Are filtered plasma proteins processed in the same way by the kidney?

In order to understand the mechanism of albuminuria we have explored how other plasma proteins are processed by the kidney as compared to inert molecules like Ficolls. When fractional clearances are plotted versus protein radius there is a remarkable parallelism between protein (molecular weight range 30-150kDa) clearance in healthy controls, in Dent's disease, in nephrotic states and the clearance of Ficolls. Although there are significant differences in the levels of fractional clearances in these states. Dent's disease results in a 2-fold increase in the fractional clearance of proteins as compared to healthy controls whereas in nephrotic states there is a further 3-fold increase in fractional clearance. Previous thinking that albumin uptake was controlled primarily by the megalin/cubilin receptor does not explain the albumin urinary excretion data and is therefore an incorrect concept. Protein clearance in nephrotic states approach the fractional clearance of inert Ficolls for a given radius. It therefore appears that there are two pathways processing these proteins. A low capacity pathway associated with megalin/cubilin that degrades filtered protein (that is inhibited in Dent's disease) and a high capacity pathway that retrieves the filtered protein and returns it to the blood supply (without retrieval nephrotic protein excretion will occur and this will account for hypoproteinemia). On the other hand low molecular weight proteins (<20kDa) are processed entirely differently by the kidney. They are not retrieved but are comprehensively degraded in the kidney with the degradation products predominantly returned to the blood supply.

Controversies in nephrology: response to 'renal albumin handling, facts, and artifacts'.

For 40 years indirect measurements of the glomerular sieving coefficient of albumin yielded very low values. The first direct measurement by 2-photon microscopy by Russo et al (Kidney Int (2007) 71, 504-513) gives values 50-times higher. This demonstrated that relatively large quantities of albumin are normally filtered based on size selectivity alone. Most of this albumin is retrieved and returned to the blood supply. These new discoveries represent a paradigm shift in our understanding of albumin processing by the kidney. They also serve to explain several anomalous aspects of previous studies on glomerular filtration and mechanism of albuminuria and support the fact that glomerular charge selectivity is not a major factor controlling glomerular permselectivity.

The normal kidney filters nephrotic levels of albumin retrieved by proximal tubule cells: retrieval is disrupted in nephrotic states.

The origin of albuminuria remains controversial owing to difficulties in quantifying the actual amount of albumin filtered by the kidney. Here we use fluorescently labeled albumin, together with the powerful technique of intravital 2-photon microscopy to show that renal albumin filtration in non-proteinuric rats is approximately 50 times greater than previously measured and is followed by rapid endocytosis into proximal tubule cells (PTCs). The endocytosed albumin appears to undergo transcytosis in large vesicles (500 nm in diameter), identified by immunogold staining of endogenous albumin by electron microscopy, to the basolateral membrane where the albumin is disgorged back to the peritubular blood supply. In nephrotic rats, the rate of uptake of albumin by the proximal tubule (PT) is decreased. This is consistent with reduced expression of clathrin, megalin, and vacuolar H(+)-ATPase A subunit, proteins that are critical components of the PT endocytotic machinery. These findings strongly support the paradigm-shifting concept that the glomerular filter normally leaks albumin at nephrotic levels. Albuminuria does not occur as this filtered albumin load is avidly bound and retrieved by PTCs. Dysfunction of this retrieval pathway leads to albuminuria. Thus, restoration of the defective endocytotic and processing function of PT epithelial cells might represent an effective strategy to limit urinary albumin loss, at least in some types of nephrotic syndrome.

Glomerular permselectivity factors are not responsible for the increase in fractional clearance of albumin in rat glomerulonephritis.

The increased fractional clearance of albumin in nephrotic states has long been attributed to glomerular permselectivity dysfunction. Using radiolabeled rat serum albumin, transferrin, IgG, and polydisperse Ficoll, this study investigated the changes in their in vivo fractional clearance in puromycin aminonucleoside nephrosis and anti-glomerular basement membrane glomerulonephritis. In control rats the lack of charge selectivity was confirmed by the demonstration that carboxymethyl Ficoll (valence approximately -39) had the same fractional clearance as uncharged Ficoll. Both diseases exhibited similar effects on fractional clearance measurements suggesting an underlying common mechanism. In disease, there was good agreement between the fractional clearance of proteins determined by radioactivity as compared to those determined by radioimmunoassay. A small increase in the fractional clearance for IgG was evident in disease as compared to controls, which mirrored the change in the equivalent size Ficoll, suggesting that the increase is because of the development of a small proportion of large pores in the glomerular capillary wall. There was no increase, however, in the fractional clearance of Ficoll of equivalent size to albumin in either disease, yet the fractional clearance of the albumin increased by 12 to 14 times as determined by radioactivity and 4500 to 6600 times as determined by radioimmunoassay. This study demonstrates that glomerulonephritis is not a disease associated with changes in glomerular permeability to albumin but is because of alterations in albumin processing by cells distal to the glomerular basement membrane. It is also apparent that approaches to glomerular pathology and proteinuria as risk factors in renal disease must be reassessed.

