Laminin trimers composed of α, β, and γ chains are major components of basal laminae (BLs) throughout the body. To date, three α chains (α1–3) have been shown to assemble into at least seven heterotrimers (called laminins 1–7). Genes encoding two additional α chains (α4 and α5) have been cloned, but little is known about their expression, and their protein products have not been identified. Here we generated antisera to recombinant α4 and α5 and used them to identify authentic proteins in tissue extracts. Immunoprecipitation and immunoblotting showed that α4 and α5 assemble into four novel laminin heterotrimers (laminins 8–11: α4β1γ1, α4β2γ1, α5β1γ1, and α5β2γ1, respectively). Using a panel of nucleotide and antibody probes, we surveyed the expression of α1-5 in murine tissues. All five chains were expressed in both embryos and adults, but each was distributed in a distinct pattern at both RNA and protein levels. Overall, α4 and α5 exhibited the broadest patterns of expression, while expression of α1 was the most restricted. Immunohistochemical analysis of kidney, lung, and heart showed that the α chains were confined to extracellular matrix and, with few exceptions, to BLs. All developing and adult BLs examined contained at least one α chain, all α chains were present in multiple BLs, and some BLs contained two or three α chains. Detailed analysis of developing kidney revealed that some individual BLs, including those of the tubule and glomerulus, changed in laminin chain composition as they matured, expressing up to three different α chains and two different β chains in an elaborate and dynamic progression. Interspecific backcross mapping of the five α chain genes revealed that they are distributed on four mouse chromosomes. Finally, we identified a novel full-length α3 isoform encoded by the Lama3 gene, which was previously believed to encode only truncated chains. Together, these results reveal remarkable diversity in BL composition and complexity in BL development.
Kidney disease affects over 20 million people in the United States alone. Although the causes of renal failure are diverse, the glomerular filtration barrier is often the target of injury. Dysregulation of VEGF expression within the glomerulus has been demonstrated in a wide range of primary and acquired renal diseases, although the significance of these changes is unknown. In the glomerulus, VEGF-A is highly expressed in podocytes that make up a major portion of the barrier between the blood and urinary spaces. In this paper, we show that glomerular-selective deletion or overexpression of VEGF-A leads to glomerular disease in mice. Podocyte-specific heterozygosity for VEGF-A resulted in renal disease by 2.5 weeks of age, characterized by proteinuria and endotheliosis, the renal lesion seen in preeclampsia. Homozygous deletion of VEGF-A in glomeruli resulted in perinatal lethality. Mutant kidneys failed to develop a filtration barrier due to defects in endothelial cell migration, differentiation, and survival. In contrast, podocyte-specific overexpression of the VEGF-164 isoform led to a striking collapsing glomerulopathy, the lesion seen in HIV-associated nephropathy. Our data demonstrate that tight regulation of VEGF-A signaling is critical for establishment and maintenance of the glomerular filtration barrier and strongly supports a pivotal role for VEGF-A in renal disease.
ABSTRACT Alport syndrome (AS) is characterized by glomerular basement membrane (GBM) abnormalities leading to progressive glomerulosclerosis. Mutations in the COL4A3, COL4A4 or COL4A5 genes encoding type IV collagen α3α4α5 cause AS. Truncated α3, α4, and α5 chains lacking an intact COOH-terminal noncollagenous domain due to a premature termination codon (PTC) cannot assemble into heterotrimers or incorporate into the GBM. Therefore, achieving full-length protein expression is a potential therapy for AS caused by truncating nonsense mutations. Small molecule-based PTC readthrough (PTC-RT) therapy has been well studied in other genetic diseases, but whether PTC-RT is applicable to AS is unexplored. To investigate the feasibility of PTC-RT therapy in AS, we made a cDNA to express COL4A5 fused to a C-terminal NanoLuc luciferase (NLuc) to monitor full-length translation. Full-length COL4A5-NLuc produces luminescence, but mutants truncated due to a PTC do not. To screen for COL4A5 nonsense mutants susceptible to PTC-RT, we introduced 49 individual nonsense mutations found in AS patients into the COL4A5-NLuc cDNA. Luciferase assays revealed that 11 mutations ( C29X, S36X, E130X, C1521X, R1563X, C1567X, W1594X, S1632X, R1683X, C1684X and K1689X ) were susceptible to PTC-RT induced by G418, which is known to have high readthrough activity. Moreover, we found that some next-generation “designer” PTC-RT drugs induced RT, and RT enhancer compounds increased the efficacy of PTC-RT in a G418-susceptible PTC mutant. These results suggest that PTC-RT therapy is a feasible approach for some patients with AS. Our luciferase-based COL4A5 translation reporter system will contribute to further development of PTC-RT therapies in a personalized medicine approach to treating AS.
