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Department of Nephrology, University Hospital of Lund, Lund, Sweden
Submitted 13 April 2006 ; accepted in final form 26 June 2006
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ABSTRACT |
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 TOP ABSTRACT EXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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) were obtained by analyzing Ficoll [mol.radius (ae) 13–85 Å] in urine and plasma after 20 and 60 min I/R. Furthermore, for human serum albumin (HSA) was estimated using a tissue uptake technique after 20 and 60 min of I/R, while clearance of HSA compared with that for neutralized HSA (nHSA) was assessed after 20 min of I/R only. Glomerular filtration rate (GFR) was measured by [51Cr]EDTA and inulin. I/R reduced GFR and increased for Ficoll molecules of ae >55 Å and for albumin. for Ficoll vs. ae, analysed using a two-pore model, demonstrated that, despite increases in , the large-pore fractional ultrafiltration coefficient (
L) was unchanged after 20 min of I/R, owing to the decline in GFR, but increased after 60 min of I/R. However, the apparent L for albumin increased already after 20 min of I/R (P < 0.005) and the nHSA/HSA clearance ratio was slightly reduced, possibly reflecting a diminished negative charge barrier. In conclusion, after 20 min of I/R, indications of a reduced charge selectivity were noted, while after 60 min of I/R, there was mainly a reduction in size selectivity, compatible with an increased formation of large pores. albumin; Ficoll; anoxia; fractional clearance; oxidative stress
The glomerular barrier is made up by three sequential layers: the fenestrated endothelium with its glycocalyx, the glomerular basement membrane (GBM), and finally, the podocytes with their interdigitating foot processes and the podocyte slit diaphragms (PSD) (6). All of these three layers are potential ischemic targets, and alterations in any of them would lead to an increased glomerular permeability. The first layer, the endothelial glycocalyx, is composed of negatively charged proteoglycans that, conceivably, contribute to the negative charge of the glomerular barrier. Morphological studies in heart capillaries have indicated that ischemia causes disruption and "clumping" of the endothelial glycocalyx (16, 30), hence suggesting that the disruption of glomerular glycocalyx may play a role during ischemia-induced proteinuria. However, the endothelial glycocalyx is not visualized by normal fixation procedures and is therefore overlooked in many pathological situations. Since ischemia does not cause any obvious ultrastructural changes of the glomerular filter, it has been proposed that the proteinuria-induced by ischemia may, at least partly, be a result of injury to the glycocalyx.
The second layer, the GBM, is composed of collagen type IV, laminin-11, entactin and heparan sulphate proteoglycans (HSPGs), such as perlecan and agrin (9). HSPGs are important components of the GBM, and their negative charge may be of both structural and functional importance. This is evident in animals overexpressing heparanase-1, an enzyme degrading HSPG chains, the ensuing alteration in the GBM resulting in proteinuria (33). Furthermore, increased concentrations of metalloproteinases have been detected in glomeruli after I/R (4) and may be a factor contributing to ischemic proteinuria, as these enzymes are important for normal degradation and remodelling of the GBM.
It has been shown that podocytes undergo flattening and spreading of their foot processes in ARF (20). These changes occur early, already during the ischemic period, and increase in severity with the ischemic duration. There is a close structural interaction between the GBM and the podocytes. In mice with altered GBM, the podocytes appear effaced with a reduced number of foot processes (33). Hence, following I/R, alterations clearly occur in all layers of the glomerular capillary wall.
