Renal Basement Membrane Research Articles

Histopathological characters of calcium salt deposits on renal papilla in patients with infectious calculi

Objective To explore the relationship between stone formation and histopathological characters of renal papilla in patients with infectious renal calculi. Methods A total of 43 patients with infectious renal calculi undergoing percutaneous nephrolithotomy (PCNL) were included in this study. Renal papilla biopsy specimens were obtained under a nephroscope during the operation, fol-lowed by staining with hematoxylin-eosin or alizarin bordeaux for light microscopy and electron mi-croscopy. The extracted stones were analyzed and proved to be composed of magnesium ammonium phosphate or carbonate apatite with a transform-infrared spectrometer. Results Renal papilla calci-um plaques were found in 24 of the 43 patients (56 % ) during PCNL. Local calcium salt deposits could be found localized in the renal tubular basement membrane and spread to the interstitial tissue. Tiny stone adherence was observed in the renal papillae where calcium salts grew into the collection system. Conclusion Calcium salt deposits exist on the renal papilla in some patients with infectious renal calculi, which may be correlated with the formation of infectious stones. Key words: Infectious renal calculus; Calcium salt deposit; Pathological conditions, anatomical

Connective tissue growth factor(CCN2), a pathogenic factor in diabetic nephropathy. What does it do? How does it do it?

Connective tissue growth factor (CTGF/CCN2) is a member of the CCN family of matricellular proteins. Its expression is induced by a number of factors including TGF-β. It has been associated with fibrosis in various tissues including the kidney. Diabetic nephropathy (DN) develops in about 30% of patients with diabetes and is characterized by thickening of renal basement membranes, fibrosis in the glomerulus (glomerulosclerosis), tubular atrophy and interstitial fibrosis, all of which compromise kidney function. This review examines changes in CTGF expression in the kidney in DN, the effects they have on glomerular mesangial and podocyte cells and the tubulointerstitium, and how these contribute to driving fibrotic changes in the disease. CTGF can bind to several other growth factors modifying their function. CTGF is also able to interact with receptors on cells, including integrins, tyrosine receptor kinase A (TrkA), low density lipoprotein receptor-related protein (LRP) and heparan sulphate proteoglycans. These interactions, the intracellular signalling pathways they activate, and the cellular responses evoked are reviewed. CTGF also induces the expression of chemokines which themselves have pharmacological actions on cells. CTGF may prompt some responses by acting through several different mechanisms, possibly simultaneously. For example, CTGF is often described as an effector of TGF-β. It can promote TGF-β signalling by binding directly to the growth factor, promoting its interaction with the TGF-β receptor; by triggering intracellular signalling on binding the TrkA receptor, which leads to the transcriptional repression of Smad7, an inhibitor of the TGF-β signalling pathway; and by binding to BMP-7 whose own signalling pathway opposing TGF-β is inhibited, leading to enhanced TGF-β signalling.

Lack of nidogen‐2 increases blood pressure, glomerular and tubulointerstitial damage in DOCA‐salt hypertension

Nidogen-2, an extracellular matrix protein, is ubiquitous in renal basement membranes linking the laminin and collagen IV networks. Nidogen-2-deficient (nidogen-2(-/-)) mice do not exhibit a phenotype, and renal basement membranes appear normal. The functional role of nidogen-2 in the adult kidney under pathological conditions however remains unclear. We tested the hypothesis that nidogen-2 mediated cell-matrix interactions are important to maintain glomerular integrity and structure in renal hyperperfusion and hypertension. Two weeks after unilateral nephrectomy (UNX), desoxycorticosterone (DOCA)-salt hypertension was induced in nidogen-2(-/-) mice and their wild type littermates for 6 weeks. Renal damage was assessed by means of semiquantitative scoring, morphometric analysis, immunohistochemistry and measurement of serum creatinine and albumin excretion. UNX alone resulted in a very mild increase in renal damage in nidogen-2(-/-) mice compared to wild type animals. Following DOCA-salt treatment, blood pressure, serum creatinine and albumin excretion were significantly higher in nidogen-2(-/-) than in wild type mice. In addition, nidogen-2(-/-) mice showed increased mesangial cell hyperplasia and matrix expansion with higher expression of fibronectin and its receptor alpha8 integrin. Glomerular capillaries were significantly reduced in size and number. We demonstrate that in both mild and severe glomerular damage, lack of nidogen-2 is associated with: (i) increased mesangioproliferation; (ii) higher mesangial matrix expansion; and (iii) reduction in glomerular capillary supply. These findings suggest a critical role for nidogen-2 in the maintenance of glomerular structure in the diseased kidney.

