. Since the kidneys of both groups of rats were studied under identical conditions, differences in the physical properties of the glomerular capillary wall due to an altered ultrastructure should have become apparent. A special feature of the isolated perfused kidney is an elevated glomerular protein filtration [14]. There are, however, no reports that protein filtration per se influences glomerular hydraulic conductivity in the absence of foot process retraction. Considering the different experimental approaches, the Kf values we calculated are remarkably similar to the results obtained by other authors under in vivo conditions. For SpragueDawley rats, a value of 0.038 nl/s·mmHg [15] has been reported, and 0.025 nl/s·mmHg for non-mutant Wistar rats [11].
By measuring physical forces for glomerular ultrafiltration and GBM surface area of identical glomeruli, we were able to calculate k. The values 18.0 and 15.8 nl/s·mmHg·cm2 are lower than those calculated by more indirect methods for Wistar rats with surface glomeruli [16]. They are nonetheless >10-fold higher than k values measured in peripheral capillaries such as in rat muscle and rabbit omentum. The value of k of the glomerular capillary wall did not differ significantly between the two groups of rats investigated in spite of the fact that the thickness of the GBM differed by a factor of 2.4. This finding can be interpreted in several ways. It could be concluded that the GBM does not significantly contribute to the overall hydraulic resistance of the glomerular capillary wall if it is assumed that the specific hydraulic resistance of the GBM material does not change during the ageing process. Our findings support theoretical models of the glomerular filtration barrier which predict that the decisive hydraulic resistance is located in the epithelial slit pores [1722].
Recently it has been suggested that nephrin could represent the molecular structure of these pores [18]. The constancy of k in the presence of a large increment in GBM thickness could also be the consequence of a compensatory increase of the size of the epithelial slit pores. Since the hydraulic resistance of slit-like pores is reciprocal to the square of the slit width [23], only minor changes of the pore width would be necessary to compensate for an increase in the hydraulic resistance of other components of the capillary wall. With presently available morphological techniques, these small changes escape detection. Another possible explanation for the relatively small change of k in the presence of a large increment in GBM thickness may be that a change in the physical properties of the GBM material occurs. This in turn could be the consequence of biochemical alterations occurring during the process of ageing such that the effect of thickening is exactly compensated.
How our findings can be reconciled with current theoretical models of glomerular hydraulic conductivity [24] is beyond the scope of the present study.
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Acknowledgments |
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Conflict of interest statement. None declared.
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References |
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