The return of glomerular filtered albumin to the rat renal vein--the albumin retrieval pathway.

BACKGROUND: Recent studies have demonstrated that the normal glomerular capillary wall (GCW) is not charge selective to albumin. This means that albumin flux across the GCW is high. This has been confirmed in studies where albumin uptake by the tubules has been inhibited. Therefore, there must be a high capacity postglomerular retrieval pathway in normal kidneys that returns filtered albumin back to the blood supply. METHODS: This study identifies the presence of glomerular filtered albumin in the renal vein from the analysis of the decrease of radioactivity in the venous effluent after the injection of a pulse of tritium labeled albumin into the renal artery in vivo and in the isolated perfused kidney (IPK). RESULTS: The glomerular filtered albumin is returned to the blood supply by a high capacity pathway that transports this albumin at a rate of 1830+/-292 microg/min rat kidney (n= 14) (mean+/-SEM). This pathway has been identified under physiological conditions in vivo and in the IPK. The pathway is specific for albumin as it does not occur for horseradish peroxidase (HRP). The pathway is inhibited in a non-filtering kidney. The pathway is also inhibited by NH4Cl, an inhibitor of protein uptake. CONCLUSIONS: The high capacity retrieval pathway for albumin is most likely associated with transtubular cell transport. It is also apparent that most albuminuric states could be accounted for by the malfunctioning of this pathway without resorting to any change in glomerular permselectivity.

Immuno-unreactive albumin excretion increases in streptozotocin diabetic rats.

We previously showed that albumin is fragmented (>90%) during renal passage to low-molecular-weight (<10 kd) peptides. The aim of the present study was to document the renal handling of albumin in experimental diabetes. Tritium-labeled albumin was infused into control and streptozotocin (STZ) diabetic rats during 7 days. Urinary radioactivity, assessed by size exclusion chromatography, revealed a major peak corresponding to low-molecular-weight, albumin-derived fragments and a minor peak corresponding to intact albumin or high-molecular-weight, albumin-derived protein. The fractional clearance of albumin, calculated from total radioactivity measurements, was at least 100-fold greater than the fractional clearance of albumin determined by radioimmunoassay (RIA) for control and diabetic rats. This result was mainly because low-molecular-weight, albumin-derived fragments were not detected by RIA. The fractional clearance of high-molecular-weight, albumin-derived protein was 2- to 10-fold greater than the fractional clearance determined by RIA. The immuno-unreactive high-molecular-weight, albumin-derived protein (called ghost albumin), characterized by size exclusion chromatography and high-performance liquid chromatography, was present in control and diabetic rat urine. Ghost albumin excretion rate was enhanced 11-fold after 8 weeks of STZ diabetes as compared with aged-matched controls. This study shows that renal modification resulting in low-molecular-weight and high-molecular-weight components of albumin is a major contributor to the renal handling of albumin. The results indicate that excretion of modified albumin is increased in STZ rats as compared with albumin detected by conventional RIA. Long-term studies are necessary to evaluate the potential of ghost albumin as a new marker for the assessment of urinary albumin in diabetes.

Ramipril and aminoguanidine restore renal lysosomal processing in streptozotocin diabetic rats.