The absence of Discs-large 1 (DLG1), the mouse ortholog of the Drosophila discs-large tumor suppressor, results in congenital hydronephrosis characterized by urinary tract abnormalities, reduced ureteric bud branching, and delayed disconnection of the ureter from the common nephric duct (CND). To define the specific cellular requirements for Dlg1 expression during urogenital development, we used a floxed Dlg1 allele and Pax2-Cre, Pax3-Cre, Six2-Cre, and HoxB7-Cre transgenes to generate cell type-restricted Dlg1 mutants. In addition, we used RetGFP knockin and retinoic acid response element-lacZ transgenic mice to determine the effects of Dlg1 mutation on the respective morphogenetic signaling pathways. Mutation of Dlg1 in urothelium and collecting ducts (via HoxB7-Cre or Pax2-Cre) and in nephron precursors (via Pax2-Cre and Six2-Cre) resulted in no apparent abnormalities in ureteric bud branching or in distal ureter maturation, and no hydronephrosis. Mutation in nephrons, ureteric smooth muscle, and mesenchyme surrounding the lower urinary tract (via the Pax3-Cre transgene) resulted in congenital hydronephrosis accompanied by reduced branching, abnormal distal ureter maturation and insertion, and smooth muscle orientation defects, phenotypes very similar to those in Dlg1 null mice. Dlg1 null mice showed reduced Ret expression and apoptosis during ureter maturation and evidence of reduced retinoic acid signaling in the kidney. Taken together, these results suggest that Dlg1 expression in ureter and CND-associated mesenchymal cells is essential for ensuring distal ureter maturation by facilitating retinoic acid signaling, Ret expression, and apoptosis of the urothelium.
Alport disease is caused by mutations in genes encoding the alpha3, alpha4, or alpha5 chains of type IV collagen, which form the collagenous network of mature glomerular basement membrane (GBM). In the absence of alpha3, alpha4, alpha5 (IV) collagen, alpha1, alpha2 (IV) collagen persists, which ordinarily is found only in GBM of developing kidney. In addition to dysregulation of collagen IV, Alport GBM contains aberrant laminins, which may contribute to the progressive GBM thickening and splitting, proteinuria, and renal failure seen in this disorder. This study sought to characterize further the laminin dysregulation in collagen alpha3(IV) knockout mice, a model of Alport disease. With the use of confocal microscopy, laminin alpha1 and alpha5 abundance was quantified, and it was found that they co-distributed in significantly large amounts in areas of GBM thickening. In addition, labeling of entire glomeruli for laminin alpha5 was significantly greater in Alport mice than in wild-type siblings. Reverse transcriptase-PCR from isolated glomeruli demonstrated significantly more laminin alpha5 mRNA in Alport mice than in wild-type controls, indicating upregulated transcription of Lama5. For testing glomerular barrier function, ferritin was injected into 2-wk-old Alport and control mice, and GBM was examined by electron microscopy. Highest ferritin levels were seen in Alport GBM thickenings beneath effaced podocyte foot processes, but morphologically normal GBM was significantly permeable as well. We concluded that (1) ultrastructurally normal Alport GBM residing beneath differentiated podocyte foot processes is inherently and abnormally permeable, and (2) upregulation of Lama5 transcription and concentration of laminin alpha1 and alpha5 within Alport GBM thickenings contribute to abnormal permeabilities.
