Temporary effects of circulating Amadori products on glomerular filtration properties in the normal mouse

I. Londoño and M. Bendayan

Department of Pathology and Cell Biology, Université de Montréal, Montreal, Quebec, Canada H3C 3J7


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Previous studies have established a preferential glomerular filtration of glycated BSA (gBSA), as well as a facilitated filtration of BSA in the presence of gBSA. We intend to determine whether these modifications are permanent or transitory. gBSA was intravenously injected into anesthetized normal mice and maintained in circulation for 30 min, 1, 2, 24, and 48 h. Five minutes before death, FITC-BSA was injected. On immunocytochemical evaluations, increased glomerular filtration of FITC-BSA was found at all circulating time points. Changes at 24 and 48 h were less pronounced. Glomerular basement membrane (GBM)-to-lumen gBSA labeling ratios were similar at all time points suggesting no accumulation of gBSA in the GBM. Seventy percent of the gBSA was cleared from the circulation and the GBM after 24 h, and 95% after 48 h. This was confirmed in experiments with radiolabeled tracers. These results suggest that the alteration in GBM permeability to BSA in the normal mouse are due to the presence of gBSA and are gradually overcome along with its clearance from circulation. In early diabetes, increasing concentrations of circulating glycated proteins could be responsible for changes in glomerular permselectivity and probably for the alteration in glomerular filtration properties leading to diabetic nephropathy.

glycated albumin; glomerular basement membrane permselectivity


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

PROLONGED EXPOSURE TO HYPERGLYCEMIA constitutes the primary cause for the development of most diabetic complications (8). During the early phases of diabetes, prior to microalbuminuria, a number of alterations take place in the kidney including an increase in glomerular filtration rate (GFR) (27, 29, 36), glomerular hypertrophy and hyperplasia (45), and changes in the extracellular matrix (1, 35, 49). Recently, modifications of structural as well as circulating proteins by glycation have drawn much attention because of their potential role in the etiopathogenesis of diabetes (8, 12, 30).

Glycation is characterized by the nonenzymatic covalent attachment of glucose residues to basic and free terminal amino acids in proteins, resulting in the formation of an unstable Schiff base adduct that rapidly progresses to a stable ketoamine derivative, the Amadori product. Further irreversible chemical reactions lead to the formation of advanced glycation end-products that accumulate in long-lived proteins, in intra and extracellular spaces. Glycation of circulating serum proteins occurs during hyperglycemic stages such as those preceding diabetes (17, 24) or associated with ageing (3, 37). Nevertheless, low levels of glycated serum proteins are also detected in young normal subjects (15-17).

A participation of Amadori proteins in diabetic glomerulosclerosis has been proposed, based on various experimental data. Glycated albumin (gBSA) has been found to stimulate mRNA expression as well as protein synthesis of fibronectin and type IV collagen in glomerular endothelial cells (14) and mesangial cells (13, 51), in vivo and in vitro, under normoglycemic conditions. The introduction of a monoclonal antibody specific for glycated albumin prevented these events and was able to retard the progression of diabetic nephropathy in diabetic db/db mice, reducing proteinuria and mesangial matrix expansion (reviewed in Ref. 12). Based on physiological studies performed on normal and microalbuminuric subjects, a relationship between glycation of circulating proteins and alterations of glomerular functions has been proposed. The first piece of evidence came from the demonstration of the preferential urinary excretion of glycated serum proteins by normal subjects (20, 32). Later on, by means of renal micropuncture methods, Sabbatini et al. (41) demonstrated that the infusion of Amadori products in the circulation of normal rats induced glomerular vasodilatation and hyperfiltration, similar to those found in streptozotocin-induced diabetic animals. Other in vivo experiments, consisting of repeated intravenous injections of glycated serum proteins into normal mice, demonstrated renal glomerular changes such as diffuse thickening of the glomerular basement membrane (GBM) (34). These changes were not observed in mice injected with nonglycated serum proteins indicating that Amadori adducts could be directly involved in the etiopathology of some diabetic vascular complications. Recently, studies from our laboratory (33) have demonstrated, by quantitative immunocytochemistry, that glycated albumin infused into the circulation of normal mice is easily filtered by the GBM. In addition, native albumin was also found to cross the glomerular wall toward the urinary space, in a facilitated fashion, when the glycated form was present in circulation (33).

