Ultrastructure Of Glomerular Basement Membrane Research Articles

Ramipril therapy in integrin α1-null, autosomal recessive Alport mice triples lifespan: mechanistic clues from RNA-seq analysis.

The standard of care for patients with Alport syndrome (AS) is angiotensin-converting enzyme (ACE) inhibitors. In autosomal recessive Alport (ARAS) mice, ACE inhibitors double lifespan. We previously showed that deletion of Itga1 in Alport mice [double-knockout (DKO) mice] increased lifespan by 50%. This effect seemed dependent on the prevention of laminin 211-mediated podocyte injury. Here, we treated DKO mice with vehicle or ramipril starting at 4 weeks of age. Proteinuria and glomerular filtration rates were measured at 5-week intervals. Glomeruli were analyzed for laminin 211 deposition in the glomerular basement membrane (GBM) and GBM ultrastructure was analyzed using transmission electron microscopy (TEM). RNA sequencing (RNA-seq) was performed on isolated glomeruli at all time points and the results were compared with cultured podocytes overlaid (or not) with recombinant laminin 211. Glomerular filtration rate declined in ramipril-treated DKO mice between 30 and 35 weeks. Proteinuria followed these same patterns with normalization of foot process architecture in ramipril-treated DKO mice. RNA-seq revealed a decline in the expression of Foxc2, nephrin (Nphs1), and podocin (Nphs2) mRNAs, which was delayed in the ramipril-treated DKO mice. GBM accumulation of laminin 211 was delayed in ramipril-treated DKO mice, likely due to a role for α1β1 integrin in CDC42 activation in Alport mesangial cells, which is required for mesangial filopodial invasion of the subendothelial spaces of the glomerular capillary loops. Ramipril synergized with Itga1 knockout, tripling lifespan compared with untreated ARAS mice. © 2023 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Lysyl oxidase like-2 contributes to renal fibrosis in Col4α3/Alport mice

Lysyl oxidase like-2 (LOXL2) is an amine oxidase with both intracellular and extracellular functions. Extracellularly, LOXL2 promotes collagen and elastin crosslinking, whereas intracellularly, LOXL2 has been reported to modify histone H3, stabilize SNAIL, and reduce cell polarity. Although LOXL2 promotes liver and lung fibrosis, little is known regarding its role in renal fibrosis. Here we determine whether LOXL2 influences kidney disease in COL4A3 (-/-) Alport mice. These mice were treated with a small molecule inhibitor selective for LOXL2 or with vehicle and assessed for glomerular sclerosis and fibrosis, albuminuria, blood urea nitrogen, lifespan, pro-fibrotic gene expression and ultrastructure of the glomerular basement membrane. Laminin α2 deposition in the glomerular basement membrane and mesangial filopodial invasion of the glomerular capillaries were also assessed. LOXL2 inhibition significantly reduced interstitial fibrosis and mRNA expression of MMP-2, MMP-9, TGF-β1, and TNF-α. LOXL2 inhibitor treatment also reduced glomerulosclerosis, expression of MMP-10, MMP-12, and MCP-1 mRNA in glomeruli, and decreased albuminuria and blood urea nitrogen. Mesangial filopodial invasion of the capillary tufts was blunted, as was laminin α2 deposition in the glomerular basement membrane, and glomerular basement membrane ultrastructure was normalized. There was no effect on lifespan. Thus, LOXL2 plays an important role in promoting both glomerular and interstitial pathogenesis associated with Alport syndrome in mice. Other etiologies of chronic kidney disease are implicated with our observations.