The purpose of the present study was to investigate the functional nature of the lesions occurring in the glomerular capillary wall following acute I/R injury. To determine whether acute I/R leads to an alteration in the size-selective barrier, we investigated the urine excretion of FITC-Ficoll, a neutral polydisperse polysaccharide, allowing the assessment of sieving coefficients (fractional clearances, ) for a broad spectrum of molecular sizes in one single experiment. To get further insights into the potential charge-dependent nature of permeability changes, we also assessed the fractional clearance of native (neg.) albumin. Moreover, when permeability changes were very discrete, i.e., after 20 min I/R, we compared the clearance of native (neg. charged) albumin to that of neutralized albumin.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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Experimental protocols. Glomerular filtration rate (GFR) was measured in both kidneys separately during the preischemic resting period (20–30 min), using 51Cr-EDTA. A priming dose of 51Cr-EDTA (100 µl, 0.37 MBq, Amersham Biosciences, Buckinghamshire, UK) was administered and followed by a continuous infusion (3 ml/h) of 51CrEDTA (0.37 MBq/ml in 0.9% NaCl) throughout the experiment. Ischemia of the left kidney was induced with a temporary 20- or 60-min clamp of the left renal artery. Urine was collected from the urethers (no urine was produced from the clamped kidney during the artery obstruction). After the ischemic period (20 or 60 min), the artery clamp was removed, followed by a 20- or 60-min reperfusion period, respectively. During the reperfusion period, urine production resumed in the previously obstructed kidney. Ten minutes before the end of the reperfusion period, a bolus dose containing FITC-labeled Ficoll-70 (42 µg), FITC-Ficoll-400 (1 mg), and FITC-inulin (0.5 µg; TdB Consultancy, Uppsala, Sweden) was given followed by an infusion of FITC-Ficoll-70 (94.5 µg/min), FITC-Ficoll-400 (3 mg/min), FITC-inulin (1.5 µg/min), and 51Cr-EDTA (n = 7 at 20 min and n = 9 at 60 min). At the end of the reperfusion period, urine was collected for 5 min and a midpoint plasma sample was aspirated for analysis of Ficoll sieving coefficients.
Tissue uptake technique.
After 20 or 60 min of I/R, albumin was measured using a tissue uptake technique (17). [125I]human serum albumin (HSA) was given as a bolus (0.2 MBq, Institute for Energy Technique, Kjeller, Horten, Norway) in the tail artery (n = 11 at 20 min and n = 6 at 60 min). Six blood samples (25 µl) and one urine sample were collected during 8 min. Thereafter, a whole body washout was performed via the carotid artery (20 ml/min) for 8 min. The inferior vena cava was freed and cut open for collection of the rinse fluid. The washout fluid mixture contained equal amounts of 0.9% saline and heparinized horse serum (SVA, Uppsala, Sweden). Thereafter, the kidneys were removed and the cortex was dissected and assessed with respect to radioactivity. Sieving coefficients for neutralized human serum albumin (nHSA) were measured after 20 min of I/R (n = 6) as described above for [125I]HSA. In seven additional experiments, we simultaneously compared the clearance of [125I]HSA to that of [131I]nHSA after 20-min ischemia. The simultaneous measurement of [125I]HSA and [131I]nHSA did not allow the assessment of GFR by [51Cr]EDTA. Hence, only a clearance ratio, but not absolute values of , could be obtained in these experiments. nHSA was prepared by Dr. Olav Tenstad by a graded modification of the COOH groups using a procedure modified from Hoare and Koshland (11) and earlier described at some length (17). nHSA was labeled with 131I, using 1,3,4,6-tetrachloro-3,6-diphenylglycouril (Iodo-Gen) as described in (17). All radioactivity measurements were performed in a gamma scintillation counter (Wizard 1480, LKP Wallac, Turku, Finland). Radioactive decay and spillover from the 51Cr to the 125I channel, or from the 131I channel to the 125I channel, were appropriately accounted for.
High-performance size exclusion chromatography.
Size exclusion was achieved by using an Ultrahydrogel-500 column (Waters) and a phosphate buffer with 0.15 M NaCl (pH 7.4). Fluorescence was detected with a fluorescence detector with a 
exitation at 492 nm and a emission at 518 nm (Waters 2475). The system was controlled by Breeze software 3.2 (Waters). The column was calibrated using five narrow FITC-Ficoll standards, seven narrow FITC-dextran standards and a few protein standards. A calibration curve was achieved using the relationship: y = –475.45x3 + 1,059.4x2 – 878.78x + 294.41 and is described elsewhere (1).
Calculations.
