Synthesis of the glomerular basement membrane in the rat kidney

To study the origin and the formation of the glomerular basement membrane, autoradiographic investigations with³H-proline and³H-leucine have been performed in ultrathin and semithin sections of the glomeruli of 42 male rats. The results of this study indicate that, of the three cell types of the glomerulus, the epithelial cells (=podocytes) synthesize the proline-rich scleroproteins of the glomerular basement membrane. Our autoradiographic studies have yielded no evidence for participation of the endothelial or mesangial cells in the formation of the basement membrane. The mesangial cells appear to be responsible for the synthesis of the mesangial matrix only.     

Co-culture of glomerular epithelial cells and mesangial cells on collagen-gauze-fiber gel

A novel collagen-gauze-fiber gel was created as a scaffold for the co-culture of renal glomerular epithelial cells and mesangial cells at its opposite sides. This gauze-fiber-gel provides a mimic environment like that of renal glomeruli in vivo. The cell morphology, cell growth and cell viability were investigated and the results showed that this novel scaffold maintains cell growth and cell viability without changing cell morphology for more than 3 weeks. Interestingly, glomerular epithelial cells co-cultured with mesangial cells on the gauze-fiber gel resulted in the polarity formation which usually appears on the normal epithelial cells existing at glomerular basement membrane in vivo, but seldom appears on the epithelial cells when cultured in vitro.     

The glomerular mesangial cell: an expanding role for a specialized pericyte

The mesangial cell occupies a central position in the renal glomerulus. It has characteristics of a modified smooth muscle cell, but is also capable of a number of other functions. Among these are generation of prostaglandins (PGs) and mediators of inflammation; production and breakdown of basement membrane and other biomatrix material; synthesis of cytokines; and uptake of macromolecules, including immune complexes. In terms of its smooth muscle activity, the mesangial cell contracts or relaxes in response to a number of vasoactive agents. This ability allows the cells to modify glomerular filtration locally. The cellular mechanism of action of many agents influencing mesangial cells involves activation of phospholipase C for phosphatidylinositol 4,5-bisphosphate. This results in generation of inositol trisphosphate and release of intracellular calcium. Mesangial cell relaxation can be mediated by enhanced cAMP or cGMP generation. Many vasoactive substances also stimulate PG production by mesangial cells. This involves activation of both phospholipase C and A2, the latter being responsible for the release of arachidonic acid. Mesangial cells are also capable of endocytosis of macromolecules, including immune complexes. This is initiated by binding to a specific receptor, resulting in formation of PG, platelet-activating factor, and reactive oxygen species. Mesangial cells can generate interleukin 1 and platelet-derived growth factor and respond to these in an autocrine manner. Thus, the mesangial cell not only can control glomerular filtration, but may also be involved in the response to local injury, including cell proliferation and basement membrane remodeling.     

Light and Electron Microscopical Studies of the Renal Lesion in Dogs With Pyometra

Kidney biopsies from 23 bitches with pyometra and an entire kidney from four pyometra bitches were examined by light microscopy. Kidney tissue was also taken from three bitches at different intervals after ovariohysterectomy for pyometra. All the pyometra bitches had membranous glomerulonephritis or mixed proliferative and membranous glomerulonephritis. Two of the bitches had intraglomerular hyaline nodules resembling those seen in conjunction with diabetes in human beings. The degree of glomerular damage could be correlated with the reduction in glomerular filtration rate determined by function tests. The proximal tubules generally contained numerous hyaline droplets but the degree of this change could not be correlated to the degree of glomerular damage. A yellow pigment, a lipofuscin, was regularly present in the proximal tubules as well as epithelial proliferation and mitoses. Focal atrophy of tubules also occurred, presumably because of obliteration of glomeruli. The cortical interstitium contained collections of mature and immature plasma cells, often surrounding the glomeruli. When the kidneys from three bitches were examined after ovariohysterectomy for pyometra, the glomerular damage in two had regressed to leave only slight thickening of the capillary walls. In the third bitch, examined only 14 days after ovariohysterectomy, healing was partial. Kidney tissue from five bitches was also examined by electron microscopy. The glomerular endothelial cells were swollen and the basement membrane was grealy thickened. With more severe degrees of glomerular damage, an electron-dense material was deposited along the inner surface of the basement membrane and the swollen mesangial cells contained numerous inclusions. There was focal fusion of the foot processes of the glomerular epithelial cells; in one bitch with heavy proteinuria, the fusion was widespread. The proximal tubules contained numerous protein absorption droplets representing resorbed protein. The tubular basement membrane at all levels was thickened. Because of similarities with some other types of renal damage (nephrotoxic nephritis in dogs and acute proliferative glomerulonephritis in human beings), the possibility is broached that the renal lesion in pyometra is the result of an immunobiological process.     

