Accumulation Of Basement Membrane Material Research Articles

Psammomys obesus, a particularly important animal model for the study of the human diabetic nephropathy

The Psammomys obesus lives in natural desert habitat on low energy (LE) diet, however when maintained in laboratory conditions with high energy (HE) diet it exhibits pathological metabolic changes resembling those of type 2 diabetes. We have evaluated and correlated the histopathology, metabolic and functional renal alterations occurring in the diabetic Psammomys. Renal function determined by measuring glomerular filtration rate (GFR), protein excretion, protein/creatinine ratio and morpho-immunocytochemical evaluations were performed on HE diet diabetic animals and compared to LE diet control animals. The diabetic animals present a 54% increase in GFR after one month of hyperglycemic condition and a decrease of 47% from baseline values after 4 months. Protein excretion in diabetic animals was 5 folds increased after 4 months. Light microscopy showed an increase in glomeruli size in the diabetic Psammomys, and electron microscopy and immunocytochemical quantitative evaluations revealed accumulation of basement membrane material as well as frequent splitting of the glomerular basement membrane. In addition, glycogen-filled Armanni-Ebstein clear cells were found in the distal tubules including the thick ascending limbs of the diabetic animals. These renal complications in the Psammomys, including changes in GFR with massive proteinuria sustained by physiological and histopathological changes, are very similar to the diabetic nephropathy in human. The Psamommys obesus represents therefore a reliable animal model of diabetic nephropathy.

Expression and localization of MT1-MMP and furin in the glomerular wall of short- and long-term diabetic rats

Diabetic glomerulopathy has been linked to shifts in balance between the synthetic and degradative pathways of the glomerular basement membrane (GBM), a key player in the permselectivity properties of the glomerular wall. The goal of this study was to trace the expression and localization of membrane type-1 metalloprotease (MT1-MMP) and its activating enzyme furin, key proteins involved in basement membrane turnover, in short- and long-term diabetic rat renal tissues. Quantitative immunogold was carried out for MT1-MMP and furin and their expression was evaluated in renal tissues of young and old, control and diabetic rats. To corroborate immunocytochemical findings, Western blots were performed on glomerular lysates. Electron microscopy revealed that the overall expression of MT1-MMP and furin is reduced in plasma membranes of all glomerular cell types of old normoglycemic animals, a phenomenon that is exacerbated in long-term diabetic animals. This observation supports the prevailing theory that diabetes fosters acceleration in the aging process. Interestingly, while biochemical results confirmed a decrease in MT1-MMP expression, an increase in furin was observed. Immunocytochemical studies resolved this discrepancy by tracing the increased furin expression in endoplasmic reticulum and Golgi membranes of podocytes, indicating that furin is retained in the secretory pathway in a diabetic environment. Disturbances at the molecular level of the otherwise tightly regulated MT1-MMP/furin interactions found at the cell surface must account for a lack in extracellular matrix remodeling, increased deposition of GBM material, and loss of glomerular filtration integrity.

Role of vascular basement membrane components in diabetic microangiopathy.

The complications of diabetic microangiopathy have been well documented clinically for many years; however, the specific cellular mechanisms that lead to the dysfunction of small vessels in diabetes are unknown. Histopathological studies on diabetic microangiopathy have established thickened vascular basement membrane as one of the first and foremost structural abnormalities. Evidence suggests that this fundamental structural lesion develops, at least in part, from excess accumulation of basement membrane material in the vessel walls. Several vascular basement membrane components have been identified whose expression is up-regulated in diabetes. Enzymes responsible for the degradation of basement membrane components have also been reported to exhibit reduced activity. Strategies are being developed to regulate the abnormal accumulation of extracellular matrix (ECM) components in diabetic microangiopathy. In experimental diabetes, aldose reductase inhibitors have been found to be effective in preventing vascular basement membrane thickening when applied soon after the onset of hyperhexosemia. Clinical trials have been initiated with inhibitors of protein kinase C, an enzyme that up-regulates the synthesis of ECM components in diabetes. Currently, gene therapy strategies are also being developed to inhibit specific gene expression responsible for excess ECM synthesis in diabetes. Recent studies in the authors' laboratory have shown the applicability and efficacy of antisense oligonucleotides toward reducing vascular basement membrane component synthesis. Several gene regulatory approaches are currently being developed to modulate and prevent vascular basement membrane component synthesis. Such strategies may help identify key events that underlie the complex developmental stages of diabetic microangiopathy.

