Basement membranes are thin sheets of extracellular matrix constructed from four major protein classes: laminins (LMs), type IV collagens, nidogens, and sulfated proteoglycans.1,2 LMα, LMβ, and LMγ chains assemble with a 1:1:1 stoichiometry in the endoplasmic reticulum to form covalently linked heterotrimers.3 In the glomerulus, these are secreted into the space between podocytes and endothelial cells.4 The α-, β-, and γ-chains’ laminin amino-terminal (LN) polymerization domains link LM heterotrimers together at α-β-γ “trimeric nodes” to form a planar, sheet-like network in the matrix milieu, whereas the LMα chain’s carboxyl-terminal LM globular domain links the LM network to cell surface receptors (e.g., integrins and dystroglycan).3,5 Similarly, type IV collagens assemble intracellularly to form heterotrimeric “protomers” that are secreted into the extracellular space, where distinct carboxyl-terminal and amino-terminal domain interactions promote collagen IV network formation.6
In the glomerular basement membrane (GBM), laminin α5β2γ1 (LM-521) trimers form separate networks adjacent to podocytes and endothelial cells, and collagen α3α4α5(IV) (COL4A345) protomers form a network at the center of the GBM.7 A separate collagen α1α2α1(IV) network forms adjacent to the endothelium.7,8 Nidogen, a basement membrane protein with binding sites for LMγ1 and collagen IV, links the LM and collagen networks.9 The long heparan sulfate proteoglycan agrin links the LM-521 coiled coil domain to cell surface integrins and dystroglycan.10 Collectively, these LM-cell, LM-nidogen, and nidogen-collagen IV interactions dictate the superstructure of the GBM, although the GBM contains numerous other extracellular matrix proteins.11
The GBM, the podocytes, and the endothelium interact to form the glomerular filtration barrier.12,13 The loss of podocyte slit diaphragm–associated proteins (nephrin, podocin, CD2AP, etc.) or GBM components (laminin β2 [LAMB2] or COL4A345) causes various forms of hereditary kidney disease that are always accompanied by proteinuria. Loss of LAMB2, which prevents synthesis of LM-521, causes Pierson syndrome, a congenital nephrotic syndrome with diffuse mesangial sclerosis and distinct ocular and neurologic abnormalities.14,15 Loss of any one of the collagen-α3, -α4, or -α5 chains, which prevent or reduce production of the COL4A345 network, causes Alport syndrome, a hereditary nephritis associated with hearing and eye defects.16,17 The pathophysiologic mechanisms underlying these diseases are incompletely understood.
LAMB2 mutations that cause Pierson syndrome are typically nonsense or splice site mutations leading to absent or truncated LAMB2 protein. In contrast, LAMB2 missense mutations cause the less severe nephrotic syndrome type 5 with or without ocular abnormalities (OMIM 614199).14,18 Most missense mutations cluster in or near the LN domain that mediates LM-521 polymerization.14 Such LN domain mutations are logically predicted to cause LM polymerization defects that should adversely affect GBM architecture and permselectivity, thereby causing nephrotic syndrome. However, studies of two pathogenic missense mutations in mice, LAMB2-R246Q and LAMB2-C321R, revealed a mechanism involving impaired protein folding and secretion and induction of endoplasmic reticulum stress.19,20 Such knowledge about the pathogenic mechanisms of individual mutations can be informative for devising personalized therapies.
In the interest of deciphering the pathogenic mechanisms of other LAMB2 mutations, we studied the LAMB2-S80R LN domain mutant, which was discovered as homozygous in a patient with delayed-onset, high-level albuminuria and is presumed to be pathogenic.14,21 Proteinuria at 2 g/d was observed at age 5 years old and persisted for several years. By age 11 years old, the proteinuria worsened to 4.1 g/m2 per day. A biopsy at age 11 years old showed mild diffuse mesangial sclerosis and tubular atrophy. LMβ2 immunostaining indicated that LAMB2-S80R is secreted and incorporates into the GBM similar to control. We engineered this mutation into mice in two different ways and found no evidence that it is pathogenic on its own. However, in the context of mouse models of Alport syndrome, the mutation behaved as a strong modifier allele that worsened disease progression. This provides the first genetic evidence that a variant in an LM gene can affect the progression of kidney disease in Alport mice and suggests that even clinically silent variants in GBM-related genes may be capable of affecting the severity of the Alport phenotype in patients.
Expression of LAMB2-S83R in Transgenic Mice
To validate the causality and investigate the mechanisms of pathogenicity of the human LAMB2-S80R mutation, we first generated transgenic mice expressing rat LAMB2-S83R (the analogous amino acid change in both rats and mice) in podocytes via the mouse nephrin promoter.22 Evaluation of transgene expression with an anti-rat LAMB2 antibody revealed relatively high, normal, or mosaic expression in three different lines (Supplemental Figure 1A), similar to our previous results from transgenic expression of LAMB2-C321R and LAMB2-R246Q mutant proteins.19,20 LAMB2-S83R colocalized with GBM nidogen, indicating secretion from podocytes and incorporation into the GBM (Supplemental Figure 1B) as observed in the biopsy of the patient with LAMB2-S80R.21 Transgenes that expressed were bred onto the Lamb2−/− genetic background23–25 to test the function of the S83R mutant in the absence of wild-type (WT) LAMB2. Mice with relatively “high” and “normal” levels of rat LAMB2-S83R in the GBM did not become proteinuric, even when aged to over a year, suggesting that the LAMB2-S83R mutation is not pathogenic in mice. Lamb2−/− mice with mosaic LAMB2-S83R deposition exhibited albuminuria, although not until 9 months of age (Supplemental Figure 1D). Mosaic LAMB2 expression in Lamb2−/− mice is expected to cause proteinuria due to segmental paucity of LM-521 in the GBM, and therefore, the observed proteinuria cannot be interpreted as a functional defect in LAMB2-S83R.