The gene corresponding to the putative Goodpasture antigen is present in Alport’s syndrome

Alport's syndrome is a heterogeneous group of inherited abnormalities of basement membranes that may result in progressive renal failure, defective hearing and lens abnormalities. The glomerular basement membrane (GBM) characteristically has areas of reduplication, lamellation and attenuation on electron microscopic examination. In the majority of affected males and some females, there is reduced or variable binding of serum from patients with anti-GBM disease (Goodpasture's syndrome) to these basement membranes. These sera contain antibodies directed against the Goodpasture antigen which has been thought to be located in the non-collagenous domain of the alpha3 chain of type IV collagen and is presumed to be important in cross-linking of the collagen molecules. The reduced staining for the Goodpasture antigen suggests that this structure is either absent or masked in Alport's syndrome. We have tested DNA from six unrelated individuals with Alport's syndrome. All had been transplanted for renal failure. The diagnosis of Alport's syndrome was made on the characteristic electron microscopic appearance of the renal basement membranes (n = 4), the presence of sensori-neural deafness (n = 4), a family history of Alport's syndrome (n = 5) and the presence of circulating inhibitable anti-GBM antibody activity post-transplant (n = 2). Oligonucleotides (20mers) corresponding to the 5' and 3' ends of the known 25 amino acid sequence for the putative Goodpasture antigen were used as primers for amplification of genomic DNA. The products were then blotted and probed with an intermediate 19-mer DNA. All Alport's patients contained a 75-bp fragment corresponding to the published peptide sequence for the non-collagenous domain of the alpha 3 chain of type IV collagen, suggesting that a large deletion of this region, the putative Goodpasture antigen, is unlikely to account for the defect in Alport's syndrome.

Angiopathie héréditaire avec néphropathie, anévrismes et crampes musculaires (Ahnac) : une nouvelle maladie des membranes basales associée à des mutations du gène COL4A1 (gène codant pour la chaîne α1 du collagène de type IV)

Since 2005, several neurological diseases, including porencephaly, leukoencephalopathy and intracerebral hemorrhage without hypertension, and retinal arteriolar tortuosity have been linked to mutations in the COL4A1 gene, which encodes the alpha1 chain of type IV collagen, the main constituent of basement membranes. In three families, we observed a new syndrome that we called HANAC, for hereditary angiopathy with nephropathy, aneurysms and muscle cramps, which is associated with morphological alterations of cutaneous and renal basement membranes. This novel "basalopathy" is caused by glycine mutations in COL4A1 exons 24 and 25. We discuss phenotype-genotype correlations and the implications of the HANAC syndrome for the diagnosis of autosomal dominant hematuria, cystic kidney disease, intracranial aneurysms, and muscle cramps.

Demonstration of Epithelial-Mesenchymal Transition in Kidney

Traditional Periodic Acid Schiff has been extensively used, coupled with immunohistochemistry for epithelia or mesenchymal cells, to highlight renal tubular basement membrane (TBM). We recently tried to perform such technique in a 5/6 nephrectomy model of progressive renal fibrosis to demonstrate TBM disruption as an evidence for epithelial-mesenchymal transdifferentiation. Despite excellent basement membrane staining with traditional fuchsin-Periodic Acid Schiff, the interface between epithelial and mesenchymal cells was frequently blurred when revealed with 3'3 diaminobenzidine tetrachloride-peroxidase. Also, it was inadequate when revealed with alkaline phosphatase-fast red. We devised a triple staining method with Periodic Acid-Thionin Schiff to highlight basement membrane in blue, after double immunostaining for epithelium and mesenchymal cells. Blue basement membrane rendered a brisk contrast and highlighted boundaries between epithelial-mesenchymal interfaces. This method was easy to perform and useful to demonstrate the TBM, yield a clear demonstration of the very focal TBM disruption found in this model of progressive renal fibrosis.