Focal segmental glomerulosclerosis (FSGS) is a syndrome that involves kidney podocyte dysfunction and causes chronic kidney disease. Multiple factors including chemical toxicity, inflammation, and infection underlie FSGS; however, highly penetrant disease genes have been identified in a small fraction of patients with a family history of FSGS. Variants of apolipoprotein L1 (APOL1) have been linked to FSGS in African Americans with HIV or hypertension, supporting the proposal that genetic factors enhance FSGS susceptibility. Here, we used sequencing to investigate whether genetics plays a role in the majority of FSGS cases that are identified as primary or sporadic FSGS and have no known cause. Given the limited number of biopsy-proven cases with ethnically matched controls, we devised an analytic strategy to identify and rank potential candidate genes and used an animal model for validation. Nine candidate FSGS susceptibility genes were identified in our patient cohort, and three were validated using a high-throughput mouse method that we developed. Specifically, we introduced a podocyte-specific, doxycycline-inducible transactivator into a murine embryonic stem cell line with an FSGS-susceptible genetic background that allows shRNA-mediated targeting of candidate genes in the adult kidney. Our analysis supports a broader role for genetic susceptibility of both sporadic and familial cases of FSGS and provides a tool to rapidly evaluate candidate FSGS-associated genes.
The organization of the lining of the gastrointestinal tube is similar for the stomach, small intestine and colon. It is composed of a continuous simple epithelium and underlying mesenchyme, including blood vessels, fibroblasts/myofibroblasts and immune cells. Individual organs contain modifications of this mucosa that are critical for their functions. We hypothesize that changes in the composition of the epithelial basement membrane are a critical determinant for structure and function of the local mucosa. Laminins are excellent candidates for such a role, as they comprise a large family of αβ γ heterotrimeric glycoproteins that demonstrate developmental, organ and cell type specificity. We have found that adult mice lacking the normal villus laminin, α5, demonstrate a compensatory increase in α1 and α4, which are normally present in the colon. This laminin switch results in colonic metaplasia of the distal small intestine, which showed deepened crypts, villus atrophy and subsequent replacement by flat surface epithelial structures, and expression of colonic markers. This change was associated with aberrant intestinal epithelial cell proliferation, differentiation and migration. Here, unlike pathologic processes in humans that result in colonic metaplasia, there was no infiltration of inflammatory cells. These observations highlight the important and distinct roles of different laminin isoforms on the patterning of intestinal crypt-villus architecture and the regulation of intestinal epithelial cell behavior. Our data also suggest that differences in laminin composition along the gut's rostrocaudal axis contribute to its regionalization into small and large intestine.
In healthy glomeruli, parietal epithelial cell (PEC)-derived extracellular matrix (ECM) proteins include laminin-β 1 , perlecan, and collagen type IV-α 2 and podocyte-specific ECM proteins include laminin-β 2 , agrin, and collagen type IV-α 4 . This study aimed to define individual ECM protein isoform expression by PECs in both experimental and human focal segmental glomerulosclerosis (FSGS) and diabetic nephropathy (DN) and to determine if changes were CD44 dependent. In experimental FSGS induced with a cytotoxic podocyte antibody and in the BTBR ob/ob mouse model of DN, PEC-derived protein staining was significantly increased in PECs. Dual staining also showed de novo expression of the podocyte-specific ECM proteins laminin-β 2 and agrin in PECs. Similar findings were observed in biopsies from patients with FSGS and DN. Increases in individual ECM proteins colocalized with CD44 in PECs in disease. To determine the role of CD44, FSGS was induced in CD44 −/− and CD44 +/+ mice. PEC staining for perlecan, collagen type IV-α 2 , laminin-β 2 , and agrin were significantly lower in diseased CD44 −/− mice compared with diseased CD44 +/+ mice. These results show that in experimental and human FSGS and DN, PECs typically in an activated state, produce both PEC-derived and podocyte-specific ECM protein isoforms, and that the majority of these changes were dependent on CD44.
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