LAMB1 is restricted to the mesangium in healthy glomeruli, but it is aberrantly localized in the Lamb2−/− GBM25 and can functionally compensate for the loss of LAMB2 when overexpressed in podocytes.26 Immunofluorescence analysis indicated that LAMB1 was restricted to the mesangium in Lamb2−/−; Neph-LAMB2-S83R mice, similar to Lamb2+/+ mice (Supplemental Figure 1C), which further supports LAMB2-S83R’s lack of pathogenicity. However, because LAMB2-S83R was expressed under the control of the highly active nephrin promoter, it may somehow be expressed and integrated into the GBM at supraphysiologic levels. By possible analogy, transgene-enhanced expression of the LAMB2-R246Q mutant overcomes the folding/secretion defect that causes congenital nephrotic syndrome in patients.19 Because the patient with LAMB2-S80R exhibits a phenotype that was initially mild, it is feasible that overexpression and overaccumulation of LAMB2-S83R may compensate for an intrinsic functional defect.
To test the pathogenicity of the LAMB2-S83R mutation in the context of normal endogenous Lamb2 gene regulation, we engineered Lamb2S83R knock-in mice via an in vivo CRISPR/Cas9 gene editing strategy. Sanger sequencing confirmed the targeted AGT to CGT codon mutation directing the serine to arginine conversion (Figure 1A). Lamb2S83R mice were crossed with Lamb2+/− (heterozygous null) mice to eventually produce Lamb2+/S83R, Lamb2S83R/S83R, and Lamb2−/S83R mice. LAMB2 deposition in the GBM (Figure 1B) and colocalization with nidogen (shown for Lamb2S83R/S83R in Supplemental Figure 2A) were similar to Lamb2+/+ mice. Regular urinalysis over 12–13 months, an age range in mice that extends well beyond the 5 years of age at which the patient homozygous for the LAMB2-S80R mutation exhibited proteinuria, indicated that the Lamb2S83R knock-in mice, as in the rat transgene studies, did not reflect the patient’s condition (Figure 1B). These knock-in mice expressing only LAMB2-S83R also lacked increased LAMB1 in the GBM (Supplemental Figure 2B).
Polymerization Properties of LAMB2-S83R In Vitro
Previous biochemical studies of LMβ1 short-arm (NH2-terminal) fragments indicate that the LAMB1-S68R mutant, which is analogous to the LAMB2-S83R mutant, is polymerization defective.27 In light of our genetic studies presented above, we tested the polymerization capacity of both the LAMB1-S68R and LAMB2-S83R mutants in vitro using a Schwann cell–based assay. Schwann cells do not express LMs in culture, rendering basement membrane assembly dependent on ectopic addition of LMs and other BM components.28 In this model system, the loss of a single LN domain of a heterotrimer reduces heterotrimer retention on the cell surface and impairs the scaffolding of a basement membrane–like structure.29 Similar effects were observed in mice expressing an LN domain–truncated LAMA2 mutant that models congenital muscular dystrophy.30
As shown in Figure 2, there was an approximately 75% reduction in retention of LAMB1-S68R heterotrimers on the cell surface compared with WT LM-111 heterotrimers but only an approximately 50% reduction in LAMB2-S83R heterotrimers on the cell surface compared with WT LM-121 heterotrimers (Figure 2B). Retention of all heterotrimers tested was further decreased by addition of netrin-4 (Figure 2B), a matrix protein that competitively inhibits LM LN domain ternary node formation. These data indicate that the pathogenic mechanism of the human LAMB2-S80R allele should involve a polymerization defect. However, the apparently normal LAMB2-S83R accumulation in the mouse GBM (Figure 1, Supplemental Figures 1 and 2) does not reflect the in vitro findings and does not support a polymerization defect. Nevertheless, we note that some potentially polymerization-defective LAMA2 missense mutations causing a relatively mild muscular dystrophy in patients result in nearly normal levels of LM-211 in muscle and Schwann cell basement membranes.5,30–32
Effect of LAMB2-S83R on Disease Progression in Alport Mice
We next hypothesized that the murine model lacks a contextual factor(s) necessary to elicit LAMB2-S83R’s pathogenicity and that Lamb2S83R might act as a modifier allele in mice. To test this, we crossed the Lamb2S83R allele onto the Col4a3−/− Alport background.33 Strikingly, Lamb2+/S83R; Col4a3−/− mice (harboring just one Lamb2S83R allele) showed a significant reduction in survival compared with Lamb2+/+; Col4a3−/− littermates, indicating a dominant pathogenic effect of the LAMB2-S83R protein (Figure 3A). Notably, a Lamb2 null allele did not affect the Alport phenotype in Lamb2+/−; Col4a3−/− mice (Supplemental Figure 3). LAMB2 expression and colocalization with nidogen in the Lamb2+/S83R; Col4a3−/− GBM at postnatal days 7–11 (P7–P11) (Figure 4) were similar to those observed in Lamb2+/+; Col4a3−/− littermates. Thus, the reduced survivability of Lamb2+/S83R; Col4a3−/− mice cannot be attributed to reduced LAMB2 levels but is likely due to defective function of LAMB2-S83R within the GBM.