AIMS/HYPOTHESIS: We aimed to examine the time course for the diabetes-related changes in renal lysosomal processing and to determine whether these changes can be prevented by aminoguanidine or ramipril treatment. METHODS: The percentage desulphation of intravenously injected tritium labelled dextran sulphate ([3H]DSO4) in the urine, as determined by ion-exchange chromatography, was used as a marker of lysosomal sulphatase activity. Sulphatase activity was determined 1, 2, 3 and 4 weeks after the onset of diabetes in rats as well as in rats treated with either aminoguanidine or ramipril for twelve weeks. RESULTS: The amount of totally desulphated [3H]DSO4 in urine collected from control rats was 65.6 +/- 0.8%. This was significantly reduced in diabetic rats two (57.4 +/- 1.4% desulphated), three (56.8 +/- 1.3 % desulphated) and four (52.9 +/- 2.2% desulphated) weeks after the onset of diabetes. The significant decrease in the amount of totally desulphated [3H]DSO4 in the urine also found at 12 weeks after the onset of diabetes was not affected by drug treatment. There was no significant difference in the amount of partially desulphated [3H]DSO4 in the urine between all the study groups. However, the increase in totally sulphated [3H]DSO4 in the urine collected from diabetic rats (8.7 +/- 1.7 % sulphated) compared with that of control rats (2.2 +/- 0.5% sulphated) was normalised by treatment with both aminoguanidine (4.8 +/- 1.6% sulphated) or ramipril (4.5 +/- 0.8% sulphated). CONCLUSIONS/INTERPRETATION: These results raise the possibility that the diabetes-induced changes in renal lysosomal processing may be one of the initial events in the development of diabetic nephropathy. Aminoguanidine and ramipril, known for their different mechanism of action, seem to prevent diabetes-induced changes in lysosomal processing either through their effects on enzyme activity within the lysosome or through their effects on the trafficking of molecules to and from the lysosome.

Variability of standard clinical protein assays in the analysis of a model urine solution of fragmented albumin.

OBJECTIVES: This study investigates the sensitivity of various standard clinical techniques in the detection of albumin fragments. The significance of this work is in the detection of urinary proteins, such as albumin, which has recently been discovered to be excreted as mainly peptide fragments as a result of filtered albumin undergoing degradation during renal passage. All filtered proteins undergo a similar degradation process. DESIGN AND METHODS: Albumin digested with trypsin was used as a model urine solution. The solution was assayed for albumin concentration by various methods including the biuret assay that is known to detect urinary albumin fragments. The digest solution was also analyzed by various clinically used chromagen assays, electrophoretic and chromatographic methods to determine whether they are able to detect the fragmented protein. RESULTS: The benzethonium chloride, Coomassie blue, and pyrogallol red assays for urine protein, the immunoassay for human albumin and sodium dodecyl sulfate polyacrylamide gel electrophoresis with Coomassie blue staining were unable to detect the albumin fragments. Capillary electrophoresis was sensitive to the fragments but with low resolution. High-performance liquid chromatography gave the best results. CONCLUSIONS: Many techniques utilized to assay patient urine samples are unable to detect fragmented albumin and, hence, will severely underestimate albumin and protein excretion.

Albuminuria in patients with type 1 diabetes is directly linked to changes in the lysosome-mediated degradation of albumin during renal passage.

Previous studies by our group have shown that albumin is metabolized in rodents during renal passage and excreted in the urine as a mixture of intact protein and albumin-derived fragments. The aim of this study was to examine whether albumin is metabolized during renal passage in nondiabetic volunteers and in type 1 diabetic patients with varying levels of albuminuria. Nine nondiabetic normoalbuminuric volunteers and 11 type 1 diabetic patients with albumin excretion rates varying from normoalbuminuria to macroalbuminuria were studied. Each subject received an intravenous injection of tritium-labeled albumin ([3H]-albumin). Urine was collected at 4 h and 24 h after injection and analyzed by size exclusion chromatography. The amount of intact and fragmented albumin was quantified, and each fraction was analyzed by radioimmunoassay (RIA) for albumin. [3H]-albumin in nondiabetic volunteers was metabolized during renal passage to small peptide fragments not detectable by conventional RIA (only 0.05-3.8% of the total urinary radioactivity was associated with intact albumin). The process responsible for albumin fragmentation was similar in diabetic patients with normoalbuminuria (intact albumin represented 0.01-4.0% of total urinary radioactivity). However, there was a reduction in the fragmentation ratio (fragmented:intact) in diabetic patients with micro- or macroalbuminuria (intact albumin represented 2.7-55.5%, P = 0.048). This change in the fragmentation ratio was directly related to the degree of albuminuria. These results have important implications for understanding the mechanisms underlying albuminuria in nondiabetic volunteers and type 1 diabetic patients. In nondiabetic volunteers, the renal processing of albumin involves a relatively rapid and comprehensive degradation of albumin to small fragments (range 1-15 kDa). The degradation process is inhibited in diabetic nephropathy in proportion to the level of albuminuria detected by RIA.

Characteristics of albumin processing during renal passage in anti-Thy1 and anti-glomerular basement membrane glomerulonephritis.