The absence of one or both nidogens does not alter basement membrane composition in adult murine kidney.

Nidogen-1 and nidogen-2 are major components of all basement membranes and are considered to function as link molecules between laminin and collagen type IV networks. Surprisingly, the knockout of one or both nidogens does not cause defects in all tissues or in all basement membranes. In this study, we have elucidated the appearance of the major basement membrane components in adult murine kidney lacking nidogen-1, nidogen-2, or both nidogens. To this end, we localized laminin-111, perlecan, and collagen type IV in knockout mice, heterozygous (+/-) or homozygous (-/-) for the nidogen-1 gene, the nidogen-2 gene, or both nidogen genes with the help of light microscopic immunostaining. We also performed immunogold histochemistry to determine the occurrence of these molecules in the murine kidney at the ultrastructural level. The renal basement membranes of single knockout mice contained a similar distribution of laminin-111, perlecan, and collagen type IV compared to heterozygous mice. In nidogen double-knockout animals, the basement membrane underlying the tubular epithelium was sometimes altered, giving a diffuse and thickened pattern, or was totally absent. The normal or thickened basement membrane of double-knockout mice also showed a similar distribution of laminin-111, perlecan, and collagen type IV. The results indicate that the lack of nidogen-1, nidogen-2, or both nidogens, plays no crucial role in the occurrence and localization of laminin-111, collagen type IV, and perlecan in murine tubular renal basement membranes.

Contribution of Proteoglycans Towards the Integrated Functions of Renal Glomerular Capillaries: A Historical Perspective

Since the original colloidal iron tracer study by Farquhar and Palade almost 50 years ago,1 the permeability characteristics of mammalian glomerular capillary wall have been the subject of extensive investigations.2,3 In the ensuing 2 to 3 decades, various studies were performed to delineate whether glomerular basement membrane or the slit diaphragm of the capillary wall is the primary filtration barrier.4,5 Amid this contentious issue, in the mid to late 1970s various clearance and additional tracer studies indicated that the glomerular capillary wall behaves as a size- as well as charge-selective barrier, and the latter biophysical properties were attributed to the anionic sites within the basement membrane that are enriched with sulfated proteoglycans (PGs).6,7,8,9 The article by Harvey et al10 in this issue of The American Journal of Pathology suggests that agrin, a basement sulfated proteoglycan, imparts an electronegative charge to the glomerular basement membrane (GBM), but it may not be responsible for its charge-selective permeability properties. In light of these findings, we deemed it necessary to revisit the tracer work performed during the past few decades and reconcile with recent observations made in various mutant mice keeping in perspective the complex ultrastructure and integrated functions of the glomerular capillary wall.11

A Cumulative Shear Mechanism for Tissue Damage Initiation in Shock-Wave Lithotripsy

Evidence suggests that inertial cavitation plays an important role in the renal injury incurred during shock-wave lithotripsy. However, it is unclear how tissue damage is initiated, and significant injury typically occurs only after a sufficient dose of shock waves. Although it has been suggested that shock-induced shearing might initiate injury, estimates indicate that individual shocks do not produce sufficient shear to do so. In this paper, we hypothesize that the cumulative shear of the many shocks is damaging. This mechanism depends on whether there is sufficient time between shocks for tissue to relax to its unstrained state. We investigate the mechanism with a physics-based simulation model, wherein the basement membranes that define the tubules and vessels in the inner medulla are represented as elastic shells surrounded by viscous fluid. Material properties are estimated from in-vitro tests of renal basement membranes and documented mechanical properties of cells and extracellular gels. Estimates for the net shear deformation from a typical lithotripter shock (∼0.1%) are found from a separate dynamic shock simulation. The results suggest that the larger interstitial volume (∼40%) near the papilla tip gives the tissue there a relaxation time comparable to clinical shock delivery rates (∼1 Hz), thus allowing shear to accumulate. Away from the papilla tip, where the interstitial volume is smaller (∼20%), the model tissue relaxes completely before the next shock would be delivered. Implications of the model are that slower delivery rates and broader focal zones should both decrease injury, consistent with some recent observations. (E-mail: jbfreund@uiuc.edu)