Urinalysis showed a significant increase in the urinary albumin-to-creatinine ratio (ACR) in Lamb2+/S83R; Col4a3−/− mice versus Lamb2+/+; Col4a3−/− and control (non-Alport) littermates as early as P7–P11 (Figure 3B). Although ACRs remained high through 7 weeks, ACRs decreased as BUN levels increased at 5–7 weeks (Figure 3C) and renal failure occurred (Figure 3A). Statistically insignificant differences in ACR and BUN between Lamb2+/+; Col4a3−/− and control littermates at these ages further showed the dramatic effect of the Lamb2S83R allele on the Alport phenotype.
The Alport phenotype presents histologically with a mix of focal segmental and global glomerulosclerosis, sometimes with crescents. In contrast, diffuse mesangial sclerosis is the histologic hallmark of Pierson syndrome. Periodic acid–Schiff (PAS) staining of Lamb2+/S83R; Col4a3−/− kidneys revealed normal features at P7–P11 (Figure 5A) but pronounced histopathologic features in 6- to 8-week-old mice (Figure 5B). In comparison, Lamb2+/+; Col4a3−/− kidneys exhibited less severe pathology at 6–8 weeks of age (Figure 5B). Furthermore, PAS analysis of P7 homozygous Lamb2S83R/S83R; Col4a3−/− kidneys revealed a phenotype more consistent with 6- to 8-week-old Lamb2+/S83R; Col4a3−/− kidneys (Figure 5C). Blinded quantification of glomerular lesions showed significantly accelerated pathology in Lamb2+/S83R; Col4a3−/− mice versus Lamb2+/+; Col4a3−/− and control mice (Figure 5D). Thus, histologic analysis also indicates that a single Lamb2S83R allele is sufficient to worsen the Alport phenotype.
Alport syndrome features ectopic deposition of LAMA2, LAMB1, and COL4A112 in the GBM, which could be compensatory and/or pathogenic.8,34,35Lamb2+/S83R; Col4a3−/− mice exhibited these matrix constituents in the GBM at levels higher than age-matched Lamb2+/+; Col4a3−/− mice (Figure 6, Supplemental Figures 4 and 5). Elevated expression of LAMB2-S83R between 6–10 weeks of age (Figure 6, Supplemental Figure 4) indicates that accelerated disease progression in Lamb2+/S83R; Col4a3−/− mice is not due to loss of LAMB2 expression. Transmission electron microscopy revealed GBM splitting and bulging typical of Alport syndrome at early stages in Lamb2+/S83R; Col4a3−/− mice, which eventually worsened to become extremely thick and electron lucent compared with those in Lamb2+/+; Col4a3−/− mice (Figure 7). At P7–P11, Lamb2+/+; Col4a3−/− mice exhibited mild foot process effacement, and by comparison, Lamb2+/S83R; Col4a3−/− mice showed a statistically insignificant trend of increased effacement (Figure 7B). By 5 weeks, foot process effacement in Lamb2+/S83R; Col4a3−/− mice was significantly worse than that in Lamb2+/+; Col4a3−/− littermates. Consistent with the histologic data, P7–P11 Lamb2S83R/S83R; Col4a3−/− mice exhibited effacement that was more consistent with that of 5- and 7-week-old Lamb2+/S83R; Col4a3−/− mice, and the P7–P11 Lamb2S83R/S83R; Col4a3−/− GBM’s ultrastructure resembled that of 2.5-day-old Lamb2−/−; Col4a3−/− mice (Supplemental Figure 3).
We next tested the Lamb2S83R allele in a less severe Alport syndrome model. The Col4a5 gene resides on the X chromosome, accounting for the increased frequency of Alport syndrome in males. Because of random X inactivation, Col4a5+/− females show mosaic, discontinuous deposition of COL4A345 in the GBM and a mild Alport phenotype. We crossed Lamb2S83R mice with Col4a5 mutant mice36 to generate Lamb2+/S83R; Col4a5+/− females and compared COL4A345 and LAMB2 expression, albuminuria levels, and histology with those of control littermates at 2–12 weeks of age. Lamb2+/+; Col4a5+/− and Lamb2+/S83R; Col4a5+/− GBMs exhibited the expected mosaic pattern of COL4A345 staining (Figure 8A). LAMB2 deposition in COL4A345-negative segments of the Col4a5+/− GBM was enhanced (Figure 8A), similar to the homogeneous increase in LAMB2 observed in the Col4a3−/− GBM (Figure 6, Supplemental Figure 4). Urinalysis revealed significantly increased albuminuria in Lamb2+/S83R; Col4a5+/− versus Lamb2+/+; Col4a5+/− mice between 4 and 6 weeks of age (Figure 8B). Curiously, the average ACR of Lamb2+/+; Col4a5+/− mice was not different from that of Lamb2+/S83R; Col4a5+/− mice at 8 weeks, but it was significantly higher in Lamb2+/S83R; Col4a5+/− mice at 12 weeks, indicating accelerated disease progression. Histologic analysis revealed enhanced glomerulosclerosis and protein casts in Lamb2+/S83R; Col4a5+/− kidneys compared with Lamb2+/+; Col4a5+/− kidneys as early as 8 weeks of age (Figure 8C). Collectively, urinalysis and histology both showed accelerated disease progression in female Col4a5+/− Alport mice harboring the Lamb2S83R allele, similar to the effects of Lamb2S83R in Col4a3−/− mice.