Localization in situ of type VI collagen protein and its mRNA in mesangial proliferative glomerulonephritis using renal biopsy sections

 Extracellular matrix accumulation is crucial in the pathogenesis of glomerulosclerosis in mesangial proliferative glomerulonephritis (GN). In an attempt to explore the distribution of type VI collagen and its synthesizing cells in normal and diseased glomeruli, we investigated mRNA and protein expression of type VI collagen in renal biopsy sections, histologically diagnosed as mesangial proliferative GN. Five renal biopsies from patients diagnosed as having minor glomerular abnormalities and one surgical renal tissue were also simultaneously examined as controls. Immunohistochemical studies revealed type VI collagen immunostaining in the mesangium and glomerular basement membrane of the control glomeruli. Compared to the control, increased deposition of type VI collagen was noted in the mesangial proliferative and sclerotic lesions in GN. To identify the cells responsible for the synthesis of type VI collagen mRNA, renal sections were hybridized in situ with digoxigenin-labeled antisense oligo-DNA probe complementary to a part of α1 (VI) mRNA. Occasionally intraglomerular cells hybridized with digoxigenin-labeled antisense pro α1 (VI) oligo-DNA in control glomeruli. An increased number of intraglomerular cells (mostly epithelial cells) were, however, positive for α1 (VI) mRNA expression in GN sections. The present study documents the distribution of type VI collagen in the normal glomeruli and provides further evidence of accelerated synthesis of this collagen in mesangial proliferative GN. Accepted: 21 July 1998     

Role of TGF-β in chronic kidney disease: an integration of tubular,glomerular and vascular effects

Transforming growth factor beta (TGF-β) has been recognized as an important mediator in the genesis of chronic kidney diseases (CKD), which are characterized by the accumulation of extracellular matrix (ECM) components in the glomeruli (glomerular fibrosis, glomerulosclerosis) and the tubular interstitium (tubulointerstitial fibrosis). Glomerulosclerosis is a major cause of glomerular filtration rate reduction in CKD and all three major glomerular cell types (podocytes or visceral epithelial cells, mesangial cells and endothelial cells) participate in the fibrotic process. TGF-β induces (1) podocytopenia caused by podocyte apoptosis and detachment from the glomerular basement membrane; (2) mesangial expansion caused by mesangial cell hypertrophy, proliferation (and eventually apoptosis) and ECM synthesis; (3) endothelial to mesenchymal transition giving rise to glomerular myofibroblasts, a major source of ECM. TGF-β has been shown to mediate several key tubular pathological events during CKD progression, namely fibroblast proliferation, epithelial to mesenchymal transition, tubular and fibroblast ECM production and epithelial cell death leading to tubular cell deletion and interstitial fibrosis. In this review, we re-examine the mechanisms involved in glomerulosclerosis and tubulointerstitial fibrosis and the way that TGF-β participates in renal fibrosis, renal parenchyma degeneration and loss of function associated with CKD.     

Prostaglandin synthesis by human glomerular cells in culture

PG synthesis by cultured human glomerular mesangial and epithelial cells incubated with [1- 14C] arachidonic acid was determined by radioimmunoassay (RIA) after high performance liquid chromatography purification. Both dissociated cells and cell monolayers were studied under basal conditions. PG synthesis by epithelial cells was undetectable. Mesangial cells produced low amounts of PGE2, PGF2 alpha and 6 keto-PGF1 alpha and no TXB2. We also examined the effects of several agents on PG synthesis in these two types of cells scraped away from their flasks using direct RIA. Arachidonic acid produced a slight stimulation only with mesangial cells whereas angiotensin II, cyclic AMP and calcium ionophore were inactive with both cell lines. Homogenization of the cells did not enhance the stimulatory effect of arachidonic acid. Alkalinization of the incubation medium produced an increase of PG production by mesangial cells. These results suggest that two types of human glomerular cells, particularly epithelial cells, possess low cyclooxygenase activity. The low capacity of human mesangial and epithelial cells to produce PG may have consequences for the endocrine control of the glomerular microcirculation in man.     