In the present study, we analyzed the changes in glomerular basement membrane filtration of albumin in normal mice, at different fixed intervals after the administration of early glycated albumin, to determine whether the alterations induced by this modified protein are permanent or transitory. Changes in filtration properties were evaluated through changes in immunolabeling distributions of native albumin molecules across the GBM.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Tracers. Crystallized BSA (fraction V, Sigma, St. Louis, MO) was covalently tagged to FITC (Sigma) according to previously described procedures (5, 21). On the other hand, BSA was glycated (gBSA) in vitro according to Murtiashaw and Winterhalter (39). The gBSA was separated by boronate-affinity chromatography by using immobilized boronic acid (5, 33; Pierce, Rockford, IL). After dialysis and concentration, the gBSA was dinitrophenylated according to previously described procedures (22, 33). The monomeric fractions of haptenized BSA and gBSA molecules [FITC-BSA and dinitrophenylated native glycated albumins (DNP-gBSA), respectively] were isolated by using a centriprep-200 device (Amicon, Beverly, MA) that allows for the removal of molecules heavier than 200,000 molecular weight (albumin aggregates). Concentration of tracer solutions with centriprep-30, yielded 80-120 mg/ml solutions, devoid of free hapten molecules. FITC-BSA and DNP-gBSA were finally characterized by polyacrylamide gel electrophoresis and isoelectric focusing (33). Glycation of BSA (2-3 residues of glucose per mol of BSA) slightly affects molecular weight and charge whereas haptenization (7-10 mol hapten/mol of BSA) results in a heavier and more anionic BSA molecule (33). Nevertheless, previous studies have demonstrated that hapten-tagged BSA, once injected in normal mice, yields a GBM immunolabeling distribution identical to that obtained for endogenous albumin (33).

To prepare a BSA fraction totally depleted of gBSA molecules for control experiments, native BSA was passed twice through the boronate-affinity column. This fraction is referred as nonglycated BSA.

Native and glycated BSA were iodinated by using 125I-Na, carrier free (ICN Biochemicals, Mississauga, ON) and Iodo-beads (Pierce), according to manufacturer's instructions. Final specific activities for BSA and gBSA were 6,500 and 6,300 cpm/µg protein, respectively.

Animals. Animals used in this study were CD-1 male mice, 30-35 g body wt. These animals are normoglycemic (4.7 ± 0.3 mmol glucose/l) as determined by measurements of blood glucose levels, by using Dextrostix reagent strips (Miles, Ames, ON) before the experiments.

Experimental protocols. To evaluate the changes in relative concentrations of gBSA in the capillary lumen and the GBM with time, 10 mg of DNP-gBSA were injected into the circulation of anesthetized mice, through the cava vein. At 30 min, 24 h, and 48 h, animals (n = 4 for each time point) were anesthetized and the fixative (1% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2) was introduced in the abdominal cavity to begin the fixation process in situ. Renal cortex samples were then removed, cut in small blocks and kept immersed in the fixative for 2 h at room temperature. Tissues were postfixed with OsO4, dehydrated in ethanol, and embedded in Epon. Thin tissue sections were finally cut and mounted onto nickel grids.

Aiming to evaluate the handling of native BSA in the absence and the presence of gBSA, anesthetized animals were intravenously injected with (1) 20 µl (2.5 × 106 cpm) of either 125I-BSA or 125I-gBSA mixed with 10 mg (100 µl) of the respective cold protein, or (2) 20 µl of 125I-BSA mixed with 10 mg of cold gBSA, where cpm is counts per minute. Blood was sampled at the tail vein at various time points and counted in a Miniaxi gamma counter (Packard).

To evaluate the effect of circulating gBSA on the permselectivity properties of the GBM, anesthetized mice (n = 4, for each time point) were intravenously injected with 10 mg of gBSA. Five minutes before precise circulating time points (30 min, 1, 2, 24, and 48 h), 10 mg of FITC-BSA were intravenously injected. Renal cortexes were sampled and prepared for electron microscopy according to the conditions described above. Renal tissues were also embedded in Lowicryl K4M for the detection of endogenous serum proteins. For control groups, anesthethized mice were injected with (1) 10 mg of FITC-BSA, left in circulation for 5 min, or (2) 10 mg of nonglycated BSA, left in circulation for 60 min followed by 10 mg of FITC-BSA, 5 min before death. Renal tissues were sampled and processed for electron microscopy as described above.

Immunocytochemistry. Immunocytochemical labeling was performed by means of specific anti-hapten antibodies followed by protein A-gold. Thin tissue sections were hydrated by floating grids on distilled water for 5 min and then transferred on an aqueous saturated solution of sodium metaperiodate for 30 min. Grids were rinsed in distilled water (3 washes, 5 min each) and quenched with 150 mM glycine in PBS for 30 min and 1% ovalbumine in PBS for 30 min. Grids were then transferred to a drop of rabbit anti-DNP (1/500, Dakopatts, Dimension Laboratories, Mississauga, ON) or anti-FITC (1/50, Dakopatts) and incubated overnight at 4°C. After three washes of 5 min with PBS, grids were transferred to a drop of protein A-gold (10 nm, OD530 = 0.5) for 30 min. Finally, grids were washed with PBS and distilled water and stained with uranyl acetate before observation with a Philips 410 electron microscope.

Transferrin, like albumin, is excreted in the urine of diabetic patients at early stages (31). Thus, in another set of experiments, Lowicryl-embedded renal tissues from gBSA-injected and noninjected mice were used for the detection of endogenous circulating albumin and transferrin. A similar immunocytochemical protocol to the one described above was performed, by using anti-mouse albumin (1:200, Cappel, Organon Teknika, West Chester, PA) or anti-transferrin antibodies (1:100, Cappel), respectively.

