The kidney's glomerular filtration barrier consists of two cells–podocytes and endothelial cells–and the glomerular basement membrane (GBM), a specialized extracellular matrix that lies between them. ...Like all basement membranes, the GBM consists mainly of laminin, type IV collagen, nidogen, and heparan sulfate proteoglycan. However, the GBM is unusually thick and contains particular members of these general protein families, including laminin-521, collagen α3α4α5(IV), and agrin. Knockout studies in mice and genetic findings in humans show that the laminin and type IV collagen components are particularly important for GBM structure and function, as laminin or collagen IV gene mutations cause filtration defects and renal disease of varying severities, depending on the nature of the mutations. These studies suggest that the GBM plays a crucial role in establishing and maintaining the glomerular filtration barrier.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
CRISPR/Cas9-mediated transcriptional activation (CRISPRa) is a powerful tool for investigating complex biological phenomena. Although CRISPRa approaches based on the VP64 transcriptional activator ...have been widely studied in both cultured cells and in animal models and exhibit great versatility for various cell types and developmental stages in vivo, different dCas9-VP64 versions have not been rigorously compared. Here, we compared different dCas9-VP64 constructs in identical contexts, including the cell lines used and the transfection conditions, for their ability to activate endogenous and exogenous genes. Moreover, we investigated the optimal approach for VP64 addition to VP64- and p300-based constructs. We found that MS2-MCP-scaffolded VP64 enhanced basal dCas9-VP64 and dCas9-p300 activity better than did direct VP64 fusion to the N-terminus of dCas9. dCas9-VP64+MCP-VP64 and dCas9-p300+MCP-VP64 were superior to VP64-dCas9-VP64 for all target genes tested. Furthermore, multiplexing gRNA expression with dCas9-VP64+MCP-VP64 or dCas9-p300+MCP-VP64 significantly enhanced endogenous gene activation to a level comparable to CRISPRa-SAM with a single gRNA. Our findings demonstrate improvement of the dCas9-VP64 CRISPRa system and contribute to development of a versatile, efficient CRISPRa platform.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
African Americans have a heightened risk of developing chronic and end-stage kidney disease, an association that is largely attributed to two common genetic variants, termed G1 and G2, in the APOL1 ...gene. Direct evidence demonstrating that these APOL1 risk alleles are pathogenic is still lacking because the APOL1 gene is present in only some primates and humans; thus it has been challenging to demonstrate experimental proof of causality of these risk alleles for renal disease. Here we generated mice with podocyte-specific inducible expression of the APOL1 reference allele (termed G0) or each of the risk-conferring alleles (G1 or G2). We show that mice with podocyte-specific expression of either APOL1 risk allele, but not of the G0 allele, develop functional (albuminuria and azotemia), structural (foot-process effacement and glomerulosclerosis) and molecular (gene-expression) changes that closely resemble human kidney disease. Disease development was cell-type specific and likely reversible, and the severity correlated with the level of expression of the risk allele. We further found that expression of the risk-variant APOL1 alleles interferes with endosomal trafficking and blocks autophagic flux, which ultimately leads to inflammatory-mediated podocyte death and glomerular scarring. In summary, this is the first demonstration that the expression of APOL1 risk alleles is causal for altered podocyte function and glomerular disease in vivo.
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IJS, NUK, SBMB, UL, UM, UPUK
Synaptopodin (Synpo) is an actin-associated protein in podocytes and dendritic spines. Many functions in regulating the actin cytoskeleton
RhoA and other pathways have been ascribed to Synpo, yet no ...pathogenic mutations in the
gene have been discovered in patients. Naturally occurring Synpo isoforms are known (Synpo-short and -long), and a novel truncated version (Synpo-T) is upregulated in podocytes from
mutant mice. Synpo-T maintains some Synpo functions, which may prevent a podocyte phenotype from emerging in unchallenged mutant mice.
Novel mouse models were generated to further investigate the functions of Synpo. In one, CRISPR/Cas9 deleted most of the
gene, preventing production of any detectable Synpo protein. Two other mutant strains made truncated versions of the protein. Adriamycin injections were used to challenge the mice, and Synpo functions were investigated in primary cultured podocytes.