Mesangioproliferative Glomerulonephritis in Mink with Encephalitozoonosis

Renal specimens from 6 mink with encephalitozoonosis were studied by light and electron microscopy and immunohistochemistry. The glomeruli of affected kidneys had a mesangioproliferative glomerulonephritis which was characterized by an increase in mesangial cells and matrix in most glomeruli. Some glomeruli were partially or completely sclerosed. There were protein or granular casts in the cortical and medullary tubules. Interstitial nephritis, vasculitis and tubular cysts were found. Electron microscopy demonstrated extensive matrix and increased cellularity in the mesangial areas. Glomeruli showed segmentally thickened or wrinkled capillary basement membranes. Electron dense deposits were found in the glomerular basement membranes and mesangium. Peroxidase-anti-peroxidase immunohistochemistry demonstrated that IgG and IgM positive material was present as granular deposits in the glomerular basement membrane and occasionally in the mesangium.     

Ultrastructural distribution of endogenous IgGs in the glomerular wall of control and diabetic rats

Summary Endogenous IgG molecules were revealed with high resolutionem over the glomerular wall in renal tissues sampled from short and longterm control and streptozotocin induced diabetic rats by applying the protein A-gold immunocytochemical approach. In tissues from control animals, IgG antigenic sites were revealed on the subendothelial side of the basement membrane, the epithelial side being only weakly labelled. In contrast, in longterm diabetic animals IgG antigenic sites were present throughout the entire thickness of the basement membrane, and in patches closely associated with the plasma membrane of the epithelial cells. Deposits of basement membrane-like material present in the mesangial area were also highly labelled for IgG. Numerous intensely labelled lysosome-like structures were present in the epithelial cells. Morphometrical evaluation of the distribution of the labelling over the basement membrane confirmed these observations. In control animals a peak of labelling was found at 30 nm from the endothelial cell region corresponding to the subendothelial side of the lamina densa. In longterm diabetic animals the labelling was more uniformly distributed throughout the entire thickness of the basement membrane. These data were correlated to biochemical determinations of proteinuria and IgG excretion in urine samples from the control and the diabetic animals. These results suggest that in normal conditions the lamina densa may represent the main barrier for the restriction of the passage of IgGs through the glomerular wall. Modifications at that level occur during diabetes leading to or participating in the loss of the selective permeability of the basement membrane.     

The long pentraxin PTX3 is synthesized in IgA glomerulonephritis and activates mesangial cells

The long pentraxin PTX3 has been recently involved in amplification of the inflammatory reactions and regulation of innate immunity. In the present study we evaluated the expression and role of PTX3 in glomerular inflammation. PTX3 expression was investigated in the IgA, type I membranoproliferative, and diffuse proliferative lupus glomerulonephritis, which are characterized by inflammatory and proliferative lesions mainly driven by resident mesangial cells, and in the membranous glomerulonephritis and the focal segmental glomerular sclerosis, where signs of glomerular inflammation are usually absent. We found an intense staining for PTX3 in the expanded mesangial areas of renal biopsies obtained from patients with IgA glomerulonephritis. The pattern of staining was on glomerular mesangial and endothelial cells. Scattered PTX3-positive cells were also detected in glomeruli of type I membranoproliferative glomerulonephritis. The concomitant expression of CD14 suggests an inflammatory origin of these cells. Normal renal tissue and biopsies from patients with the other glomerular nephropathies studied were mainly negative for PTX3 expression in glomeruli. However, PTX3-positive cells were detected in the interstitium of nephropathies showing inflammatory interstitial injury. In vitro, cultured human mesangial cells synthesized PTX3 when stimulated with TNF-alpha and IgA and exhibited specific binding for recombinant PTX3. Moreover, stimulation with exogenous PTX3 promoted mesangial cell contraction and synthesis of the proinflammatory lipid mediator platelet-activating factor. In conclusion, we provide the first evidence that mesangial cells may both produce and be a target for PTX3. The detection of this long pentraxin in the renal tissue of patients with glomerulonephritis suggests its potential role in the modulation of glomerular and tubular injury.     

Distribution of αvβT3, αvβTB5 Integrins and the Integrin Associated Protein — IAP (CD47) in Human Glomerular Diseases

The av integrins present on the membrane of numerous cells, mediate attachment to matrix proteins, cell proliferation, migration and survival. We studied the expression of av integrins and CD47 (a 03 chain integrin associated protein) in various forms of glomerulonephritis (GN) characterized by mesangial proliferation and/or increased mesangial matrix. In normal glomeruli, epithelial cells expressed αvβT3, αvβT5 and CD47; endothelial cells expressed α5βT1 and CD47; mesangial cells expressed αvβT5, CD47, and to a less extent αvβT3. In acute post infectious GN (APIGN), membranoproliferative GN (MPGN) and diabetic nephropathy (DN), we observed that the βT3 chain, normally expressed by mesangial cells, was not detectable in the mesangium while its expression by epithelial cells was not modified. Parallel to the disappearance of αvβT3, the CD47 expression was decreased on the mesangial cells in MPGN, APIGN and DN. The expression of αvβT5 was clearly increased on podocytes and on proliferating mesangial cells in APIGN. By contrast, the mesangial expression of αvβT5 normal or decreased in DN. The α5 chain of integrin, absent on normal mesangial cell, was expressed on proliferating mesangial cells in MPGN and APIGN.