To evaluate the specificity of the different immunolabelings, the specific antibodies were 1) omitted, 2) substituted by diluted normal rabbit serum, and 3) tested on tissue sections from noninjected animals.

Morphometrical evaluation. Video-recorded images (100-150 frames at 155,000 final magnification) from at least three renal glomeruli per animal (n = 4 animals for each time point), were evaluated by morphometry by using a Videoplan 2 image-processing system (Carl Zeiss, Toronto, ON). Labeling densities were evaluated on capillary lumen and GBM, by direct planimetry and gold particle counting. To estimate variations in amount of tracers in each compartment occurring with time, ratios of labeling densities between compartments were calculated at each time point. Labeling distributions throughout the GBM were obtained by evaluating the distances between each gold particle and the abluminal endothelial membrane and the thickness of the GBM at this very same point. The ratio values between these two measurements, varying between 0 and 1, were represented as histogram distributions in function of the gold particles frequency (4, 33). Three to five hundred gold particles were evaluated per animal.

Statistical analysis. The t-test was performed to compare labeling densities between time points. The Mann-Whitney rank sum test, at 95% level of confidence, was used to compare the means of labeling distributions between different experimental time points, and the Kruskal-Wallis test was used to test the uniformity between labeling distributions at each time point.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Labeling by gold particles revealed antigenic sites for the injected hapten-tagged albumin molecules in the blood vessel lumen, the interstitial space and, intracellularly, in endocytic compartments of endothelial and epithelial cells of renal tissues. Similar labeling patterns were observed for both tracers, the DNP-gBSA and the FITC-BSA. These patterns were also found to be similar to those of endogenous albumin as reported previously (4, 33).

Figure 1 shows the labeling for FITC-BSA in the absence (Fig. 1A) and presence (Fig. 1B) of circulating gBSA. In the absence of gBSA, labeling for FITC-BSA was found mainly on the subendothelial side of the GBM. This reflects the restrictive filtration properties of the GBM. On the other hand, in the presence of circulating gBSA (Fig. 1B), gold particles revealing FITC-BSA were distributed through the entire thickness of the GBM, some particles also being detected in the urinary space, between podocytes. In contrast to the normal condition, this distribution indicates the passage of BSA through the glomerular wall and its presence in the primary urine, reflecting alterations in the filtration properties of the GBM.


View larger version (152K):
[in this window]
[in a new window]
 
Fig. 1.   Immunolabeling for FITC-BSA in the glomerular wall. A: in the absence of circulating glycated albumin, gold particles, revealing FITC-BSA antigenic sites, are restricted to the subendothelial side of the glomerular basement membrane (GBM). B: in the presence of circulating glycated albumin (30 min after injection), FITC-BSA is distributed through the entire thickness of the GBM. CL, capillary lumen; E, endothelial cell; P, podocyte. Magnifications: ×70,000 (A), ×40,000 (B).

Kinetics of injected gBSA. Labeling for DNP-gBSA decreased with time in both the capillary lumen and the GBM (Fig. 2). On the basis of morphometrical determinations, this reduction was estimated to 70% at 24 h and 95% at 48 h, compared with the value at 30 min (Table 1). Intravenously injected iodinated gBSA was rapidly removed from the circulation in the first 4 h and more slowly after 12 and 24 h. Elimination curves follow similar patterns for 125I-BSA, for 125I-gBSA, and for 125I-BSA in the presence of gBSA (not shown). The remaining radioactivity for 125I-gBSA at 24 h was 25-28% of the amount present at 30 min. These data are consistent with those obtained by morphometrical evaluations (Table 1).


View larger version (96K):
[in this window]
[in a new window]
 
Fig. 2.   Immunolabeling for DNP-gBSA in the glomerular wall at different time points of circulation. A: 30 min; B: 24 h; C: 48 h. At 30 min (A), labeling by gold particles is intense in the CL and the GBM. Labeling intensities decrease in both compartments after 24 (B) and 48 h (C). Magnification: ×28,000 (A), ×32,000 (B), ×30,000 (C).


                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Changes of dinitrophenylated glycated albumin labeling densities in the capillary lumen and the glomerular basement membrane with time

Similar GBM-to-capillary lumen labeling density ratios were found for DNP-gBSA at 30 min, 24 and 48 h (Table 1). This indicates similar proportions of DNP-gBSA in these two compartments at the time points evaluated. Moreover, similar labeling density ratios between circulation time points (30% in the lumen vs. 29% in the GBM at 24 h and 5% in the lumen vs. 6% in the GBM at 48 h, compared with values at 30 min) were also detected, indicating that gBSA disappears at similar rates from blood and GBM.