For the tissue uptake technique, renal tracer protein clearance was calculated from the amount of tracer radioactivity accumulated in both kidneys (cortex) plus the TCA-precipitable urine tracer activity (collected during the tracer infusion period) divided by the area under the curve of the plasma tracer concentration vs. time function. Protein values were calculated by dividing the measured protein clearance by the simultaneously assessed GFR (17, 27). Ficoll were obtained by analyzing the HPSEC-curves obtained from the urine and the plasma sample (CpF) for each experiment. The urine concentration vs. ae curve was divided by the inulin concentration, to obtain the primary urine concentrations (CuF). for each ae was calculated by dividing CuF by CpF (CuF/CpF). The two-pore model (17, 22, 27) was used to analyze the data for Ficoll (15–80Å). A nonlinear least squares regression analysis was used to obtain the best curve fit, using scaling multipliers as described previously (17). An apparent L was calculated for the HSA data, as obtained from the following equation [see Eq. 4 in (27) and Eq. 25 in (22)]:
 | (1) |
L) is very close to unity we have:
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was set at 28 mmHg, and hydraulic conductance (LpS) was calculated assuming a filtration pressure of 9 mmHg. Statistical analysis. Values are presented as means ± SE. Differences between groups were calculated using one-way ANOVA with Bonferroni's multiple-comparison correction. A paired t-test was used for comparisons between HSA and nHSA in the same kidney. Significance levels were set at *P < 0.05, **P < 0.001, and ***P < 0.005.
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RESULTS |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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45%) after the reperfusion period in the ischemic kidney (Table 1). From the relationship of log for Ficoll vs. SE- radius (15–80 Å), it is evident that for FITC-Ficoll increased for molecules >55Å in the postischemic kidney (2.7-fold) compared with the control kidney, as shown in Fig. 1. The best curve fit of vs. ae according to the two-pore theory was obtained for the parameters listed in Table 3. Even though was significantly higher for large size Ficoll (ae>55Å) after 20 min of I/R, the fractional ultrafiltration coefficient accounted for by the large pores (L) was not significantly increased, indicating that the reduced GFR postischemia may have been the prime factor responsible for the increase in Ficoll (Fig. 2) (see Ref. 23).
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for neutralized HSA was ninefold higher than that for native HSA in the control kidney (P < 0.005; Table 2). Both HSA and nHSA increased after the I/R insult, the increase however not reaching statistical difference for nHSA (Table 2). In the control kidney 99 ± 0.083% of the nHSA and 88 ± 2.8% of the native HSA was reabsorbed in the proximal tubules, while in the ischemic kidney the fractional reabsorption was 97 ± 0.49% and 81 ± 3.4%, respectively.
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L for HSA according to the two pore theory (assuming a large pore radius of 100 Å). After 20 min I/R, there was an increase in this parameter (Table 2 and Fig. 2). An increase in apparent L for native HSA, but not for the uncharged Ficoll, may thus reflect a change in the glomerular charge barrier characteristics (Fig. 3). The small pore radius (rs) and large pore radius (rL) were unchanged after the I/R insult (Table 3).
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75%; Table 1). The for Ficoll molecules of ae>55Å increased 5-fold in the I/R kidney, the increase being 2.4-fold larger than for 20 min I/R (Fig. 1). The rs and rL were not affected by 60 min I/R. However, there was significant increases in L, indicating that 60 min of I/R increased the number of large pores in the glomerular filter (Fig. 2 and Table 3), without affecting the large pore radius. A0/X was reduced threefold (P < 0.005) after 60 min of I/R, i.e., largely in parallel with the fall in GFR (Table 3), indicating a reduced effective pore area for solute diffusion after 60 min of I/R. |
DISCUSSION |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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The effects of I/R on glomerular barrier selectivity have only been rarely investigated. Hence, this is the first study, to our knowledge, in which a detailed analysis has been performed to evaluate the nature of the glomerular permeability changes occurring after acute I/R in vivo. The major result of the present study is that acute I/R markedly reduces the size selectivity of the glomerular barrier by the functional appearance of an increased number of large pores (cf. "shunt pathways"). This pattern of alterations of the glomerular barrier function mimics what has been previously seen in a number of different pathophysiologic disorders. The increased number of large pores in the present study after 60 min of I/R, as determined by Ficoll, was mirrored by a large increase in for albumin (ae = 36 Å; Fig. 1). According to the two-pore theory, native albumin, due to its net negative charge, is for its transglomerular passage more or less totally confined to the large pore pathway. This is because of the high size and charge selectivity of the glomerular barrier to proteins (21, 23, 27, 29), normally excluding (neg.) native albumin from the small pore pathway. An increase in the large pore number (mirrored by L) should thus be well reflected by increases in for albumin, as noted in the present study.