A mouse Col4a4 mutation causing Alport glomerulosclerosis with abnormal collagen α3α4α5(IV) trimers

A spontaneous mutation termed bilateral wasting kidneys (bwk) was identified in a colony of NONcNZO recombinant inbred mice. These mice exhibit a rapid increase of urinary albumin at an early age associated with glomerulosclerosis, interstitial nephritis, and tubular atrophy. The mutation was mapped to a location on Chromosome 1 containing the Col4a3 and Col4a4 genes, for which mutations in the human orthologs cause the hereditary nephritis Alport syndrome. DNA sequencing identified a G to A mutation in the conserved GT splice donor of Col4a4 intron 30, resulting in skipping of exon 30 but maintaining the mRNA reading frame. Protein analyses showed that mutant collagen α3α4α5(IV) trimers were secreted and incorporated into the glomerular basement membrane (GBM), but levels were low, and GBM lesions typical of Alport syndrome were observed. Moving the mutation into the more renal damage-prone DBA/2J and 129S1/SvImJ backgrounds revealed differences in albuminuria and its rate of increase, suggesting an interaction between the Col4a4 mutation and modifier genes. This novel mouse model of Alport syndrome is the only one shown to accumulate abnormal collagen α3α4α5(IV) in the GBM, as also found in a subset of Alport patients. These mice will be valuable for testing potential therapies, for understanding abnormal collagen IV structure and assembly, for gaining better insights into the mechanisms leading to Alport syndrome and to the variability in the age of onset and associated phenotypes.

Loss of the BMP antagonist USAG-1 ameliorates disease in a mouse model of the progressive hereditary kidney disease Alport syndrome

The glomerular basement membrane (GBM) is a key component of the filtering unit in the kidney. Mutations involving any of the collagen IV genes (COL4A3, COL4A4, and COL4A5) affect GBM assembly and cause Alport syndrome, a progressive hereditary kidney disease with no definitive therapy. Previously, we have demonstrated that the bone morphogenetic protein (BMP) antagonist uterine sensitization-associated gene-1 (USAG-1) negatively regulates the renoprotective action of BMP-7 in a mouse model of tubular injury during acute renal failure. Here, we investigated the role of USAG-1 in renal function in Col4a3-/- mice, which model Alport syndrome. Ablation of Usag1 in Col4a3-/- mice led to substantial attenuation of disease progression, normalization of GBM ultrastructure, preservation of renal function, and extension of life span. Immunohistochemical analysis revealed that USAG-1 and BMP-7 colocalized in the macula densa in the distal tubules, lying in direct contact with glomerular mesangial cells. Furthermore, in cultured mesangial cells, BMP-7 attenuated and USAG-1 enhanced the expression of MMP-12, a protease that may contribute to GBM degradation. These data suggest that the pathogenetic role of USAG-1 in Col4a3-/- mice might involve crosstalk between kidney tubules and the glomerulus and that inhibition of USAG-1 may be a promising therapeutic approach for the treatment of Alport syndrome.

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.

Effects of calcium dobesilate on glomerulus TIMP1 and collagen IV of diabetic rats.

To observe the effects of calcium dobesilate on the expression of glomerular tissue inhibitor of metalloproteinase 1 (TIMP1), collagen IV, and ultrastructure of glomerular basement membrane in diabetic rats, rats model of diabetes was established by unilateral nephrectomy and intraperitoneal injection of 1% STZ (55 mg/kg), and rats were administered calcium dobesilate 100 mg/ kg (DD group) or distilled water (DM group) respectively. 12 weeks later, the changes in the renal ultrastructure and creatinine clearance rate (Ccr) were examined in each group. The expression of glomerular TIMP1 and collagen IV were studied by immunohistochemical staining. Our results showed that after 12 weeks, the Ccr in DD group increased and was significantly higher than that in DM group. Electron microscopy showed that thickness of glomerular capillary basement membrane (GBM) in Group DD was less than that of DM group. No hyperplasia of collagen fibers was found, and the distance between the holes of endothelial cells in DD group was not as even as that in the normal group, but more even than that of DM group, and podocyte processes was still in order. Immunohistochemical staining of glomeruli showed that expression of TIMP1 and collagen IV in DD group were significantly less than those of DM group DM. It is concluded that calcium dobesilate can improve diabetic nephropathy by inhibiting the over- accumulation of collagen IV and calcium dobesilate may also contribute to diabetes by inhibiting the expression of TIMP1.