Thus, we observed modifications of avp3 and avp5 expression during human GN. The modulations of αvβT3 and αvβT5 expression differed according to the different glomerular cell types and were not parallel in glomerular cells: avp3 was decreased (and αvβT5 unchanged) on proliferating mesangial cells and αvβT5 was increased (and αvβT3 unchanged) in podocytes. This may reflect the existence of two distinct regulatory pathways.     

The relation between connective tissue cells and intercellular substances, including basement membranes.

Basement membranes are distributed widely in the body forming an extracellular matrix for epithelial and endothelial cells. The collagenous and glycoprotein constituents of basement membranes are synthesized by these two cell types. Disturbance of the interactions between basement membranes and their associated epithelial and endothelial cells can lead to the pathological changes seen in diseases involving basement membranes. These changes are illustrated here by reference to glomerulonephritis induced by the deposition of immune complexes in the glomerulus of the kidney, and chronic inflammatory changes occurring in the lung after inhalation of asbestos. In these diseases basement membrane changes can occur in several ways. Hydrolytic enzymes released from inflammatory cells degrade basement membranes while other constituents by epithelial and endothelial cells. Alternatively the physical separation of epithelial and endothelial cells from their basement membrances by space-occupying substances such as immune complexes can interfere with feedback mechanisms leading to synthesis of basement membrane constituents and cell proliferation. Studies of these pathological changes at a cellular level should shed new light on the ways in which cells interact with their pericellular environment.     

Integrins and tumor invasion

Cell-extracellular matrix interactions are important in the process of tumor cell invasion and metastasis. In particular, the interactions of tumor cells with basement membranes of tissue epithelial, as well as vascular endothelial, cells are likely to represent key steps in the metastatic process. The interactions between cells and the connective tissue matrix are mediated by a large family of cell surface receptors, the integrins, which represent multiple receptors for extracellular matrix and basement membrane components. Here, I review recent progress in elucidating the roles of integrins in tumor cell invasion. Altered expression of this large family of receptors on invasive tumor cells, as compared with non-invasive cells, may represent a fundamental step in the progressive expression of the invasive phenotype.     

Alteration of glomerular anionic sites by the development of subepithelial deposits in experimental glomerulonephritis in the rat

Using highly cationic polyethleneimine, alteration of glomerular anionic sites were evaluated ultrastructurally in two types of rat glomerulonephritis (GN); chronic serum sickness GN and heterologous (passive) or autologous (active) Heymann's GN. Daily i.v. injections of egg white lysozyme in physiologic saline into presensitized rats led to the formation of numerous mesangial and subepithelial deposits. In the non-proteinuric period in which immune deposits were localized predominantly in the mesangium, anionic sites of the laminae rarae and the epithelial cell coat were clearly observed. In the subsequent proteinuric period in which numerous subepithelial deposits were superimposed, a broad loss of anionic sites in the epithelial cell coat was seen. Splitting and focal loss of anionic sites on the lamina rara externa adjacent to the subepithelial deposits were commonly observed both in passive and active Heymann's GN and in lysozyme GN. These findings indicate that the subepithelial deposits are closely involved in the development of proteinuria by injuring the anionic sites, especially those on lamina rare externa of the glomerular basement membrane.     

Kidney glomerular explants in serum-free media. Sequential morphologic and quantitative analysis of cell outgrowths

Guinea pig glomeruli were grown in vitro for 22 days in a serum-free medium composed of Waymouth's MB 752/1 supplemented with sodium pyruvate, nonessential amino acids, antibiotics, insulin, transferrin, selenium, triiodothyronine, and fibronectin (FN), and sequential morphologic and quantitative studies of cell outgrowth were performed. Glomeruli grown in serum-free medium showed preservation of glomerular visceral epithelial cells but extensive necrosis of endocapillary cells (endothelial and mesangial cells). Morphologic analysis demonstrated progressive morphologic changes in cultured glomerular cells; however, most cell types observed in culture appeared to grow from the epithelial side of the glomerular basement membrane. Mitosis was a prominent component of glomerular cell outgrowth in vitro, and total DNA increased slightly during glomerular culture. FN was required for glomerular cell outgrowth, and studies using FN fragments demonstrated that the carboxy-terminal portion of FN was required for whole glomerular attachment. These results are used to develop a model for glomerular cell outgrowth in vitro.     