Effect of the presence of gBSA on FITC-BSA distribution across the GBM in normal mice. Figures 3 and 4 show the labeling distribution for FITC-BSA and that for endogenous transferrin, at different time points of gBSA circulation. The control experiments (no gBSA in circulation), consisting in the intravenous injection of either FITC-BSA (Fig. 3A) or nonglycated BSA for 1 h followed by FITC-BSA for 5 min (Fig. 3B), show asymmetrical distributions with peaks of labeling on the subendothelial side of the GBM that decrease slowly toward the epithelial side. These distributions resemble that of endogenous albumin in normal mice (33). After 30 min of circulation of gBSA, a shift in the labeling distribution for FITC-BSA was observed, with higher percentages of gold particles toward the epithelial side (Fig. 3C). These changes in labeling distribution are statistically significant (P < 0.05) according to the Mann-Whitney nonparametric test. After 1 and 2 h of circulation time, the distributions remained shifted to the right, the one at 2 h displaying the most significant changes (P < 0.05, Mann-Whitney U-test), as illustrated in Fig. 3, D and E. This is also stressed in Fig. 5, where the mean R values of the different labeling distributions are reported as a function of time. After 24 and 48 h, FITC-BSA labeling distributions still differed from the control (P < 0.05) but were less modified than the one at 2 h (Fig. 3, E-G, and 5).


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 3.   FITC-BSA labeling distributions in the GBM of mice injected with glycated and nonglycated BSA at different circulation time points. The histograms represent the percentage of gold particles found at each interval of the ratio value R. R, varying between 0 and 1, is defined by the following formula: R = (distance between gold particle and endothelial plasma membrane)/(distance between endothelial and epithelial plasma membranes). The mean value of R for each distribution is reported in the abscissa. Controls (no gBSA in circulation) correspond to mice injected with FITC-BSA for 5 min (Control A), and mice injected with nonglycated BSA, left in circulation for 1 h, then injected with FITC-BSA for 5 min (Control B). The other labeling distributions correspond to mice injected with gBSA maintained in circulation for 30 min (C), 1 h (D), 2 h (E), 24 h (F), and 48 h (G) and then injected with FITC-BSA 5 min before death (n = 4 for each time point).



View larger version (21K):
[in this window]
[in a new window]
 
Fig. 4.   Endogenous transferrin labeling distributions in the GBM of control mice and mice injected with gBSA for 30 min and 24 h. R values are defined as in legend of Fig. 3. A: transferrin labeling distribution in noninjected animals; B and C: transferrin labeling distributions of animals injected with gBSA for 30 min and 24 h, respectively.



View larger version (17K):
[in this window]
[in a new window]
 
Fig. 5.   Changes of mean R values derived from distribution histograms for FITC-BSA and endogenous transferrin in relation to time of circulation of gBSA. R values, reflecting changes in GBM permselectivity to native albumin and endogenous transferrin, increase rapidly at 30 min and remain high until 2 h. At 24 and 48 h, R values are still significantly different from the control. Statistical significances are as follows: *P < 0.05 compared with the control (no gBSA in circulation); §P < 0.05 with respect to the 30-min value; dagger P < 0.05 with respect to the 2-h value. The frame in dots is an enlarged view of the curves in the interval from 0 to 2 h.

Effect of the presence of gBSA on endogenous transferrin distribution across the GBM in normal mice. The glomerular distributions of endogenous transferrin were also evaluated to determine whether the presence of circulating glycated albumin could affect the renal handling of other serum proteins. A significant change in labeling distribution across the GBM was detected for serum transferrin at 30 min (P < 0.05) compared with the control (normal animal, noninjected with gBSA, Figs. 4, A and B, and 5). The transferrin labeling distribution at 24 h of circulation of gBSA (Fig. 4C) is not statistically different from that of the control, suggesting a return to a normal condition.

The observed changes in labeling distribution cannot be associated with an effect of increasing blood protein concentration, because 20 mg of native albumin (twice the concentration of the modified BSA) injected into circulation were unable to reproduce the alteration in labeling distribution caused by the gBSA (results not shown).

The results obtained were consistent for all the animals in each particular group. Indeed, labeling distributions among animals in each group were not statistically different, indicating a uniform effect of gBSA on renal function.

Morphological changes in glomerular ultrastructure such as fusion of podocytes and/or GBM thickening, known to affect the glomerular permselectivity (36), were not detected in any of the renal tissues derived from the injected mice at any time point. On the other hand, evidence of reabsorption of gBSA and BSA by the proximal and distal tubular epithelial cell confirmed our previously reported data (5).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

During hyperglycemia, glycation of structural and circulating serum proteins constitutes a critical event associated with the glomerular dysfunction accompanying diabetic nephropathy (8, 12, 30). Albumin, a major serum constituent involved in osmotic regulation and acting as carrier of lipids, hormones, and drugs, undergoes glycation when exposed to increased concentrations of glucose. In fact, the initial compounds generated during albumin glycation, the Amadori adducts, are abundant in plasma and urine of hyperglycemic subjects (9, 12, 24).

In the present study, glycated albumin, prepared in vitro, was introduced into the circulation of normal mice at final concentrations (10-12%) that approach those found in moderately diabetic subjects. We performed the experiments on normal mice to circumvent alterations due to exposure to a hyperglycemic milieu such as activation of the polyol pathway, modification of cellular redox state, activation of protein kinase C, and oxygen radical formation. Changes in renal hemodynamics including glomerular hyperfiltration occurring in early diabetes, which could also affect albumin passage through the glomerular basement membrane, are also bypassed.