Recent studies have shown that glomerular-filtered albumin appears to be processed by two distinct cellular pathways. The major pathway, a high-capacity retrieval pathway, returns most of the filtered albumin to the blood supply intact. The albumin not taken up by the retrieval pathway is degraded by lysosomes during renal passage and excreted as fragments in urine. We studied the interplay of the albumin retrieval pathway and the degradation pathway in the disease models of anti-Thy1 nephritis, a model of mild proteinuria, and anti-glomerular basement membrane (anti-GBM) disease, a model of severe proteinuria. This is achieved by investigating the integrity of urinary albumin and its excretion rate. Total albumin excretion (intact plus fragments) did not change significantly in the rats with anti-Thy1 nephritis. However, it was established that intact albumin excretion had a strong positive correlation with increasing total-protein excretion, which showed that the degradation pathway was being predominantly affected in this disease. For the rats with anti-GBM disease, total protein excretion increased 26-fold compared with the control group, and intact albumin excretion increased 250-fold. The profound changes in albumin excretion in anti-GBM disease are consistent with inhibition primarily of the retrieval pathway.

Prevention of albuminuria by aminoguanidine or ramipril in streptozotocin-induced diabetic rats is associated with the normalization of glomerular protein kinase C.

This study examined whether the prevention of diabetes-related albuminuria by aminoguanidine (AG) or ramipril (RAM) may be mediated by a common post-glomerular basement membrane renal intracellular mechanism involving protein kinase C (PKC). The renal handling of albumin was examined over 24 weeks in control and streptozotocin (STZ)-induced diabetic rats. A radioimmunoassay (RIA) that measures intact albumin, and intravenously injected tritium-labeled rat serum albumin, was used to assess the proportion of intact albumin and albumin fragments in urine. Diabetes was induced in male Sprague-Dawley rats by the intravenous administration of STZ at a dose of 50 mg/kg. Age-matched control rats received buffer alone. Diabetes was characterized by an increase in blood glucose (>15 mmol/l), an increase in GHb (means at 24 weeks 29.3+/-1.1%; control 6.1+/-0.1%, P<0.005), an increase in glomerular filtration rate (GFR) (4.13+/-0.15 ml/min; control 3.54+/-0.19 ml/min, P<0.005), an increase in intact albumin excretion rate (expressed as geometric mean 11.64 times/divided by 2.11 mg/24 h; control 0.74 times/divided by 1.57 mg/24 h, P<0.005) as measured by RIA, and an increase in glomerular PKC activity (26.83+/-2.38 pmol x mg(-1) x min(-1); control 14.6+/-2.99 pmol x mg(-1) x min(-1), P<0.005). Treatment of diabetic rats with either AG or RAM prevented the rise in intact albuminuria and glomerular PKC activity. Renal lysosomal cathepsin activity decreased in diabetic rats and this was not prevented by AG or RAM. Neither drug affected glycemic control or GFR, but RAM reduced systolic blood pressure (BP), whereas AG did not. These data indicate that urinary excretion of intact albumin and albumin-derived fragments in diabetes may be modulated independently of glycemic control (AG and RAM) and systolic BP (RAM). While both drugs are known for their different mechanisms of action, the fact that both prevent diabetes-related increases in glomerular PKC activity and albuminuria supports the hypothesis that PKC plays a central role in the development of diabetic nephropathy.

Puromycin aminonucleoside nephrosis results in a marked increase in fractional clearance of albumin.

Puromycin aminonucleoside nephrosis (PAN) results in a marked increase in the fractional clearance of albumin. The increase in the fractional clearance of [(3)H]albumin to approximately 0.045, as measured both in vivo and in the isolated perfused rat kidney (IPK) with PAN, occurs without an accompanying equivalent increase in glomerular capillary wall size selectivity as previously measured with dextrans. This is very similar to the marked increase in albuminuria seen with kidneys treated with inhibitors of endocytosis by the tubular epithelium, particularly lysine (T. M. Osicka, L. M. Pratt, and W. D. Comper. Nephrology 2: 199-212, 1996). The similarity is further established that, like in the presence of lysine, [(3)H]albumin excreted in urine from rats with PAN is essentially intact whereas, in both in vivo and IPK control experiments, excreted [(3)H]albumin is heavily degraded. The same observations have also been made for (3)H-labeled anionic horseradish peroxidase. These observations suggest that the significant albuminuria that occurs in PAN is primarily post-glomerular basement membrane in origin.