The Nongenetic Diagnosis of Thin Basement Membrane Nephropathy

Three disorders, thin basement membrane nephropathy (TBMN), immunoglobulin A nephropathy (IgAN), and Alport syndrome (AS), account for the majority of children and adults with persistent glomerular hematuria. Although there is some clinical overlap between these conditions, they can be distinguished on the basis of family history, extrarenal findings, routine immunofluorescence, and glomerular basement membrane ultrastructure or type IV collagen chain composition. This distinction is important because of the very different prognoses of these conditions.

Ultrastructural Defects of the Glomerular Basement Membranes Associated with Primary Glomerular Nephropathies

It is generally accepted that a glomerular basement membrane (GBM) thinner than 200 nm should be considered below normal. When this abnormality has a global and diffuse distribution, the associated clinical condition is a benign familial hematuria related to mutations of the COL4A4/COL4A3 genes, or an Alport syndrome. More often the GBM defects display a focal and segmental pattern, too small to express a thin glomerular basement membrane disease. The aim of this study is to emphasize statistical data concerning the pathogenic link between the renal glomerular diseases and the preexisting thin and very thin GBM. A series of 487 renal biopsies from adult patients has been thoroughly investigated both for nephropathologic diagnosis and the GBM ultrastructure. It has been statistically concluded that there is a close coexistence of primary glomerulonephritis and thin glomerular basement membranes with the role of a predisposing condition for immune complex deposition.

Ultrastructure of Glomerular Basement Membrane in Active Heymann Nephritis Rats Revealed by Tissue-Negative Staining Method

Recently, we have developed a tissue-negative staining method, and successfully visualized fine meshwork structure of the glomerular basement membrane (GBM). To clarify the mechanism of proteinuria in active Heymann nephritis, we performed tissue-negative staining and investigated the ultrastructural alterations of the GBM. Active Heymann nephritis, the animal model of human membranous nephropathy, was induced in Lewis rats by the injection of proximal tubular brush border antigen, i.e. Fx1A. Urinary protein excretion was measured and histological studies were performed over 15 weeks following the Fx1A injection. Proteinuria developed at 10 weeks after injection (38.2 ± 7.4 mg/day) and progressively increased (160.2 ± 20.6 mg/day at 15 weeks). Capillary fine deposits of IgG and C3 were seen by immunofluorescence, and subepithelial electron dense deposits (EDD) by transmission electron microscopy (TEM). Using the tissue-negative staining method, regular meshwork structure consisted of fine fibrils and pores (2.5 ± 0.7 nm in short dimension) was observed in the GBM of control rats. At 10 and 15 weeks after injection, the GBM, directly facing the endothelial side of EDD, contained enlarged pores and nephrotic tunnels. Mean values of the short dimension of enlarged pores were 2.9 ± 0.5 nm at 10 weeks and 3.1 ± 0.4 nm at 15 weeks, which were significantly larger than that of control rats (p < 0.01). The rest area of the GBM, including newly produced GBM covering the epithelial side of EDD, had no significant difference in size of the pores from control GBM and no tunnels. Although there was no significant difference in the size of enlarged pores between 10 and 15 weeks, the percentage area of GBM with impaired size barrier increased at 15 weeks (51.4 ± 8.1%) compared with 10 weeks (24.0 ± 8.3%) and related to severity of proteinuria. The density of the tunnels also increased at 15 weeks. In conclusion, immune deposits may affect the GBM biosynthesis and induce the defect of size barrier of the GBM, which is responsible for proteinuria in active Heymann nephritis.

Ultrastructural study of glomerular basement membrane in diabetic rats by the quick-freezing and deep-etching method

The three-dimensional ultrastructure of glomerular basement membrane (GBM) and mesangium in very early stage of streptozotocin-induced diabetic rat kidney was examined by the quick-freezing and deep-etching method. In control rats GBM was composed of three layers. Outer and inner layers consisted of files of fibrils that connected endothelial or epithelial cells perpendicularly with meshwork of a middle layer that also consisted of dense meshwork structure composed of fibrils. In diabetic rats, perpendicular fibrils of the inner layer were irregular and disrupted, and the layer was enlarged. The meshwork structure of the middle layer was irregularly enlarged, and matrix fibrils were disrupted. Early insulin treatment prevents these morphological changes. Furthermore, early administration of beraprost sodium, a PGI 2 analogue, might have beneficial effects in preventing the progression of these changes.