Previous studies using the immunocytochemical approach have revealed endogenous albumin in the glomerular wall, particularly restricted to the subendothelial side of the GBM (4, 40). Based on morphometrical evaluation, this labeling pattern on the GBM can be represented by histograms which, in normal conditions, display distributions with a peak of labeling on the subendothelial side of the GBM. This reflects the retention of circulating proteins at this level, which is characteristic of a normal glomerular permselectivity function. When gBSA was introduced into the circulation of normal mice, the labeling was detected over the entire thickness of the GBM, yielding a labeling histogram with a more centered peak, significantly different from the left-sided distribution obtained for injected native albumin (33). We proposed that these changes are consistent with an enhanced glomerular permeability to macromolecules (33). When monitoring the native and the nonglycated albumins injected into the circulation of mice, in the presence of gBSA, the nonmodified albumin also produced centered labeling distributions indicating that it penetrates deep into the GBM. In fact, under this condition, nonmodified albumin was also detected in the urinary space and in the endocytic compartment of renal tubules (4, 33). In the present study, similar altered passage through the GBM was also observed for native albumin in the presence of gBSA, at all circulating time points. The most significant changes took place 2 h after introduction of the gBSA into the circulation. According to data in Figs. 3 and 5, after 2 h of gBSA in circulation, changes in glomerular filtration for BSA show a gradual regression toward a normal distribution. This suggests a partial recovery of GBM permselectivity properties. However, because labeling distribution at 48 h still differed from the control, we cannot assure total recovery. Labeling densities of circulating gBSA decreased 70% after 24 h and 95% after 48 h, according to morphometrical determinations. The value at 24 h was found to be similar to the one obtained by using radioiodinated tracers. We thus propose that the alterations induced by gBSA on the GBM filtrating properties are transitory and are related to the levels of circulating gBSA. This result is consistent with the fact that controlling the levels of Amadori adducts by specific antibodies (12) or by strict control of hyperglycemia (usually evaluated by HbA1c levels, an Amadori adduct of hemoglobin) is associated with a reversal of proteinuria (45).

Disappearance rates of gBSA from circulation and from the GBM are similar indicating that there is no accumulation of gBSA in the GBM. Previous studies have demonstrated similar amounts of BSA and gBSA in the mesangial matrix of renal glomeruli of injected mice, which also coincide with values obtained for the endogenous albumin detected in this compartment (steady-state amounts) (33). Thus deposition of gBSA in the GBM or in the mesangial matrix at the time points evaluated cannot explain alterations on glomerular filtration.

Glycation induces changes in the functional properties of serum albumin. In fact, glycation alters the albumin binding affinity for bilirubin and fatty acids (43), decreases its antioxidant properties (7), its hypoagreggating effect on red blood cells (10), and increases its calcium-binding capacity (46). During glycation, glucose reacts covalently with basic amino acids, predominantly lysine residues (16, 24), which can be involved in ligand recognition (43). This reaction induces a decrease in the isoelectric point leading to a more anionic molecule (20, 32, 33). However, the enhanced filtration of glycated albumin through the GBM cannot be explained by changes in its physicochemical properties (19, 20, 32, 48) because the modified molecule is heavier and more anionic than the nonmodified one. Structural changes in the protein have thus been proposed to explain its altered handling by the GBM (19, 32) as well as changes in ligand recognition (43). Some authors suggest that factors such as protein hydration would affect the protein flexibility and hydrodynamics and its ability to interact with other proteins (6, 26, 50), and likely with interstitial components. Watala et al. (47) reported a glycation-induced change in the tryptophan residue of albumin, which becomes more exposed to the water surrounding solution. How spatial changes in this hydrophobic residue can affect the behavior of albumin in its passage through the GBM is unclear.

The small amounts of circulating albumin that cross the GBM in normal conditions could correspond to the glycated forms of albumin present in normoglycemic subjects (3-8% of total circulating albumin). According to this and previous studies (20, 32, 33, 41, 48), the presence of the glycated form in circulation also allows the nonglycated albumin to traverse the GBM more easily. In normal subjects, native albumin is rapidly reabsorbed by the proximal tubules, whereas the filtered gBSA seems to be excluded from tubular reabsorption (5, 20, 32). At very early stages of diabetes, the efficient tubular reabsorption prevents the excretion of albumin. However, early tubular dysfunction (1, 44) or tubular saturation in prediabetic subjects, combined to the glomerular protein hyperfiltration that could be induced by glycated albumin, must contribute to the pathogenesis of diabetic nephropathy. Therefore, the presence of the glycated albumin in circulation may contribute to the pathogenesis of diabetic nephropathy. In the course of diabetes, production of albumin Amadori adducts as early as on two days of hyperglycemia (16) could thus contribute to the early filtration of glycated and nonglycated forms of albumin.