The return of glomerular-filtered albumin to the rat renal vein.

BACKGROUND: Recent studies have demonstrated that the normal glomerular capillary wall (GCW) is not charge selective to albumin. This means that albumin flux across the GCW is high, and this has been confirmed in studies in which albumin uptake by the tubules has been inhibited. Therefore, there must be a high-capacity postglomerular retrieval pathway in normal kidneys that returns filtered albumin back to the blood supply. METHODS: This study identifies the presence of glomerular-filtered albumin in the renal vein from the analysis of the decrease of radioactivity in the venous effluent after the injection of a pulse of tritium-labeled albumin into the renal artery in vivo and in the isolated perfused kidney. RESULTS: The postglomerular filtered albumin is returned to the blood supply by a high-capacity pathway that transports this albumin at a rate of 1830 +/- 292 micrograms/min.rat kidney (N = 14, mean +/- SEM). This pathway has been identified under physiological conditions in vivo and in the isolated perfused kidney. The pathway is specific for albumin, as it does not occur for horseradish peroxidase. The pathway is inhibited in a nonfiltering kidney. The pathway is also inhibited by ammonium chloride (an agent that inhibits tubular protein uptake but does not alter glomerular size selectivity) and by albumin peptides (which compete for the tubular albumin receptor). CONCLUSIONS: The high-capacity retrieval pathway for albumin is most likely associated with transtubular cell transport. It is also apparent that most albuminuric states could be accounted for by the malfunctioning of this pathway without resorting to any change in glomerular permselectivity.

Fractional clearance of high molecular weight proteins in conscious rats using a continuous infusion method.

BACKGROUND: The purported existence of "large pores" in the glomerular capillary wall has been derived primarily from studies using dextrans and Ficolls. Systematic studies using high molecular weight proteins have not been performed. One of the difficulties is that recent studies have demonstrated that albumin and other proteins undergo degradation during renal passage. Our study took into account this renal degradation in measuring the fractional clearance of various high molecular weight proteins (the hydrodynamic radii range was between 48 to 70 A). METHODS: Fractional clearances of tritium-labeled proteins were measured using ALZET osmotic pumps, which are designed to release a slow continuous infusion of tracer. Blood and urine collections were taken at 24-hour intervals over seven days and were counted for radioactivity, and glomerular filtration rate was measured by a creatinine assay. RESULTS: Steady-state levels of [3H]protein in plasma were obtained by day 6. The [3H]proteins in the plasma showed no degradation. The fractional clearances (mean +/- sd, N = 5) of the various proteins were albumin (radius = 36 A; 0.0023 +/- 0.0009), transferrin (48 A; 0.0046 +/- 0.0007), lactoperoxidase (58 A; 0. 0045 +/- 0.0005), immunoglobulin G (62 A; 0.0043 +/- 0.0009), lactate dehydrogenase (64 A; 0.0041 +/- 0.0009), and glucose oxidase (70 A; 0.0036 +/- 0.0011). CONCLUSIONS: These values suggest a weak dependence of fractional clearance on size-selective filtration, except for albumin, which undergoes a specific type of postglomerular processing. The fractional clearances were higher than expected from previous data on dextrans and Ficolls of equivalent hydrodynamic radius, and thus demonstrate that "large pores" may already exist in normal glomerular capillary walls.

Tubular inhibition destroys charge selectivity for anionic and neutral horseradish peroxidase.

The fractional clearance of [3H]anionic HRP and [3H]neutral HRP in the isolated perfused kidney as determined by radioactivity analysis was 0.0160+/-0.0028 (n=6) and 0.0388+/-0.0076 (n=8) respectively. The apparent charge selectivity for both the anionic and neutral forms of HRP observed was destroyed with the inclusion of the tubular uptake inhibitors, 150 mM lysine and 10 mM NH4Cl, in the perfusate. In the presence of 150 mM lysine, the clearances of [3H]anionic HRP and [3H]neutral HRP were 0.0645+/-0.0110 (n=4) and 0. 0784+/-0.0120 (n=4) respectively, and 0.0564+/-0.0035 (n=4) and 0. 0694+/-0.0054 (n=4) respectively in the presence of 10 mM NH4Cl. The clearance for both the anionic and neutral HRP probes in these tubular uptake inhibited systems fits precisely the size selective characteristics of the glomerular capillary wall as determined by transport probes calibrated for hydrodynamic size by size exclusion chromatography. The tubular uptake inhibitors were observed not to alter glomerular permselectivity as determined using polydisperse dextran fractions and the behaviour of neutral HRP. This study demonstrates that charge selectivity for differently charged proteins is not as great as originally thought and suggests that the clearance differences between anionic and neutral forms may be due to differential handling by the tubules.