Anti-Glycated Albumin Therapy Ameliorates Early Retinal Microvascular Pathology in db/db Mice

Hyperglycemia plays a primary causal role in the early vascular damage leading to diabetic retinopathy, but the intermediate biochemical mechanisms involved are not known. Because albumin modified by Amadori glucose adducts has been implicated in the pathogenesis of diabetic nephropathy, we investigated whether or not glycated albumin plays a similar role in diabetic retinopathy. We observed basement membrane thickening and an accumulation of basement membrane material in the capillaries of the outer plexiform layer of retinae from diabetic db/db mice compared with their nondiabetic db/m littermates. Both of these abnormalities were ameliorated by chronic (8 week) treatment with monoclonal antibodies that specifically recognize Amadori-modified glycated albumin (and not other glucose-modified or advanced glycation endproducts-modified proteins), despite the fact that the administration of these antibodies did not alter the glycemic status of the diabetic animals. Thus, albumin containing Amadori glucose adducts contributes to the pathogenesis of diabetic retinal vascular disease, and agents that neutralize or prevent the formation of excess glycated albumin in diabetes may offer prophylaxis against the early changes of diabetic retinopathy.

Collagen distribution in human membranous glomerulonephritis.

In membranous glomerulonephritis (MGN), thickening of the glomerular basement membrane (GBM) is partly due to the accumulation of basement membrane material between and around immune deposits located on the epithelial aspect of the GBM. We investigated the distribution of type IV collagen chains (alpha 1/alpha 2, alpha 3, alpha 4, alpha 5, alpha 6) and of types I, III, V, and VI collagen in the glomeruli from 16 patients, by indirect immunofluorescence in 13 and the high-resolution immunogold technique in 6. No changes were detected in stage I MGN. The spiky projections of the GBM in stage II MGN and the basement membrane layers encircling immune deposits in stage III contained the alpha 3, alpha 4, and alpha 5 chains of type IV collagen. In contrast, the alpha 1/alpha 2 chains of type IV, as well as type VI collagen accumulated in the subendothelial aspect of the GBM. No significant staining for types I, III, and V collagens or for the alpha 6 chain of type IV collagen was detected. The results show that, as in the normal glomeruli, the different chains of type IV collagen are not co-distributed in the glomerular extracellular matrix in MGN. They also indicate that type IV collagen chains and type IV collagen play an important role in the thickening of the GBM in human MGN.

The nature of large vessel disease in diabetes mellitus

Reprint requests: Thomas Ledet, MD, Research Laboratory for Biochemical Pathology, Aarhus Kommunehospital, DK-8000, Aarhus C, Denmark. The problem of cardiovascular disease and diabetes mellitus can be subdivided into one distinct zone where large-scale research work has provided a number of well documented pieces of information from epidemiologic and clinical studies, and another, large and indeterminate grey region of unasked questions and unposed problems, which is inhabited by borderline problems between diabetes and cardiovascular research. The epidemiologist has told us that heart disease occurs 3-5 times more frequently among diabetic patients. At present, it is not possible to explain this very high frequency as a consequence of the usual risk factors in atherosclerosis, such as increased serum LDL.’ Likewise, various experimental investigations have not been able to provide an explanation through analyses of non-enzymatic glycosylated lipoproteins.* In our laboratory, the study of large vessel disease in diabetes mellitus is guided by a hypothesis that postulates the existence of a diabetic macroangiopathy which develops independent of the usual atherosclerotic risk factors, but is a result of diabetic intermediary metabolism and endocrinopathy.3s4 Since deposition of a strong PAS-positive substance in the wall of the smaller blood vessels is a characteristic feature of diabetic microangiopathy, we decided to investigate the extent of this PAS-positive substance in the larger coronary arteries from a group of NIDDM patients.5 The data obtained from the diabetic patients were compared with the results from a group of age and gender matched nondiabetic subjects. An area of the tunica media was selected, based on the presence or absence of atherosclerotic plaques in the tunica intima. Using a traditional point counting procedure, we estimated the area occupied by the PAS-positive material in the tunica media. It appeared that a significantly larger area was occupied by the PAS-positive substance in the coronary arteries from diabetic individuals than was the case in nondiabetics. Moreover, it appeared that the size of the PAS-positive area was completely independent of the presence of atherosclerotic plaques (Figure 1). The same pattern was observed when the amount of collagen was evaluated using a histologic staining technique and the same point counting. In other histologic sections taken from the same samples of the coronary arteries, the area occupied by acid mucopolysaccharides (glycosaminoglycans) was evaluated by identification with alcian blue, again with the point counting procedure. It could be shown that the area containing acid mucopolysaccharides was substantially reduced in coronary arteries from diabetic patients, independent of the presence of atherosclerotic plaques. The basement membrane in the tunica media is a PAS-positive structure surrounding individual arterial smooth muscle cells.6s7 Since the basement membrane is abnormal in many organs in diabetic patients, we have focused on the elaboration of that structure in a tissue culture system of rabbit aortic smooth muscle cells. Using normolipemic fasting serum from a group with IDDM and a group with NIDDM, we have evaluated the elaboration of basement membrane, and found that sera from the diabetic patients significantly enhanced the accumulation of basement membrane material compared with sera from normolipemic nondiabetic individuals of the same age and gender (Figure 2).* In the same culture system, we tested the effect of hyperlipemic serum obtained from rabbits fed a high cholesterol diet for more than 6 weeks, but found no influence on basement membrane accumulation.