An interesting aspect is the consideration of critical concentrations of circulating glycated albumin affecting GBM properties. We were not able to detect alterations in GBM permselectivity at the concentrations of circulating glycated albumin present in normal subjects (3-8%). GBM labeling distributions of endogenous and injected native albumin in normal animals, showed, accordingly, retention at the subendothelial side of GBM. Similarly, the injection of a nonglycated albumin fraction, free of Amadori products, before the injection of native albumin, did not alter the GBM permselectivity. However, the concentrations of injected gBSA remaining after 24 and 48 h in our experiments seem to be sufficient to affect GBM properties. Whether this effect is mediated by the glycated albumin itself or through stimulation of signaling events in glomerular cells (12) remains to be resolved. Glycated albumin has been shown to enhance nitric oxide (NO) synthase activity and NO gene expression in vascular endothelial cells (2) and smooth muscle cells (VSMC) (25). Because NO is an important regulator of the vascular function, local changes in the glomerular wall could affect the GBM permselectivity function. Hattori et al. (25) have demonstrated that the gBSA-induced NO release from cultured VSMC is potentiated by interferon and results from the induction of inducible-NO synthase gene transcription by activation of the transcription factor nuclear factor kappa B (NFkappa B). NFkappa B has a critical role in inducing genes involved in cell activation and in inflammatory responses. It has thus been proposed that glycated albumin could constitute an inflammatory mediator in diabetic vasculopathies (11, 25). In addition, studies performed with diabetic patients have demonstrated that Amadori albumin correlate with markers of endothelial dysfunction (42). Glycated proteins could thus induce the release of mediators that can locally affect the vascular hemodynamics (2, 23, 28, 38, 42) leading to glomerular dysfunction.

In early diabetic patients, changes in hemodynamic factors such as increased glomerular plasma flow and transcapillary pressure-gradient driving forces have been proposed to be associated with an enhanced protein filtration across the GBM (29). Furthermore, in particular physiological conditions such as exercise and fever, the temporary loss of glomerular permselectivity, with induction of proteinuria, seems to also be related to changes in hemodynamic factors. In the case of circulating glycated albumin, previous studies by Sabbatini et al. (41) have demonstrated that normal animals injected with glycated serum proteins manifested changes in hemodynamic parameters, mimicking those found in diabetic animals. That changes in hemodynamic parameters would be responsible for the temporary effect that glycated albumin exerts on GBM permselectivity of normal mice as well as of the facilitated passage of other circulating serum proteins, concomitantly with the glycated form, remains to be demonstrated.

In conclusion, we have demonstrated that the presence of glycated albumin in circulation alters the GBM filtration properties in a temporary fashion, inducing an enhanced passage of not only the native albumin but also that of other circulating proteins.


    ACKNOWLEDGEMENTS

The authors thank Dr. P. Vinay, Faculty of Medicine, for support, Dr. Lucian Ghitescu for collaboration in the experiment with iodinated tracers, and Diane Gingras for excellent technical assistance.


    FOOTNOTES

The Medical Research Council of Canada supported this work.

Address for reprint requests and other correspondence: M. Bendayan, Dept. Pathology and Cell Biology, Université de Montréal, C.P. 6128, Succ. Centre-Ville, Montréal, Québec, H3C 3J7, Canada (E-mail: moise.bendayan{at}umontreal.ca).

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.

Received 13 March 2000; accepted in final form 1 September 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Abrass, CK. Diabetic proteinuria: glomerular or tubular in origin? Anal Nephrol 4: 337-346, 1984.

2.   Amore, A, Cirina P, Mitola S, Peruzzi L, Gianoglio B, Rabbone I, Sacchetti C, Cerutti F, Guillo C, and Copp R. Non-enzymatically glycated albumin (Amadori adducts) enhances nitric oxide synthase activity and gene expression in endothelial cells. Kidney Int 51: 27-35, 1997[ISI][Medline].

3.   Bakala, H, Verbeke P, Perichon M, Corman B, and Schaeverbeke J. Glycation of albumin with aging and diabetes in rats: changes in its renal handling. Mech Ageing Dev 78: 63-71, 1995[ISI][Medline].

4.   Bendayan, M, Gingras D, and Charest P. Distribution of endogenous albumin in the glomerular wall of streptozotocin-induced diabetic rats as revealed by high-resolution immunocytochemistry. Diabetologia 29: 868-875, 1986[ISI][Medline].

5.   Bendayan, M, and Londoño I. Reabsorption of native and glycated albumin by renal proximal tubular epithelial cells. Am J Physiol Renal Fluid Electrolyte Physiol 271: F261-F268, 1996[Abstract/Free Full Text].

6.   Bone, S, and Pethig R. Dielectric studies of protein hydration and hydration-induced flexibility. J Mol Biol 181: 323-326, 1985[ISI][Medline].

7.   Bourdon, E, Loreau N, and Blache D. Glucose and free radicals impair the antioxidant properties of serum albumin. FASEB J 13: 233-244, 1999[Abstract/Free Full Text].

8.   Brownlee, M, Cerami A, and Vlassara H. Advanced glycosylation end products in tissue and the biochemical basis of diabetic complications. N Engl J Med 318: 1315-1321, 1988[ISI][Medline].