Loss of LAMB2 causes Pierson syndrome, a rare but catastrophic congenital disease with both renal and extrarenal phenotypes that leads to death, usually within the first year of life.14 Missense mutations clustered in or near the LAMB2 LN domain have been discovered in patients who exhibit phenotypes that are, in general, less severe than Pierson syndrome,14 suggesting that a protein with at least partial function is produced in these instances. Here, we generated two different mouse models to investigate the pathogenicity of the LAMB2S80R missense mutation, which was discovered as homozygous in a girl with an unusually late-onset nephrotic-range proteinuria at age 6 years old.14,21 The failure of the analogous murine Lamb2S83R mutation to cause proteinuria when either homozygous or opposite a Lamb2 null allele was surprising, because the patient also exhibited a retinal phenotype consistent with the Pierson syndrome spectrum.21 This discrepancy may have resulted from either differences in mouse and human glomerular physiology or an unknown second genetic or environmental hit in the patient. Previous analysis of the patient ruled out potential contributions by mutations in NPHS1, NPHS2, and WT1,21 but it is possible that the patient harbors mutations in other genes that affect the phenotype.
Strong candidates for a potential second hit that also associate with GBM defects are COL4A3 and COL4A4, because approximately 1% of the population carries mutations that can cause Alport syndrome when homozygous or compound heterozygous. Moreover, COL4A3 and COL4A4 heterozygosity can present either with no clinical phenotype or with features as variable as hematuria, thin basement membrane nephropathy, proteinuria, focal segmental glomerulosclerosis, and even renal failure.37–42 Although we did not find a phenotype in Lamb2S83R/S83R; Col4a3+/− mice, the severe detrimental effect of Lamb2S83R heterozygosity on the Alport phenotype in both Col4a3−/− and Col4a5+/− mutant mice is noteworthy. These findings provide support for the existence of modifier genes that encode GBM proteins and could contribute to the well known variability in phenotypes of patients with Alport syndrome and identical COL4 mutations. This variability could result from second hits in modifier genes that might not otherwise be overtly pathogenic. To our knowledge, this is the first report describing a genetic modifier of Alport syndrome that involves a GBM component; previous studies have shown roles for Itga1, Itga2, Ddr1, Trp53, and Alb in modulating kidney disease progression in Alport mice.43–47 In addition, a variant in NPHS2 (podocin) has been associated with proteinuria in patients with thin basement membrane nephropathy caused by a heterozygous mutation in COL4A3 or COL4A4.48
Histology revealed features consistent with Alport syndrome that were accelerated in Lamb2+/S83R; Col4a3−/− mice. The localization of ectopic matrix protein isoforms, including LAMA2, LAMB1, and COL4A1, to the GBM was similar in Lamb2+/+; Col4a3−/− and Lamb2+/S83R; Col4a3−/− mice, although expression levels seemed to correlate with progression of pathology (Figure 5). At the ultrastructural level, we observed an acceleration of foot process effacement in Lamb2+/S83R; Col4a3−/− compared with Lamb2+/+; Col4a3−/− mice, but similar GBM splitting that is typical of Alport syndrome was observed as early as P7–P11 in both (Figure 7). However, by 7 weeks, the Lamb2+/S83R; Col4a3−/− GBM exhibited a severe thickening and unusual podocyte disorganization that resembled the Lamb2S83R/S83R; Col4a3−/− GBM at P7 and the Lamb2−/−; Col4a3−/− GBM at P2.5 (Supplemental Figure 3). Collectively, we interpret these data to suggest that the Lamb2S83R allele sped kidney disease progression in Alport mice by disrupting the LM network, thereby worsening GBM ultrastructure, eventually leading to lesions resembling those observed in the total absence of LAMB2 shortly after birth.