Diagnosis of Alport syndrome without biopsy?

Alport syndrome (AS) is genetically heterogeneous. The gene COL4A5 is mutated in the more frequent X-linked dominant form of the disease whereas COL4A3 or COL4A4 are mutated in the autosomal recessive and dominant forms. Diagnosis of AS and determination of the mode of transmission are important because of the differences in prognosis and genetic counselling attached to these different forms. Recently, promising results have been obtained in Col4a3-null mice, an animal model for AS, with different therapeutic trials when administered early in the course of the disease, an additional reason for making early diagnosis of AS in children. Since the identification of the molecular basis of the disease, mutation screening is theoretically the best diagnostic approach, avoiding the use or renal or skin biopsy. However, for many reasons linked to the genetic heterogeneity of the disease, the large size of the three genes and the random distribution of the mutations all along these huge genes, this method is tedious, expensive and time consuming. Moreover, its sensitivity is reduced. For these reasons, evaluation of the expression of type IV collagen chains in the skin, and if necessary in the renal basement membrane, remains a useful tool for AS diagnosis. At this time, the indication for these different approaches, which are not mutually exclusive but complementary, depends on the patient clinical presentation and family history.

Role for Macrophage Metalloelastase in Glomerular Basement Membrane Damage Associated with Alport Syndrome

Alport syndrome is a glomerular basement membrane (GBM) disease caused by mutations in type IV collagen genes. A unique irregular thickening and thinning of the GBM characterizes the progressive glomerular pathology. The metabolic imbalances responsible for these GBM irregularities are not known. Here we show that macrophage metalloelastase (MMP-12) expression is >40-fold induced in glomeruli from Alport mice and is markedly induced in glomeruli of both humans and dogs with Alport syndrome. Treatment of Alport mice with MMI270 (CGS27023A), a broad spectrum MMP inhibitor that blocks MMP-12 activity, results in largely restored GBM ultrastructure and function. Treatment with BAY-129566, a broad spectrum MMP inhibitor that does not inhibit MMP-12, had no effect. We show that inhibition of CC chemokine receptor 2 (CCR2) receptor signaling with propagermanium blocks induction of MMP-12 mRNA and prevents GBM damage. CCR2 receptor is expressed in glomerular podocytes of Alport mice, suggesting MCP-1 activation of CCR2 on podocytes may underlie induction of MMP-12. These data indicate that the irregular GBM that characterizes Alport syndrome may be mediated, in part, by focal degradation of the GBM due to MMP dysregulation, in particular, MMP-12. Thus, MMP-12/CCR2 inhibitors may provide a novel and effective therapeutic stra-tegy for Alport glomerular disease.

The clearance of BNP modeled using the NT-proBNP–BNP relationship

Background The ventricular myocardium simultaneously secretes two natriuretic peptides useful in the evaluation of heart failure: BNP, hormonally active, and NT-proBNP, the N-terminal end, non-hormonally active, but ultimately their concentrations differ and their clearance patterns are poorly defined. Methods We measured NT-proBNP and BNP in patients with and without heart failure and compared their concentrations using regression analysis. Results The relationship between NT-proBNP with BNP is nonlinear. Between 45 and 70 pmol of BNP/L (class II heart failure) the slope is much higher than in other ranges and the NT-proBNP/BNP ratio reaches its maximum in patients with class II NYHA heart failure. Conclusions The difference in concentration for NT-proBNP and BNP can be related to the diffusion across the renal basement membrane. Their ratio is nonlinear because BNP is cleared faster than in patients with class II heart failure than other classes or normal, suggesting a change in a non-renal mode of clearance.