Just a minor (nonsignificant) increase in the fractional ultrafiltration coefficient accounted for by the large pores (L) was observed after 20 min of I/R for the Ficoll sieving data. By contrast, L for Ficoll increased significantly (4-fold) after 60 min of I/R (P < 0.05; Fig. 2). Assessed from for native albumin, however, an increase in apparent L was observed already at 20 min of I/R, but no further increase was observed after 60 min I/R. Since Ficoll data predicted only a minor, non-significant increase in large pore number after 20 min of I/R, the increase in apparent L for HSA may be due to a small charge defect in the glomerular barrier appearing after 20 min of I/R, making albumin partly permeable also through the small pore pathway. This was further corroborated by the fact that the clearance ratio of nHSA to HSA was slightly reduced at 20 min I/R (Fig. 3). However, after 60 min of I/R, the increase in large pore number, as interpreted from the increase in L for Ficoll, apparently superseded the small charge defect observed at 20 min I/R, because L for albumin (in absolute and relative terms) did not differ from that calculated from Ficoll data in this situation.
There were no changes in Ficoll among small Ficoll molecules (ae<50 Å) after the I/R injury. The reduction in GFR following upon I/R would per se lead to an increase in for these Ficoll molecules provided that the capillary pore area (A0/x) and small pore radius (rs) had remained unchanged, leading to a "steeper" sieving curve (23). However, this effect was canceled by the simultaneous parallel reduction in A0/x, thus leaving the Peclet numbers (ratio of GFR to A0/x), and hence, the sieving coefficients in the 20–50Å ae-interval, unchanged. The parallel decrease in A0/x and GFR could be attributed to alterations in renal hemodynamics. After ischemia, swelling of endothelial cells and a reduction of the capillary lumen have been reported. The ensuing decline in glomerular blood flow has been denoted the "no-reflow" phenomenon (26). There are also indications of a reduced NO production in the ischemic endothelium, related to the formation of ROS (1), also contributing to the "no-reflow" phenomenon. Indeed, intravital videomicroscopy of glomerular vessels during reperfusion of a previously obstructed kidney has demonstrated an instantaneous recovery of glomerular blood flow, followed by an oscillating flow pattern of cessation and partial recovery of glomerular perfusion (31).
Although the increment in for native (neg. charged) albumin (ae=36 Å) in the present study largely paralleled the increase in for large MW (ae>55 Å) Ficoll molecules, for Ficoll of 36Å radius (Ficoll36Å), remained largely unchanged. This may be explained by the fact that Ficoll36Å was two orders of magnitude more permeable than native albumin and one order of magnitude more permeable than neutralized albumin of equivalent molecular radius across the glomerulus, as discussed at some length previously (23, 29). The relative "hyperpermeability" of Ficoll36Å will make this molecule relatively insensitive to changes in large pore number and large pore volume flow (23, 29). In the present study 60 min of I/R caused the large pore fractional fluid flow (JvL/GFR) to increase from 3.6·10–4 to 16·10–4, i.e., by 0.0012. Such changes would theoretically alter for Ficoll36Å insignificantly, i.e., by only 0.5% (from 0.10 to 0.1005), assuming an unchanged small and large pore radius. At the same time, one would observe a twofold increase in for HSA (from 0.0005 to 0.001), provided that no charge alterations had occurred and that albumin transport is confined to large pores. Again, the fact that alb increased more than twofold may be ascribed to slight reductions in microvascular charge barrier.
Although Ficoll seems to overestimate the clearance of proteins in the ae interval of 25–55 Å, there is a remarkable similarity in sieving coefficients for proteins and Ficoll in the ae interval 55–75Å. As discussed in a previous paper, this probably is due to the fact that the ae/rs ratio for this molecular size range is approximately <0.5–0.7, in which the anomalous glomerular permeation behavior of Ficoll is not manifested (2, 23). Because both proteins and Ficoll>55Å can be used to assess the large pore radius and L, we have tried to reconcile albumin with those for Ficoll55–75Å. This analysis yielded a slightly higher value for L and a slightly lower value for the large pore radius than that obtained from Ficoll data alone (Fig. 4). It thus seems that for albumin may have been (slightly) higher than expected from just extrapolating Ficoll55–75Å data back to 36 Å. The reason for this discrepancy is not obvious. It may be due to a slight overestimation of for albumin, especially in the ischemic situation, partly due to postischemic interstitial trapping of tracer that may not be easily washed out (7). could also be slightly overestimated due to trapping of proteins in the endothelium and in the mesangium. However, we believe that the fraction of trapped proteins/polysaccharides is, after all, very small since our data comply with a recent, very carefully performed micropuncture study (28). Alternatively, the large pore radius was slightly overestimated from Ficoll data alone. One reason for such an overestimation could be the predicted Ficoll hyperpermeability, for very large Ficoll molecules, i.e., when ae/rL>0.7 (for ae>70–75Å).