Why is the LAMB2-S83R mutant protein innocuous on its own in mice but detrimental on the Alport background? Existing biochemical studies27 and our cell biologic studies (Figure 2) of mutant LAMB1 and LAMB2 chains show that the conserved Ser in the LN domain is necessary for proper ternary node formation with LMα and LMγ LN domains and efficient LM trimer polymerization and accumulation at cell surfaces.29 Our data showing that LAMB2-S83R incorporates into the GBM (Figures 1, 3, and 5, Supplemental Figures 1 and 3) together with the presence of LAMB2-S80R in the patient’s GBM21 indicate that the mutant LM-521 (LM-52*1) is somehow stabilized in vivovia a mechanism not active in vitro. We propose that the macromolecular complex that forms by integrin-LM-nidogen-COL4 interactions in the confined space between podocytes and endothelial cells facilitates retention of LM-52*1 within the GBM, despite the proven defect in LM polymerization. In contrast, in Alport GBM, the LM-52*1’s defective polymerization apparently synergizes with the COL4 defect to destabilize the GBM and hasten its demise and the resulting decrease in kidney function. The fact that LAMB2-S83R is pathogenic, despite the presence of WT LAMB2 presumably expressed at a 1:1 ratio in Lamb2+/S80R; Col4a3−/− mice, suggests that even modestly modulating the efficiency of LM polymerization can drastically affect progression of Alport syndrome. Finally, we speculate that methods of improving WT LM-521 polymerization and strengthening its interactions with other GBM components in the Alport context exist, and they can be exploited to slow damage to the GBM and attenuate the resulting decline in kidney function. In this regard, various designer basement membrane proteins have been used to slow or prevent muscular dystrophy due to Lama2 mutation.10,30,49,50
Transgenes and Mice
All animal experiments conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Washington University Institutional Animal Care and Use Committee. For transgenic expression, the S80R mutation (S83R in rat and mouse) was engineered into the previously described rat Lamb2 cDNA24 by site-directed mutagenesis (QuickChange XL; Stratagene, La Jolla, CA) and placed under control of the mouse 4.2-kb nephrin promoter22 for podocyte-specific expression. Founders were mated with Lamb2 mutant mice25,51 for functional analysis of the nephrin-LAMB2-S83R transgene on the Lamb2−/− background. Lamb2−/− mice also carried the Muscle Creatine Kinase-rat LAMB2 transgene to prevent lethal neuromuscular junction defects.24
To generate the mouse Lamb2S83R knock-in allele via CRISPR/Cas9, a guide RNA (5′-aggtgacagacaatacagta-3′) was designed by the Washington University Transgenic Vectors Core and microinjected into B6CBAF2/J pronuclei along with Cas9 mRNA and a 196-base donor DNA oligonucleotide that was engineered to introduce the desired c.247A>C mutation by homology-directed repair. Founders were mated with Lamb2+/− mice on a mixed B6CBA background51 to produce Lamb2S83R/S83R homozygotes or Lamb2−/S83R compound heterozygotes. For Alport studies, Lamb2S83R mice were mated with Col4a3 and Col4a5 mutant mice on mixed B6CBA backgrounds.33,36 Mice with Lamb2+/+, Lamb2+/−, Col4a3+/+, and Col4a3+/− genotypes were used as controls.
Urinalyses and BUN Assays
Urine creatinine levels were determined with the Quantichrome creatinine detection kit. Blood was collected from cheek puncture–induced bleeds and centrifuged at 5500 rpm for 5 minutes to collect serum. BUN was assayed with the Quantichrome Urea detection kit. To determine albumin levels, creatinine-normalized urines were subjected to SDS-PAGE and Coomassie blue staining with BSA standards, quantified by densitometry with ImageJ software, and extrapolated from a best fit curve of the BSA standards.
PAS staining of paraffin kidney sections and transmission electron microscopy were performed by standard methods. Glomerulosclerosis was quantified by an observer blinded to genotypes. For immunofluorescence, kidneys were frozen in OCT and cryosectioned at 7 μm. Antibodies, including rabbit anti-mouse LAMB1 and LAMB2,52 mouse anti-rat LAMB2,24 rat anti-mouse LAMA2 (clone 4H8–2; Axxora),53 rat anti-mouse nidogen (Clone ELM1), mouse anti-bovine COL4A345,54 and rabbit anti-mouse agrin,55 were diluted into PBS containing 1% BSA.
LM Polymerization Assay
Schwann cells were grown as described.29 Cells were incubated at 37°C with recombinant LMs (14 nM) plus 28 nM recombinant nidogen-1 and 14 nM type IV collagen without or with a tenfold molar excess of recombinant netrin4ΔC for 1 hour followed by washing with PBS and fixation with 3% paraformaldehyde for 15 minutes at room temperature. After blocking with 5% goat serum and 0.5% BSA in PBS, wells were incubated with rat anti-LMγ1 (MAB1920; Millipore), washed, and then incubated with secondary antibody. Schwann cells were viewed by indirect immunofluorescence, and digital images were recorded (six to seven fields per condition, each 1300×1030 pixels) using a ×10 microscope objective with controlled exposure times. Fluorescence levels were estimated with ImageJ.29 Nuclei were stained with 4′,6-diamidino-2-phenylindole to determine the number of cells in each field. The average background per pixel was determined using untreated cultures. The summed intensities from each field were divided by the area followed by background subtraction and division by the number of counted nuclei. Data were expressed as the mean±SD of normalized net summed intensities per cell, with plots and statistical calculations prepared in SigmaPlot 12.5 (Systat). Significance was determined by one-way ANOVA followed by Holm–Sidak pairwise comparisons.
We thank Gloriosa Go and Jennifer Richardson for technical assistance, the Transgenic Vectors Core for design and validation of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 guideA, the Mouse Genetics Core for nucleic acid microinjections and mouse husbandry, the Pulmonary Morphology Core and Advanced Imaging and Tissue Analysis Core (supported by National Institutes of Health [NIH] grant P30DK052574) for histology, and the Washington University Center for Cellular Imaging for electron microscopy. We are grateful to Takako Sasaki, Yoshikazu Sado, and Dorin-Bogdan Borza for gifts of antibodies.