Increased endogenous antioxidants and reduced basement membrane thickening in Ames dwarf mouse kidney

Experimental evidence suggests that disease- and age-related thickening of glomerular (GBM) and tubular (TBM) basement membranes may be related to increases in reactive oxygen species (ROS). Studies also suggest that antioxidants may function as renoprotectants against such ROS-induced damage. The current study focuses on metallothionein (MT) and glutathione (GSH), both of which exhibit potent antioxidant functions. Ames dwarf mice, which exhibit elevated tissue antioxidant levels (catalase, Cu/Zn superoxide dismutase, GSH) and an average life span significantly greater (50–65%) than wild type controls, were used to test our hypothesis that increased levels of renal MT and GSH are concomitant with decreased rates of renal basement membrane thickening. A competitive ELISA assay was developed to quantitate both endogenously polymerized and “total” (glutaraldehyde-polymerized) renal MT while renal GSH levels were determined by fluorimetry. At 90, 300, 600, and 720 days, dwarf renal MT and GSH levels were significantly higher than controls. Interestingly, TEM morphometry data demonstrated that with increasing age, both the GBMs and TBMs of dwarf animals were thinner (1.1–15.7% and 28.8–45.5%, respectively) than those of age-matched controls. Since MT and GSH are well-documented ROS scavengers, it seems possible that in the Ames dwarf model of delayed aging, increased endogenous antioxidant levels provide renoprotection from oxidative damage and lead to reduced rates of age-related GBM and TBM thickening.

The Role of Type IV Collagen and Basement Membranes in Cancer Progression and Metastasis

The basement membrane acts as a barrier separating the epithelium from the surrounding stroma. While the structural role of the basement membrane in defining tissue architecture and compartmentalization has been appreciated for decades, its dynamic role in the regulation of cell behavior and recruitment of signaling pathways is only now emerging.1 The basement membrane further defines the tumor microenvironment and provides significant host-derived regulatory signals during progression of tumor growth and metastasis. The major component of the basement membrane is known to be type IV collagen.1 Nevertheless, a precise role for type IV collagen in its assembled suprastructural form versus its unassembled/degraded form is primarily unknown. In this issue of The American Journal of Pathology, Ikeda and colleagues2 demonstrate the effect of promoter hypermethylation on the expression levels of COL4A5/COL4A6 collagen genes. They suggest that such hypermethylation leads to diminished levels of α5(IV) and α6(IV) collagen in colorectal cancer tissue and colon cancer cells. The normal production and assembly of the basement membrane is disrupted in this malignant process. This data is consistent with evidence emerging from recent studies examining different cancer tissues, including prostate cancer, using antibodies generated in the Sado Ninomiya laboratory.3–8 However, a lack of free access to these antibodies to all investigators has limited independent evaluation. In vitro studies demonstrate that the expression of α5(IV) and α6(IV) collagen is down-regulated in association with hypermethylation of the bidirectional promoter of COL4A5/COL4A6 genes in colon cancer cell lines.2 These results suggest that normal assembly and organization of the basement membrane is disrupted in cancer progression.2 In an earlier study, Tanaka and colleagues9 analyzed the expression of the α1(IV) to α6(IV) collagen chain assembly by immunohistochemical analysis in colorectal cancer tissue, revealing normal expression of α1(IV)/α2(IV) and α5(IV)/α6(IV) chains but lost expression of α5(IV)/α6(IV) chains upon cancer invasion. These authors suggested that loss of α5(IV)/α6(IV) expression in the invasive cancer may be attributable to remodeling of basement membrane assembly, which in turn may be regulated by gene expression. The present article by Ikeda and colleagues2 now further demonstrates that such loss may be at the gene expression level.