Accumulation of laminin and type IV collagen in the kidney in congenital nephrosis

The aim of this study was to evaluate qualitatively the occurrence of the basement membrane proteins laminin and type IV collagen in the kidneys of ten infants with congenital nephrotic syndrome of the Finnish type (CNF) aged from 3 to 23 months and to compare the results with those for age-matched controls. A slow accumulation of basement membrane (BM) material occurred in the glomerular mesangium, the peripheral capillaries, around atrophied tubules, and the renal vessels in the course of the disease. The staining pattern of accumulated material depended on the duration of the disease and subsequent renal parenchymal damage. Young CNF patients with slight morphological changes in the kidney had only focal and minimal increases in the amounts of mesangial matrix, but as the disease advanced, so the BMs of the glomerular capillaries, renal arteries, and atrophied tubules also became involved and were thicker than normal. The staining reaction was in all patients similar with antibodies against the fragment P1 of laminin and the 7-S domain of type IV collagen. The accumulation of BM material in CNF kidneys is regarded as a secondary phenomenon induced by an unknown pathogenetic defect in the metabolism of some BM component.

Diabetic glomerulopathy. Structural characteristics of the early and advanced stages.

Studies of the glomerular structure in diabetes mellitus have helped to elucidate the basis for some functional abnormalities. The decline in glomerular filtration occurring in many long-term diabetics is a functional disorder of great clinical importance. Quantitative structural studies of the glomeruli in diabetics at different stages of disease are necessary to learn about the development of structural changes leading to the end-stage kidney disease. Preliminary results of a study of glomeruli from long-term diabetics with clinical nephropathy are compared with those obtained in control subjects and in diabetics within the first 5 yr of disease. In the long-term diabetics the peripheral basement membrane thickness was doubled. On the average, mesangial regions occupied nearly 60% of the total tuft volume as compared with 33% in the early stages. A marked accumulation of basement membrane material in the mesengial regions had taken place so that 85% of the total basement membrane material of the tufts (i.e., peripheral basement membrane in the capillary walls plus mesangial basement membrane-like material) was localized within the mesangial regions. In the early stages equal amounts were found at these two different sites. The distribution of the lesions within the kidney is under investigation in a light microscopic study of autopsy material from long-term diabetics with varying degrees of glomerulopathy. The severity of the diabetic glomerulopathy was quantitated separately within the superficial and the deep cortical zones. The results showed that there was no tendency toward increased severity in the deep glomeruli.(ABSTRACT TRUNCATED AT 250 WORDS)

Fast accumulation of basement membrane material and the rate of morphological changes in acute experimental diabetic glomerular hypertrophy.

Renal glomerular structures have been studied during the initial phases of renal and glomerular hypertrophy in streptozotocin diabetic rats. -- After four days of diabetes significant increases were found in the following absolute structural quantities: the tuft volume (25%), the surface area (42%) and volume (46%) of peripheral basement membrane, and capillary luminal volume (29%). No further changes in these quantities took place over the succeeding 47 days. The geometry of the capillaries, however, changed over this period: at 4 days the total capillary length had increased significantly (32%) with an unchanged average cross sectional area, whereas at 47 days the length had reverted to normal, but average cross sectional area had increased (30%). The increased surface of filtering area may be the morphological counterpart of increased glomerular filtraton rate. The changes in glomerular volume were thus accompanied by changes in glomerular structural composition which after a few weeks returned to normal, in balance with the increased volume.

Reduced susceptibility of nonenzymatically glucosylated glomerular basement membrane to proteases: is thickening of diabetic glomerular basement membranes due to reduced proteolytic degradation?

Glomerular basement membrane (GBM) preparations were enzymatically glucosylated and applied to proteolytic degradation by several enzymes. The split products were then characterized and quantitatively estimated by high pressure liquid chromatography. For this purpose, GBMs were isolated by a sieving and sonication method, incubated with glucose and digested with the proteases trypsin, chymotrypsin, papain, pepsin and a lysosomal preparation. Comparison of the concentrations of split products obtained by proteolytic degradation of normal and nonenzymatically glucosylated membranes showed a remarkable reduced susceptibility of the nonenzymatically glucosylated membranes, possibly due to steric hindrance or altered electrical charge of the glucosylated membrane proteins. This could be interpreted as an additional factor for accumulation of basement membrane material in the diabetic state, that not only increased basement membrane synthesis may occur but also reduced catabolism could possibly contribute to the diabetic changes.

Early functional and morphologic vascular renal consequences of the diabetic state

Renal hyperfunction and hypertrophy are characteristic findings in the early diabetic state, both in diabetes mellitus and in experimental diabetes. A number of structure-function relationships and their likely mechanisms are discussed. The metabolically induced hypertrophy of glomerular capillaries possibly plays a central role. Its cause is not known, but recent results on its time-course emphasize the probable long-term consequences of the irreversibility of the accumulation of basement membrane material.