S.D.F. was supported by NIH grant T32DK007126. This work was funded by NIH grants R01DK036425 (to P.D.Y.) and R01DK078314 (to J.H.M.). Production of transgenic and knock-in mice was supported by Diabetes Research Center Transgenic and Embryonic Stem Cell Core grant P30DK020579 and Digestive Diseases Research Core Center Murine Models Core grant P30DK052574.
1. Miner JH: Renal basement membrane components. Kidney Int 56: 2016–2024, 199910594777
2. Yurchenco PD, Amenta PS, Patton BL: Basement membrane assembly, stability and activities observed through a developmental lens. Matrix Biol 22: 521–538, 200414996432
3. Yurchenco PD, Cheng YS: Self-assembly and calcium-binding sites in laminin
. A three-arm interaction model. J Biol Chem 268: 17286–17299, 19938349613
4. Miner JH: Building the glomerulus: A matricentric view. J Am Soc Nephrol 16: 857–861, 200515728777
5. Yurchenco PD: Integrating activities of laminins that drive basement membrane assembly and function. Curr Top Membr 76: 1–30, 201526610910
6. Hudson BG: The molecular basis of Goodpasture and Alport syndromes: Beacons for the discovery of the collagen IV family. J Am Soc Nephrol 15: 2514–2527, 200415466256
7. Suleiman H, Zhang L, Roth R, Heuser JE, Miner JH, Shaw AS, Dani A: Nanoscale protein architecture of the kidney glomerular basement membrane. eLife 2: e01149, 201324137544
8. Kashtan CE, Kim Y: Distribution of the alpha 1 and alpha 2 chains of collagen IV and of collagens V and VI in Alport syndrome. Kidney Int 42: 115–126, 19921635341
9. Ho MS, Böse K, Mokkapati S, Nischt R, Smyth N: Nidogens-extracellular matrix linker molecules. Microsc Res Tech 71: 387–395, 200818219668
10. Meinen S, Barzaghi P, Lin S, Lochmüller H, Ruegg MA: Linker molecules between laminins and dystroglycan ameliorate laminin
-alpha2-deficient muscular dystrophy at all disease stages. J Cell Biol 176: 979–993, 200717389231
11. Lennon R, Byron A, Humphries JD, Randles MJ, Carisey A, Murphy S, Knight D, Brenchley PE, Zent R, Humphries MJ: Global analysis reveals the complexity of the human glomerular extracellular matrix. J Am Soc Nephrol 25: 939–951, 201424436468
12. Miner JH: Glomerular basement membrane composition and the filtration barrier. Pediatr Nephrol 26: 1413–1417, 201121327778
13. Miner JH: The glomerular basement membrane. Exp Cell Res 318: 973–978, 201222410250
14. Matejas V, Hinkes B, Alkandari F, Al-Gazali L, Annexstad E, Aytac MB, Barrow M, Bláhová K, Bockenhauer D, Cheong HI, Maruniak-Chudek I, Cochat P, Dötsch J, Gajjar P, Hennekam RC, Janssen F, Kagan M, Kariminejad A, Kemper MJ, Koenig J, Kogan J, Kroes HY, Kuwertz-Bröking E, Lewanda AF, Medeira A, Muscheites J, Niaudet P, Pierson M, Saggar A, Seaver L, Suri M, Tsygin A, Wühl E, Zurowska A, Uebe S, Hildebrandt F, Antignac C, Zenker M: Mutations in the human laminin
beta2 (LAMB2) gene and the associated phenotypic spectrum. Hum Mutat 31: 992–1002, 201020556798
15. Zenker M, Aigner T, Wendler O, Tralau T, Müntefering H, Fenski R, Pitz S, Schumacher V, Royer-Pokora B, Wühl E, Cochat P, Bouvier R, Kraus C, Mark K, Madlon H, Dötsch J, Rascher W, Maruniak-Chudek I, Lennert T, Neumann LM, Reis A: Human laminin
beta2 deficiency causes congenital nephrosis with mesangial sclerosis and distinct eye abnormalities. Hum Mol Genet 13: 2625–2632, 200415367484
16. Kashtan CE: Alport syndrome. An inherited disorder of renal, ocular, and cochlear basement membranes. Medicine (Baltimore) 78: 338–360, 199910499074
17. Miner JH, Baigent C, Flinter F, Gross O, Judge P, Kashtan CE, Lagas S, Savige J, Blatt D, Ding J, Gale DP, Midgley JP, Povey S, Prunotto M, Renault D, Skelding J, Turner AN, Gear S: The 2014 international workshop on Alport syndrome. Kidney Int 86: 679–684, 201424988067
18. Hasselbacher K, Wiggins RC, Matejas V, Hinkes BG, Mucha B, Hoskins BE, Ozaltin F, Nürnberg G, Becker C, Hangan D, Pohl M, Kuwertz-Bröking E, Griebel M, Schumacher V, Royer-Pokora B, Bakkaloglu A, Nürnberg P, Zenker M, Hildebrandt F: Recessive missense mutations in LAMB2 expand the clinical spectrum of LAMB2-associated disorders. Kidney Int 70: 1008–1012, 200616912710
19. Chen YM, Kikkawa Y, Miner JH: A missense LAMB2 mutation causes congenital nephrotic syndrome by impairing laminin
secretion. J Am Soc Nephrol 22: 849–858, 201121511833