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in our study, the measured was insensitive to the differences in proximal tubular reabsorption among the different tracers.
The endothelial glycocalyx is thought to represent the major charge barrier in renal and systemic capillaries (10). Modifying the glycocalyx by enzymatic digestion using hyaluronidase, increased the glomerular permeability to both albumin and large size Ficoll molecules in vivo (12). Furthermore, treatment with another glycocalyx-degrading enzyme, chondroitinase, in the cooled isolated perfused kidney (cIPK), apparently reduced the thickness of the glycocalyx while increasing the permeability () to albumin. However, in those experiments the increases in for large size Ficolls were not significant (13). The results mimic the changes observed in the present study after 20 min of I/R (mild ischemia), suggesting that this relatively short I/R period may have led to alterations that could, at least partly, be attributed to alterations in the endothelial glycocalyx. The reactive oxygen species (ROS) formed during the reperfusion period (8) can alter molecular structures and induce an inflammatory response. According to a previous study infusion of H2O2 for a period of 1 h, indeed induced an increase in urine albumin excretion and in for large (>42 Å) dextrans, without causing significant changes in GFR, again indicating the induction of an increased number of large pores in the glomerular filter (32). Although the authors did not find any apparent alterations in the anionic barrier of the GBM, assessed by staining with polyethyleneimine, there could still have been marked alterations in the endothelial glycocalyx in addition to the reduced size selectivity.
The pathophysiology of the permeability changes occurring after severe ischemia (60 min of I/R) is not exactly known, but could be ascribed to a number of different mechanisms. One scenario could be a remodelling of the GBM. In isolated renal endothelial cells from rats which had undergone 30 min of ischemia, there was an increase in the proteolytic activity of matrix metalloproteinases (MMPs) (3), enzymes responsible for matrix (collagen) remodelling. Furthermore, after renal I/R both mRNA and protein levels of MMP-9 increased (4), indicating that renal ischemia could lead to degradation of the GBM, which in turn could be responsible for the increase in glomerular permeability. HSPGs are important structural contributors in the GBM, and gene modifications leading to either loss of HSPGs, or their degradation, indeed leads to proteinuria (18, 33). Thus both HSPGs and collagen may be targets of ischemic damage.
Ischemia has been shown to cause flattening and spreading of the podocyte foot processes, a condition that occurs early in the ischemic response (20). There are tight connections between the GBM and the podocytes. The architecture of the foot processes is dependent on integrin- (3
1) and -dystroglycan-mediated interactions with the GBM, -dystroglycan being linked to laminin and agrin. Recently, it has been shown that these linkages could be disrupted by ROS in in vitro experiments (14). Hence, the increased amount of ROS associated with ischemia could, in part, explain the detachment of the podocyte processes from the GBM. Whether the detachment is a primary phenomenon or a secondary consequence occurring due to remodelling of the GBM is not clear. In mice overexpressing heparanase-1, the podocytes appear flattened and have a reduced number of foot processes (33), probably secondary to the degradation of the GBM. Likewise, mice lacking the 3-integrin are born with disorganized GBM and nondifferentiated podocytes lacking foot-processes (15). Thus it seems that any condition in which GBM and the podocytes loose their interconnections may lead to alterations in the glomerular size selective barrier.
In conclusion, 60 min of I/R induced an increased formation of large pores in the glomerular filter, reflected by increases in L for both Ficoll and native albumin. However, after 20 min of I/R, the insult to the glomerular filter was less pronounced and compatible with reductions in charge selectivity, and to a lesser extent, in size selectivity, reflected by a larger increment in the glomerular permeability to albumin than to Ficoll.
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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, ischemia20 as 
, control60 as 
, and ischemia60 as open pentagons.