Alterations in renal degradation of albumin in early experimental diabetes in the rat: a new factor in the mechanism of albuminuria.

1. Albumin is normally excreted as a mixture of intact protein and fragments that are produced during renal passage. The purpose of this study was to investigate the ratio of intact versus degraded forms of excreted albumin to ascertain whether changes in this ratio could account for the apparent increase in albumin excretion seen in diabetes, as measured by standard radioimmunoassay techniques. 2. Four-week male Sprague-Dawley rats with streptozotocin-induced diabetes and age-matched control rats were intravenously injected with [3H]albumin. Urine collected over 2 h was analysed by size exclusion chromatography and radioimmunoassay. A standard radioimmunoassay found a 7-fold increase in albumin excretion rate in diabetic rats, whereas there was only a 2-fold increase in albumin excretion (intact plus fragments). Urine analysed by size exclusion chromatography showed severe degradation for control rats (% monomer=4+/-2%); in diabetic rats there was a significant amount of monomer albumin excreted, along with moderately degraded and heavily degraded albumin (% monomer=17+/-5%). 3. This study has shown that the radioimmunoassay, which specifically detects intact albumin, considerably underestimates the amount of total urinary albumin which consists of intact and degraded material. The increase in albumin excretion rate observed in diabetes as measured by radioimmunoassay is mainly due to a change in the amount of intact albumin excreted and this is specifically due to the inhibition of albumin degradation at a post-glomerular site and not due to the onset of any type of glomerular 'shunt' pathway.

Anomalous decrease in dextran sulfate clearance in the diabetic rat kidney.

The anomalous increase in charge selectivity as previously observed with reduced dextran sulfate clearances in diabetic rats (L. D. Michels, M. Davidman, and W. F. Keane. Kidney Int. 21: 699-705, 1982) was confirmed in 4-wk streptozotocin (STZ) diabetic Sprague-Dawley rats using the isolated perfused kidney technique. The apparent charge selectivity in both control and diabetic rats could be abolished by increasing the dextran sulfate concentration to 200 micrograms/ml in the perfusate. This was demonstrated by a high rate of processing of dextran sulfate (approximately 1,700 ng.min-1.kidney-1) by glomeruli in both control and diabetic kidneys and by the fact that charge interaction could not explain the concentration dependence. The amount of urinary desulfation of dextran sulfate was also found to be significantly less in the diabetic kidney as was glomerular sulfatase activity compared with controls. Dextran sulfate glomerular processing is therefore altered in the STZ diabetic rat kidney but could be rationalized in terms of previous models of endothelial cell receptor-mediated uptake of dextran sulfate. The results are consistent with recent work demonstrating that there is little or no electrostatic charge interaction operating on dextran sulfate or other negatively charged molecules at the glomerular capillary wall.

Changes in sulfated macromolecules produced in vivo during normal and indomethacin-delayed ulcer healing in rats.

The aim of this study was to examine the synthesis of sulfated glycosaminoglycans during normal healing of experimental acetic acid-induced gastric ulcer in rats and to investigate the effect of indomethacin, a drug known to delay ulcer healing, on this synthesis using an in vivo labelling system. Analysis revealed the presence of two major sulfated species in control tissue; a population of sulfated mucins and glycosaminoglycans, predominantly galactosaminoglycans. The incorporation of [35S]sulfate label into glycosaminoglycans synthesized in the granulation tissue of healing ulcers increased significantly (P < 0.05) as compared to day 0 and control levels at day 14. Treatment of animals with indomethacin (1 mg/kg daily) resulted in a further significant (P < 0.01) rise in sulfated glycosaminoglycan synthesis in indomethacin-treated ulcer tissue compared to that found in healing ulcers at day 14. The increased glycosaminoglycan synthesis was due to increased levels of chondroitin sulfate and dermatan sulfate. Glycosaminoglycan synthesis is elevated at the ulcer site during healing of experimental gastric ulcers; however, indomethacin treatment, which delays ulcer healing, significantly increases the synthesis of glycosaminoglycans above that seen in healing ulcers. Changes in the sulfated glycosaminoglycan content of the ulcer may play a role in the healing process and may give further insight into the mechanisms by which indomethacin delays ulcer healing.