9.   Brownlee, M, Vlassara H, and Cerami A. Non-enzymatic glycosylation and the pathogenesis of diabetic complications. Ann Intern Med 101: 527-537, 1984[ISI][Medline].

10.   Candiloros, H, Muller S, Ziegler O, Donner M, and Drouin P. Role of albumin glycation on the erythrocyte aggregation: an in vitro study. Diabet Med 13: 646-650, 1996[ISI][Medline].

11.   Cohen, MP, Clements RS, Cohen JA, and Shearman CW. Glycated albumin promotes a generalized vasculopathy in the db/db mouse. Biochem Biophys Res Commun 218: 72-75, 1996[ISI][Medline].

12.   Cohen, MP, and Ziyadeh FN. Role of Amadori-modified nonenzymatically glycated serum proteins in the pathogenesis of diabetic nephropathy. J Am Soc Nephrol 7: 183-190, 1996[Abstract].

13.   Cohen, MP, and Ziyadeh FN. Amadori glucose adducts modulate mesangial cell growth and collagen gene expression. Kidney Int 45: 475-484, 1994[ISI][Medline].

14.   Cohen, MP, Wu VY, and Cohen JA. Glycated albumin stimulates fibronectin and collagen IV production by glomerular endothelial cells under normoglycemic conditions. Biochem Biophys Res Commun 239: 91-94, 1997[ISI][Medline].

15.   Day, JF, Thorpe SR, and Baynes JW. Nonenzymatically glucosylated albumin. In vitro preparation and isolation from normal human serum. J Biol Chem 254: 595-597, 1979[Abstract].

16.   Day, JF, Thornburg RW, Thorpe SR, and Baynes JW. Non-enzymatic glucosylation of rat albumin. Studies in vitro and in vivo. J Biol Chem 254: 9394-9400, 1979[ISI][Medline].

17.   Dolhofer, R, and Wieland OH. Increased glycosylation of serum albumin in diabetes mellitus. Diabetes 29: 417-422, 1980[Abstract].

18.   Donnelly, SM. Accumulation of glycated albumin in end-stage renal failure: evidence for the principle of "physiological microalbuminuria." Am J Kidney Dis 173: 62-66, 1996.

19.   Ghiggeri, GM, Candiano G, Delfino G, and Queirolo C. Conformational mediated renal selectivity towards albumin in diabetes mellitus. Diabetes Metab 12: 68-73, 1986[ISI].

20.   Ghiggeri, GM, Candiano G, Delfino G, Bianchini F, and Queirolo C. Glycosyl albumin and diabetic microalbuminuria: demonstration of an altered renal handling. Kidney Int 25: 565-570, 1984[ISI][Medline].

21.   Ghitescu, L, and Bendayan M. Hapten-tagged plasma proteins as immunocytochemical probes for the study of vascular permeability. Microsc Res Tech 22: 392-401, 1992[ISI][Medline].

22.   Ghitescu, L, and Bendayan M. Transendothelial transport of serum albumin: a quantitative immunocytochemical study. J Cell Biol 117: 742-755, 1992.

23.   Giugliano, D, Ceriello A, and Paolisso G. Diabetes mellitus, hypertension, and cardiovascular disease: which role for oxidative stress. Metabolism 44: 363-368, 1995[ISI][Medline].

24.   Guthrow, CE, Morris MA, Day JF, Thorpe SR, and Baynes JW. Enhanced non-enzymatic glucosylation of human serum albumin in diabetes mellitus. Proc Natl Acad Sci USA 76: 4258-4261, 1979[Abstract].

25.   Hattori, Y, Banba N, Gross SS, and Kasai K. Glycated serum albumin-induced nitric oxide production in vascular smooth muscle cells by nuclear factor kappaB-dependent transcriprional activation of inducible nitric oxide synthase. Biochem Biophys Res Commun 259: 128-132, 1999[ISI][Medline].

26.   Helms, V, and Wade RC. Hydration energy landscape of the active site cavity in cytochrome P450cam. Proteins 32: 381-396, 1998[ISI][Medline].

27.   Hostetter, TH. Pathogenesis of diabetic glomerulopathy: hemodynamic considerations. Semin Nephrol 10: 219-227, 1990[ISI][Medline].

28.   Ido, Y, Kilo C, and Williamson JR. Interactions between the sorbitol pathway, non-enzymatic glycation, and diabetic vascular dysfunction. Nephrol Dial Transplant 11: 72-75, 1996[ISI][Medline].

29.   Jensen, PK, Christiansen JS, Steven K, and Parving HH. Renal function in streptozotocin-diabetic rats. Diabetologia 21: 409-414, 1981[ISI][Medline].

30.   Kennedy, L, and Baynes JW. Non-enzymatic glycosylation and the chronic complications of diabetes: an overview. Diabetologia 26: 93-98, 1984[ISI][Medline].

31.   Konen, J, Shihabi Z, and Newman J. The association of non-insulin-dependent diabetes mellitus and hypertension with urinary excretion of albumin and transferrin. Am J Kidney Dis 22: 791-797, 1993[ISI][Medline].