Apport de l'immunohistochimie dans le diagnostic du syndrome d'Alport

Alport syndrome (AS) is an hereditary disease characterised by the association of progressive hematuria nephritis. The diagnosis is based on clinical genetic and ultrastructural findings. Nowadays, immunohistochemical technique is of great interest. It enables us to analyze the distribution of the different chains of the type IV collagen in renal basement membrane (RBM) and epidermal basement membrane (EBM) which appeared to be abnormal in 70% of cases. We report a prospective study of five families affected with AS. Six patients were investigated by immunohistochemical studies of kidney (3 cases) and skin (6 cases) frozen specimens. Monoclonal antibodies recognizing the collagenous domain of alpha1 (MAB1), alpha3 (MAB3) and alpha5 (MAB5) chains of type IV collagen were used. Two methods were performed: direct immunofluorescence and immunohistochemical (ultravision) analysis. The different chains distribution of type IV collagen in the EBM and RBM was normal in four cases (4 men), abnormal in two patients (1 man and woman). Based on the clinical, genetical and immunohistochemical findings we established three transmission modes: autosomal recessive in two families, dominant X linked in two other familiales, and autosomal dominant in one family. Immunohistochemical studies is a simple technique of an easy interpretation accomplished on kidney frozen specimen, or even on a simple cutaneous biopsy. It could be very useful for the diagnosis and enables us in addition to determine the mode of transmission of AS.

Experimental evidence for a direct cytotoxicity of Loxosceles intermedia (brown spider) venom in renal tissue.

Brown spider (Loxosceles genus) venom causes necrotic lesions often accompanied by fever, hemolysis, thrombocytopenia, and acute renal failure. Using mice exposed to Loxosceles intermedia venom, we aimed to show whether the venom directly induces renal damage. The experimental groups were composed of 50 mice as controls and 50 mice that received the venom. Light microscopic analysis of renal biopsy specimens showed alterations including hyalinization of proximal and distal tubules, erythrocytes in Bowman's space, glomerular collapse, tubule epithelial cell blebs and vacuoles, interstitial edema, and deposition of eosinophilic material in the tubule lumen. Electron microscopic findings indicated changes including glomerular epithelial and endothelial cell cytotoxicity as well as disorders of the basement membrane. Tubule alterations include epithelial cell cytotoxicity with cytoplasmic membrane blebs, mitochondrial changes, increase in smooth endoplasmic reticulum, presence of autophagosomes, and deposits of amorphous material in the tubules. We also found that the venom caused azotemia with elevation of blood urea levels but did not decrease C3 complement concentration or cause hemolysis in vivo. Confocal microscopy with antibodies against venom proteins showed direct binding of toxins to renal structures, confirmed by competition assays. Double-staining immunofluorescence reactions with antibodies against type IV collagen or laminin, antibodies to venom toxins, and fluorescent cytochemistry with DAPI revealed deposition of toxins in glomerular and tubule epithelial cells and in renal basement membranes. Two-dimensional electrophoresis showed venom rich in low molecular mass and cationic toxins. By immunoblotting with antibodies to venom toxins on renal extracts from venom-treated mice, we detected a renal binding toxin at 30 kD. The data provide experimental evidence that L. intermedia venom is directly involved in nephrotoxicity.

Immunodetection of surfactant proteins in human organ of Corti, Eustachian tube and kidney.

The presence of surfactant proteins was investigated in the human organ of Corti, Eustachian tube and kidney tissues. It has previously been shown that lamellar bodies are present in hairy cells of organ of Corti, in the cytoplasm of secretory and lumen of tubal glands of Eustachian tube and kidney renal basement membrane. No evidence for the presence of surfactant proteins in the organ of Corti and kidney has been presented until now. The aim of this study was to find out if surfactant proteins were expressed in other epithelia such as organ of Corti, Eustachian tube and kidney. Surfactant proteins were identified using one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. On one-dimensional Western blots, bands for surfactant protein A in human Eustachian tube (SP-A, 34 kDa) and in kidney extracts, and for surfactant protein D (SP-D, 43 kDa) in Eustachian tube and in kidney extracts (SP-D, 86 kDa), and for surfactant protein B (SP-B, 8 kDa) in human Eustachian tube and organ of Corti extracts were detected. Bands corresponded to monomeric forms of lung surfactant proteins. These results indicate the presence of SP-A and SP-D in kidney epithelium, SP-A, SP-B and SP-D in Eustachian tube and SP-B in the organ of Corti.