Thyroid antigen-antibody nephritis: possible involvement of fucosyl-GM1 as the antigen

Hyperthyroidism, microscopic hematuria, and proteinuria developed in an 11-year-old girl. Proteinuria decreased during treatment of hyperthyroidism with an antithyroid drug. On admission, serum anti-thyroglobulin antibody, antimicrosomal antibody, and immune complex were present. The thyrotropin binding inhibitory immunoglobulin (TBII) level was low. On the other hand, an antibody to the ganglioside component (fucosyl-GM1) was detected by an enzyme linked immunosolvent assay (ELISA). A thyroid biopsy specimen showed massive lymphocytic infiltration and interstitial fibrosis. A renal biopsy specimen showed marked proliferation of mesangial cells and increased mesangial matrix with focal segmental capillary wall abnormality. Electron microscopec studies demonstrated mild paramesangial dense deposits. By indirect immunofluorescence, granular glomerular basement membrane and mesangial staining were not detected with rabbit antibody to thyroglobulin, but were detected with rabbit antibody to fucosyl GM1. Fucosyl GM1 was also seen along the basilar aspect of the thyroid follicular epithelial cells. These observation suggests the development of glomerulonephritis mediated by thyroid antigen, particularly ganglioside component.     

Changes in renal morphology and renin secretion in the golden-mantled ground squirrel (Spermophilus lateralis) during activity and hibernation

Summary Chronological changes in renal glomerular morphology and plasma renin activity were investigated during active and hibernating periods in the golden-mantled ground squirrel Spermophilus lateralis. The objective of this study was to determine whether the glomerular endothelium, visceral epithelium (podocytes), basement membrane, mesangial cells, proximal convoluted tubule cells and plasma renin activity exhibit measurable sequential differences between as well as within active and hibernating states at various time points. Limitations in the size of the experimental population prevented an evaluation of changes in these parameters during other important periods such as periodic arousal between hibernation bouts. In this study, glomerular endothelial pore number and epithelial filtration slit number significantly decreased by early hibernation when compared to those during summer activity, and then they increased back toward summer levels by late hibernation. In contrast, podocytic pedicel width along the glomerular basement membrane increased from summer activity to early hibernation, before significantly decreasing again by late hibernation. Mesangial cell and proximal convoluted tubule cell activity appeared increased during hibernation as compared to summer activity, whereas the width of the glomerular basement membrane showed no significant alterations throughout. Plasma renin activity significantly increased during early hibernation and mid-hibernation when compared to summer levels but had decreased by late hibernation toward summer values. The glomerular and plasma renin activity changes observed in this study clearly illustrate the drastic structural and functional adjustments which hibernating species make during torpor and also correlate well with the reported decrease in renal perfusion pressure and urine formation during hibernation. The observed morphological changes during hibernation do not appear to be temperature-dependent, because significant alterations in most of the parameters studied occurred during this period despite the fact that cold-room temperatures were kept constant throughout. The chronological approach to this study and its morphometric evaluation represent a pilot attempt at accurately documenting these changes during two critical states in the hibernator's cycle and may eventually lead to the characterization of these changes during the entire circannual cycle.     

Cellular aspects of rabbit Masugi nephritis V. Ultrastructural cytochemistry of monocytic cells

Ultrastructural cytochemistry for nonspecific esterase (NSE) was performed on normal and nephritic rabbit kidneys. In normal glomeruli distinct reaction deposits appearing as electron-dense granules were present in visceral epithelial cells (podocytes) and epithelial cells of Bowman's capsule, but only a few small or minute deposits were seen in some endothelial and mesangial cells. In acute proliferative glomerulonephritis (Masugi nephritis), many reaction deposits occurred in mononuclear cells accumulating in glomerular tufts which presented the characteristic features of monocytic cells. Macrophages which had migrated into subendothelial space as well as epithelioid cells and multinucleated giant cells, all of which are known to be derived from monocytes, also exhibited the reaction product. The NSE granules in mesangial and endothelial cells were much smaller and fewer in number than those in monocytic cells. The present method may contribute to the more precise differentiation of monocytic cells from mesangial and endothelial cells in proliferative glomerulonephritis.