Mice that could not make detectable Synpo (
) did not develop any kidney abnormalities up to 12 months of age. However,
mice were more susceptible to Adriamycin nephropathy. In cultured primary podocytes from mutant mice, the absence of Synpo caused loss of stress fibers, increased the number and size of focal adhesions, and impaired cell migration. Furthermore, loss of Synpo led to decreased RhoA activity and increased Rac1 activation.
In contrast to previous findings, podocytes can function normally
in the absence of any Synpo isoform. Synpo plays a protective role in the context of podocyte injury through its involvement in actin reorganization and focal adhesion dynamics.
The glomerular basement membrane (GBM) is the central, non-cellular layer of the glomerular filtration barrier that is situated between the two cellular components--fenestrated endothelial cells and ...interdigitated podocyte foot processes. The GBM is composed primarily of four types of extracellular matrix macromolecule--laminin-521, type IV collagen α3α4α5, the heparan sulphate proteoglycan agrin, and nidogen--which produce an interwoven meshwork thought to impart both size-selective and charge-selective properties. Although the composition and biochemical nature of the GBM have been known for a long time, the functional importance of the GBM versus that of podocytes and endothelial cells for establishing the glomerular filtration barrier to albumin is still debated. Together with findings from genetic studies in mice, the discoveries of four human mutations affecting GBM components in two inherited kidney disorders, Alport syndrome and Pierson syndrome, support essential roles for the GBM in glomerular permselectivity. Here, we explain in detail the proposed mechanisms whereby the GBM can serve as the major albumin barrier and discuss possible approaches to circumvent GBM defects associated with loss of permselectivity.
The glomerular basement membrane (GBM) is an especially thick basement membrane that contributes importantly to the kidney’s filtration barrier. The GBM derives from the fusion of separate podocyte ...and endothelial cell basement membranes during glomerulogenesis and consists primarily of laminin-521 (α5β2γ1), collagen α3α4α5(IV), nidogens-1 and -2, and agrin. Of these nine proteins, mutations in the genes encoding four of them (
LAMB2
,
COL4A3
,
COL4A4
, and
COL4A5
) cause glomerular disease in humans as well as in mice. Furthermore, mutation of a fifth (
Lama5
) gene in podocytes in mice causes proteinuria, nephrotic syndrome, and progression to renal failure. These results highlight the importance of the GBM for establishing and maintaining a properly functioning glomerular filtration barrier.
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DOBA, EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, IZUM, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, SIK, UILJ, UKNU, UL, UM, UPUK, VKSCE, VSZLJ, ZAGLJ
Malone et al. performed next-generation sequencing on 70 families with focal segmental glomerulosclerosis (FSGS) and discovered that 10% had variants in surprising ‘old’ genes, COL4A3 and COL4A4, ...which are involved in Alport syndrome and thin basement membrane nephropathy. These data show that a subset of renal manifestations associated with COL4A3 or COL4A4 variants cannot be distinguished from FSGS by clinical data or histopathology. Thus, a diagnosis of FSGS may sometimes fall within the spectrum of Alport syndrome.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
With the latest classification, variants in three collagen IV genes, COL4A3 , COL4A4 , and COL4A5 , represent the most prevalent genetic kidney disease in humans, exhibiting diverse, complex, and ...inconsistent clinical manifestations. This review breaks down the disease spectrum and genotype-phenotype correlations of kidney diseases linked to genetic variants in these genes and distinguishes "classic" Alport syndrome (AS) from the less severe nonsyndromic genetically related nephropathies that we suggest be called "Alport kidney diseases".
Several research studies have focused on the genotype-phenotype correlation under the latest classification scheme of AS. The historic diagnoses of "benign familial hematuria" and "thin basement membrane nephropathy" linked to heterozygous variants in COL4A3 or COL4A4 are suggested to be obsolete, but instead classified as autosomal AS by recent expert consensus due to a significant risk of disease progression.
The concept of Alport kidney disease extends beyond classic AS. Patients carrying pathogenic variants in any one of the COL4A3/A4/A5 genes can have variable phenotypes ranging from completely normal/clinically unrecognizable, hematuria without or with proteinuria, or progression to chronic kidney disease and kidney failure, depending on sex, genotype, and interplays of other genetic as well as environmental factors.
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