32.   Layton, GJ, and Jerums G. Effect of glycation of albumin on its renal clearance in normal and diabetic rats. Kidney Int 33: 673-676, 1988[ISI][Medline].

33.   Londoño, I, Ghitescu L, and Bendayan M. Glomerular handling of circulating glycated albumin in the normal mouse kidney. Am J Physiol Renal Fluid Electrolyte Physiol 268: F913-F921, 1995[Abstract/Free Full Text].

34.   McVerry, BA, Fisher C, Hopp A, and Huehns ER. Production of pseudodiabetic renal glomerular changes in mice after repeated injections of glucosylated proteins. Lancet 1: 738-740, 1980[ISI][Medline].

35.   Meyer, TW. Mechanisms of proteinuria in diabetes renal disease. Semin Nephrol 10: 194-202, 1990[ISI][Medline].

36.   Mogensen, CE, Steffes MW, Deckert T, and Christiansen JS. Functional and morphological renal manifestations in diabetes mellitus. Diabetologia 21: 89-93, 1981[ISI][Medline].

37.   Monnier, VM, Stevens VJ, and Cerami A. Maillard reactions involving proteins and carbohydrates in vivo: relevance to diabetes mellitus and aging. Prog Food Nutr Sci 5: 315-327, 1981[ISI][Medline].

38.   Mullarkey, CJ, Edelstein D, and Brownlee M. Free radical generation by early glycation products: a mechanism for accelerated atherogenesis in diabetes. Biochem Biophys Res Commun 173: 932-939, 1990[ISI][Medline].

39.   Murtiashaw, MH, and Winterhalter KH. Nonenzymatic glycation of human albumin does not alter its palmitate binding. Diabetologia 29: 366-370, 1986[ISI][Medline].

40.   Ryan, GB, and Karnosky MJ. Distribution of endogenous albumin in the rat glomerulus: role of hemodynamic factors in glomerular barrier function. Kidney Int 9: 36-45, 1976[ISI][Medline].

41.   Sabbatini, M, Sansone G, Uccello F, Giliberti A, Conte G, and Andreucci VE. Early glycosylation products induce glomerular hyperfiltration in normal rats. Kidney Int 42: 875-881, 1992[ISI][Medline].

42.   Schalkwijk, CG, Ligtvoet N, Twaalfhoven H, Jager A, Blaauwgeers HG, Schlingemann RO, Tarnow L, Parving HH, Stehouwer CD, and van Hinsbergh VW. Amadori albumin in type 1 diabetic patients: correlation with markers of endothelial function, association with diabetic nephropathy, and localization in retinal capillaries. Diabetes 48: 2446-2453, 1999[Abstract].

43.   Shaklai, N, Garlick RL, and Bunn HF. Nonenzymic glycosylation of human serum albumin alters its conformation and function. J Biol Chem 259: 3812-3817, 1979[Abstract/Free Full Text].

44.   Tucker, BJ, Rasch R, and Blantz RC. Glomerular filtration and tubular reabsorption of albumin in preproteinuric and proteinuric diabetic rats. J Clin Invest 92: 686-694, 1993[ISI][Medline].

45.   Viberti, GC, and Keen H. The pattern of proteinuria in diabetes mellitus: relevance to pathogenesis and prevention of diabetic nephropathy. Diabetes 33: 686-692, 1984[ISI][Medline].

46.   Vorum, H, Fisker K, Otagiri M, Pedersen AO, and Kragh-Hansen U. Calcium ion binding to clinically relevant chemical modifications of human serum albumin. Clin Chem 41: 1654-1661, 1995[Abstract/Free Full Text].

47.   Watala, C, Gwozdzinski K, and Malek M. Direct evidence for the alterations in protein structure and conformation upon in vitro nonenzymatic glycosylation. Int J Biochem 24: 1295-1302, 1993[ISI].

48.   Williams, SK, Devenny JJ, and Bitensky MW. Micropinocytic ingestion of glycosylated albumin by isolated microvessels: possible role in pathogenesis of diabetic microangiopathy. Proc Natl Acad Sci USA 78: 2393-2397, 1981[Abstract].

49.   Wu, F, Setty C, Mauer P, Killen SM, Nagase H, Michael AF, and Tsilibari EC. Altered kidney matrix gene expression in early stages of experimental diabetes. Acta Anat (Basel) 158: 155-165, 1997[Medline].

50.   Zanotti, JM, Bellisent-Funel MC, and Parello J. Hydration-coupled dynamics of proteins studied by neutron scattering and NMR: the case of the typical EF-hand calcium-binding parvalbumin. Biophys J 76: 2390-2411, 1999[Abstract/Free Full Text].

51.   Ziyadeh, FN, Han DC, Cohen JA, Guo J, and Cohen MP. Glycated albumin stimulates fibronectin gene expression in glomerular mesangial cells: involvement of the transforming growth factor-beta system. Kidney Int 53: 631-638, 1998[ISI][Medline].


Am J Physiol Renal Fluid Electrolyte Physiol 280(1):F103-F111
0363-6127/01 $5.00 Copyright © 2001 the American Physiological Society