20. Chen YM, Zhou Y, Go G, Marmerstein JT, Kikkawa Y, Miner JH: Laminin
β2 gene missense mutation produces endoplasmic reticulum stress in podocytes. J Am Soc Nephrol 24: 1223–1233, 201323723427
21. Lehnhardt A, Lama A, Amann K, Matejas V, Zenker M, Kemper MJ: Pierson syndrome in an adolescent girl with nephrotic range proteinuria but a normal GFR. Pediatr Nephrol 27: 865–868, 201222228401
22. Eremina V, Wong MA, Cui S, Schwartz L, Quaggin SE: Glomerular-specific gene excision in vivo. J Am Soc Nephrol 13: 788–793, 200211856786
23. Jarad G, Cunningham J, Shaw AS, Miner JH: Proteinuria precedes podocyte abnormalities inLamb2-/- mice, implicating the glomerular basement membrane as an albumin barrier. J Clin Invest 116: 2272–2279, 200616886065
24. Miner JH, Go G, Cunningham J, Patton BL, Jarad G: Transgenic isolation of skeletal muscle and kidney defects in laminin
beta2 mutant mice: Implications for Pierson syndrome. Development 133: 967–975, 200616452099
25. Noakes PG, Miner JH, Gautam M, Cunningham JM, Sanes JR, Merlie JP: The renal glomerulus of mice lacking s-laminin
beta 2: Nephrosis despite molecular compensation by laminin
beta 1. Nat Genet 10: 400–406, 19957670489
26. Suh JH, Jarad G, VanDeVoorde RG, Miner JH: Forced expression of laminin
beta1 in podocytes prevents nephrotic syndrome in mice lacking laminin
beta2, a model for Pierson syndrome. Proc Natl Acad Sci U S A 108: 15348–15353, 201121876163
27. Purvis A, Hohenester E: Laminin
network formation studied by reconstitution of ternary nodes in solution. J Biol Chem 287: 44270–44277, 201223166322
28. Li S, Liquari P, McKee KK, Harrison D, Patel R, Lee S, Yurchenco PD: Laminin
-sulfatide binding initiates basement membrane assembly and enables receptor signaling in Schwann cells and fibroblasts. J Cell Biol 169: 179–189, 200515824137
29. McKee KK, Harrison D, Capizzi S, Yurchenco PD: Role of laminin
terminal globular domains in basement membrane assembly. J Biol Chem 282: 21437–21447, 200717517882
30. McKee KK, Crosson SC, Meinen S, Reinhard JR, Rüegg MA, Yurchenco PD: Chimeric protein repair of laminin
polymerization ameliorates muscular dystrophy phenotype. J Clin Invest 127: 1075–1089, 201728218617
31. Hussain SA, Carafoli F, Hohenester E: Determinants of laminin
polymerization revealed by the structure of the α5 chain amino-terminal region. EMBO Rep 12: 276–282, 201121311558
32. Gavassini BF, Carboni N, Nielsen JE, Danielsen ER, Thomsen C, Svenstrup K, Bello L, Maioli MA, Marrosu G, Ticca AF, Mura M, Marrosu MG, Soraru G, Angelini C, Vissing J, Pegoraro E: Clinical and molecular characterization of limb-girdle muscular dystrophy due to LAMA2 mutations. Muscle Nerve 44: 703–709, 201121953594
33. Miner JH, Sanes JR: Molecular and functional defects in kidneys of mice lacking collagen alpha 3(IV): Implications for Alport syndrome. J Cell Biol 135: 1403–1413, 19968947561
34. Delimont D, Dufek BM, Meehan DT, Zallocchi M, Gratton MA, Phillips G, Cosgrove D: Laminin
α2-mediated focal adhesion kinase activation triggers Alport glomerular pathogenesis. PLoS One 9: e99083, 201424915008
35. Kashtan CE, Kim Y, Lees GE, Thorner PS, Virtanen I, Miner JH: Abnormal glomerular basement membrane laminins in murine, canine, and human Alport syndrome: Aberrant laminin
alpha2 deposition is species independent. J Am Soc Nephrol 12: 252–260, 200111158215
36. Rheault MN, Kren SM, Thielen BK, Mesa HA, Crosson JT, Thomas W, Sado Y, Kashtan CE, Segal Y: Mouse model of X-linked Alport syndrome. J Am Soc Nephrol 15: 1466–1474, 200415153557
37. Miner JH: Pathology vs. molecular genetics: (Re)defining the spectrum of Alport syndrome. Kidney Int 86: 1081–1083, 201425427084
38. Malone AF, Phelan PJ, Hall G, Cetincelik U, Homstad A, Alonso A, Jiang R, Lindsey T, Wu G, Sparks MA, Smith SR, Webb NJA, Kalra P, Adeyemo A, Shaw AS, Conlon PJ, Jennette JC, Howell DN, Winn MP, Gbadegesin RA: A high frequency of hereditary nephritis with rare COL4A3/COL4A4 variants erroneously included in a familial FSGS cohort. Kidney Int 86: 1253–1259, 2014
39. Pierides A, Voskarides K, Athanasiou Y, Ioannou K, Damianou L, Arsali M, Zavros M, Pierides M, Vargemezis V, Patsias C, Zouvani I, Elia A, Kyriacou K, Deltas C: Clinico-pathological correlations in 127 patients in 11 large pedigrees, segregating one of three heterozygous mutations in the COL4A3/ COL4A4 genes associated with familial haematuria and significant late progression to proteinuria and chronic kidney disease from focal segmental glomerulosclerosis. Nephrol Dial Transplant 24: 2721–2729, 200919357112