Immunodetection of surfactant proteins in human organ of Corti, Eustachian tube and kidney.

The presence of surfactant proteins was investigated in the human organ of Corti, Eustachian tube and kidney tissues. It has previously been shown that lamellar bodies are present in hairy cells of organ of Corti, in the cytoplasm of secretory and lumen of tubal glands of Eustachian tube and kidney renal basement membrane. No evidence for the presence of surfactant proteins in the organ of Corti and kidney has been presented until now. The aim of this study was to find out if surfactant proteins were expressed in other epithelia such as organ of Corti, Eustachian tube and kidney. Surfactant proteins were identified using one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. On one-dimensional Western blots, bands for surfactant protein A in human Eustachian tube (SP-A, 34 kDa) and in kidney extracts, and for surfactant protein D (SP-D, 43 kDa) in Eustachian tube and in kidney extracts (SP-D, 86 kDa), and for surfactant protein B (SP-B, 8 kDa) in human Eustachian tube and organ of Corti extracts were detected. Bands corresponded to monomeric forms of lung surfactant proteins. These results indicate the presence of SP-A and SP-D in kidney epithelium, SP-A, SP-B and SP-D in Eustachian tube and SP-B in the organ of Corti.

Renal Fibrosis: Extracellular Matrix Microenvironment Regulates Migratory Behavior of Activated Tubular Epithelial Cells

During progression of chronic renal disease, qualitative and quantitative changes in the composition of tubular basement membranes (TBMs) and interstitial matrix occur. Transforming growth factor (TGF)-beta(1)-mediated activation of tubular epithelial cells (TECs) is speculated to be a key contributor to the progression of tubulointerstitial fibrosis. To further understand the pathogenesis associated with renal fibrosis, we developed an in vitro Boyden chamber system using renal basement membranes that partially mimics in vivo conditions of TECs during health and disease. Direct stimulation of TECs with TGF-beta(1)/epithelial growth factor results in an increased migratory capacity across bovine TBM preparations. This is associated with increased matrix metalloproteinase (MMP) production, namely MMP-2 and MMP-9. Indirect chemotactic stimulation by TGF-beta(1)/EGF or collagen type I was insufficient in inducing migration of untreated TECs across bovine TBM preparation, suggesting that basement membrane integrity and composition play an important role in protecting TECs from interstitial fibrotic stimuli. Additionally, neutralization of MMPs by COL-3 inhibitor dramatically decreases the capacity of TGF-beta(1)-stimulated TECs to migrate through bovine TBM preparation. Collectively, these results demonstrate that basement membrane structure, integrity, and composition play an important role in determining interstitial influences on TECs and subsequent impact on potential aberrant cell-matrix interactions.

Focusing on the Glomerular Slit Diaphragm: Podocin Enters the Picture

The nature of the glomerular filtration barrier is currently one of the most intensely studied and exciting problems in nephrology research, primarily because of the relatively recent discoveries that two novel and two previously known genes are intimately involved in glomerular filtration. The two novel genes, NPHS1 and NPHS2 (encoding nephrin and podocin, respectively) were identified by positional cloning. NPHS1 is mutated in congenital nephrotic syndrome of the Finnish type, 1 and NPHS2 is mutated in steroid resistant nephrotic syndrome. 2 The other two genes, previously characterized but not known to be involved in filtration, are ACTN4, encoding α-actinin-4, and Cd2ap, encoding CD2-associated protein (CD2AP). ACTN4 was found to be mutated in several families exhibiting inherited focal segmental glomerulosclerosis, 3 and knockout mice lacking CD2AP exhibit congenital nephrotic syndrome. 4 Several recent reviews have addressed the potential roles of these molecules in glomerular filtration. 5-10