Three-dimensional ultrastructure of human glomerular basement membrane by a quick-freezing and deep-etching method.

The three-dimensional ultrastructure of glomerular basement membrane (GBM) in surgically resected human kidneys was observed by the quick-freezing and deep-etching method. In three layers of human GBM, the middle layer exhibited polygonal meshwork structures, which were composed of fine fibrils. In addition, the outer and inner layers were formed by lines of perpendicular fibrils, which connected the epithelial or endothelial cell surfaces with the meshwork structures of the middle layer. These ultrastructures might correspond to the three layers, such as lamina densa and laminae rara externa and interna, which were also observed on conventional ultrathin sections. It is concluded that the human GBM has the same meshwork structures and perpendicular fibrils as those previously observed in animals and that these structures may act functionally as size and/or charge barriers.

Kidney in Galloway-Mowat syndrome: Clinical spectrum with description of pathology

The Galloway-Mowat syndrome, a rare inherited disorder, is characterized by congenital microcephaly with hypotonia and developmental delay, often hiatus hernia, and nephrotic syndrome manifested in infancy or in early childhood. The glomerular lesion has been poorly characterized in the few previous reports of this syndrome. We studied three siblings with microcephaly and nephrotic syndrome occurring during the first two weeks of life. Hematuria, glycosuria and renal failure were also present. Renal biopsy and postmortem specimens of two patients were studied. Glomerular structure was disorganized; capillary lumina were of varying calibers, capillary walls were adherent to one another, and mesangial zones were poorly demarcated. Glomerular basement membrane ultrastructure was markedly altered. The normal trilaminar structure was obscured or replaced by flocculent material; furthermore, 6 to 8 nm fibrils of unknown nature permeated the space between endothelial and epithelial cells. Non-glomerular basement membranes were unaltered in appearance. This syndrome apparently represents, in part, a new disorder of glomerular basement membrane formation and function.

Ultrastructural study of glomerular basement membrane in diabetic rats by quick-freezing and deep-etching method.

The three-dimensional ultrastructure of glomerular basement membrane (GBM) in streptozotocin (STZ)-induced diabetic rats was examined by quick-freezing and deep-etching method. In three layers of the GBM of control rats, the outer and inner layers were formed by files of perpendicular fibrils, which connected the epithelial or endothelial cell surfaces with meshwork structures of the middle layer. In the diabetic rats, the inner layer was diffusely enlarged and the meshwork structure of the middle layer became markedly irregular due to the rupture of fine fibrils and thickening of material adherent to the fibrils. These ultrastructural changes correspond to those of subendothelial oedema, lamellation of lamina densa and fluffy material in the GBM, as revealed on conventional ultra-thin sections. It is suggested that the initial morphological change of STZ-induced diabetic nephropathy is disruption of matrix fibrils in the GBM, seemingly indicating a disturbance of size and/or charge barriers.

Ultrastructure of glomerular basement membrane by quick-freeze and deep-etch methods

The glomerular basement membrane of rat kidneys were three-dimensionally observed by quick-freeze and deep-etch replica methods at high resolution. The middle layer (lamina densa) was composed of 6 to 10 nm fibrils which formed a meshwork structure. The space between the fibrils had polygonal shape. The average long dimension of the space between fibrils was 17 nm and the short one was 13 nm. At the outer layer (lamina rara externa), fibrils connected podocytes perpendicularly with the meshwork of the middle layer. At the inner layer (lamina rara interna), similar perpendicular fibrils also connected endothelial cells with the meshwork of the middle layer. This is the first report to visualize the three-dimensional meshwork structure of the middle layer (the lamina densa) in situ. The function of anchoring podocytes to the lamina densa was suggested in the perpendicularly arranged fibrils of the outer layer. The quick-freeze and deep-etch method is useful in analyzing filamentous ultrastructure in glomeruli, and will be applied to clarifying pathological ultrastructure in kidney diseases.