40. Voskarides K, Damianou L, Neocleous V, Zouvani I, Christodoulidou S, Hadjiconstantinou V, Ioannou K, Athanasiou Y, Patsias C, Alexopoulos E, Pierides A, Kyriacou K, Deltas C: COL4A3/COL4A4 mutations producing focal segmental glomerulosclerosis and renal failure in thin basement membrane nephropathy. J Am Soc Nephrol 18: 3004–3016, 200717942953
41. Gast C, Pengelly RJ, Lyon M, Bunyan DJ, Seaby EG, Graham N, Venkat-Raman G, Ennis S: Collagen (COL4A) mutations are the most frequent mutations underlying adult focal segmental glomerulosclerosis. Nephrol Dial Transplant 31: 961–970, 201626346198
42. Deltas C, Savva I, Voskarides K, Papazachariou L, Pierides A: Carriers of autosomal recessive Alport syndrome with thin basement membrane nephropathy presenting as focal segmental glomerulosclerosis in later life. Nephron 130: 271–280, 201526201269
43. Cosgrove D, Rodgers K, Meehan D, Miller C, Bovard K, Gilroy A, Gardner H, Kotelianski V, Gotwals P, Amatucci A, Kalluri R: Integrin alpha1beta1 and transforming growth factor-beta1 play distinct roles in alport glomerular pathogenesis and serve as dual targets for metabolic therapy. Am J Pathol 157: 1649–1659, 200011073824
44. Fukuda R, Suico MA, Kai Y, Omachi K, Motomura K, Koga T, Komohara Y, Koyama K, Yokota T, Taura M, Shuto T, Kai H: Podocyte p53 limits the severity of experimental Alport syndrome. J Am Soc Nephrol 27: 144–157, 201625967122
45. Jarad G, Knutsen RH, Mecham RP, Miner JH: Albumin contributes to kidney disease progression in Alport syndrome. Am J Physiol Renal Physiol 311: F120–F130, 201627147675
46. Gross O, Girgert R, Beirowski B, Kretzler M, Kang HG, Kruegel J, Miosge N, Busse AC, Segerer S, Vogel WF, Müller GA, Weber M: Loss of collagen-receptor DDR1 delays renal fibrosis in hereditary type IV collagen disease. Matrix Biol 29: 346–356, 201020307660
47. Rubel D, Frese J, Martin M, Leibnitz A, Girgert R, Miosge N, Eckes B, Müller GA, Gross O: Collagen receptors integrin alpha2beta1 and discoidin domain receptor 1 regulate maturation of the glomerular basement membrane and loss of integrin alpha2beta1 delays kidney fibrosis in COL4A3 knockout mice. Matrix Biol 34: 13–21, 201424480069
48. Tonna S, Wang YY, Wilson D, Rigby L, Tabone T, Cotton R, Savige J: The R229Q mutation in NPHS2 may predispose to proteinuria in thin-basement-membrane nephropathy. Pediatr Nephrol 23: 2201–2207, 200818726620
49. Reinhard JR, Lin S, McKee KK, Meinen S, Crosson SC, Sury M, Hobbs S, Maier G, Yurchenco PD, Rüegg MA: Linker proteins restore basement membrane and correct LAMA2-related muscular dystrophy in mice. Sci Transl Med 9: pii:eaal4649, 201728659438
50. Moll J, Barzaghi P, Lin S, Bezakova G, Lochmüller H, Engvall E, Müller U, Ruegg MA: An agrin minigene rescues dystrophic symptoms in a mouse model for congenital muscular dystrophy. Nature 413: 302–307, 200111565031
51. Noakes PG, Gautam M, Mudd J, Sanes JR, Merlie JP: Aberrant differentiation of neuromuscular junctions in mice lacking s-laminin
beta 2. Nature 374: 258–262, 19957885444
52. Sasaki T, Mann K, Miner JH, Miosge N, Timpl R: Domain IV of mouse laminin
beta1 and beta2 chains. Eur J Biochem 269: 431–442, 200211856301
53. Ninomiya Y, Kagawa M, Iyama K, Naito I, Kishiro Y, Seyer JM, Sugimoto M, Oohashi T, Sado Y: Differential expression of two basement membrane collagen genes, COL4A6 and COL4A5, demonstrated by immunofluorescence staining using peptide-specific monoclonal antibodies. J Cell Biol 130: 1219–1229, 19957657706
54. Heidet L, Borza DB, Jouin M, Sich M, Mattei MG, Sado Y, Hudson BG, Hastie N, Antignac C, Gubler MC: A human-mouse chimera of the alpha3alpha4alpha5(IV) collagen protomer rescues the renal phenotype in Col4a3-/- Alport mice. Am J Pathol 163: 1633–1644, 200314507670
55. Harvey SJ, Jarad G, Cunningham J, Rops AL, van der Vlag J, Berden JH, Moeller MJ, Holzman LB, Burgess RW, Miner JH: Disruption of glomerular basement membrane charge through podocyte-specific mutation of agrin does not alter glomerular permselectivity. Am J Pathol 171: 139–152, 200717591961