Pathogenicity of a Human Laminin β2 Mutation Revealed in Models of Alport Syndrome

Abstract

Pierson syndrome is a congenital nephrotic syndrome with eye and neurologic defects caused by mutations in laminin β2 (LAMB2), a major component of the glomerular basement membrane (GBM). Pathogenic missense mutations in human LAMB2 cluster in or near the laminin amino-terminal (LN) domain, a domain required for extracellular polymerization of laminin trimers and basement membrane scaffolding. Here, we investigated an LN domain missense mutation, LAMB2-S80R, which was discovered in a patient with Pierson syndrome and unusually late onset of proteinuria. Biochemical data indicated that this mutation impairs laminin polymerization, which we hypothesized to be the cause of the patient’s nephrotic syndrome. Testing this hypothesis in genetically altered mice showed that the corresponding amino acid change (LAMB2-S83R) alone is not pathogenic. However, expression of LAMB2-S83R significantly increased the rate of progression to kidney failure in a Col4a3^−/− mouse model of autosomal recessive Alport syndrome and increased proteinuria in Col4a5^+/− females that exhibit a mild form of X-linked Alport syndrome due to mosaic deposition of collagen α3α4α5(IV) in the GBM. Collectively, these data show the pathogenicity of LAMB2-S80R and provide the first evidence of genetic modification of Alport phenotypes by variation in another GBM component. This finding could help explain the wide range of Alport syndrome onset and severity observed in patients with Alport syndrome, even for family members who share the same COL4 mutation. Our results also show the complexities of using model organisms to investigate genetic variants suspected of being pathogenic in humans.

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.

Results

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 background^23–25 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 Lamb2^S83R 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). Lamb2^S83R mice were crossed with Lamb2^+/− (heterozygous null) mice to eventually produce Lamb2^+/S83R, Lamb2^S83R/S83R, and Lamb2^−/S83R mice. LAMB2 deposition in the GBM (Figure 1B) and colocalization with nidogen (shown for Lamb2^S83R/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 Lamb2^S83R 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).

Figure 1.: LAMB2-S83R protein resulting from CRISPR/Cas9 mediated gene editing accumulates in the GBM but is not pathogenic. (A) Sanger sequencing shows the heterozygous A to C mutation (asterisk) resulting in an Ser to Arg conversion; the boxed sequence is the WT, and the lower sequence is the mutant. (B) SDS-PAGE analysis of urine shows the lack of elevated albuminuria in Lamb2 ^−/S83R mice versus the WT at up to 13 months (n=3–5). (C) Immunofluorescence analysis of LAMB2 in the GBMs of Lamb2 ^+/+ (10 months), Lamb2 ^+/S83R (10 months), Lamb2 ^S83R/S83R (9 months), and Lamb2 ^−/S83R (10 months) mice. LAMB2 levels in any S83R-containing mice were similar to the WT at any age up to 13 months; n=3–4 for each genotype.

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

Figure 2.: LAMB2-S83R impairs LM polymerization on Schwann cells in vitro. (A) Schwann cells were incubated with LM-111 or LM-121 heterotrimers (either the WT or with the indicated mutations), collagen IV, and nidogen with or without the LM polymerization competitor netrin-4 (concentrations shown in B). Immunofluorescence for LAMC1 was performed to assess retention of LM heterotrimers. (B) The immunofluorescence intensity of the anti-LAMC1 signal was normalized to the number of nuclei and calculated as the net summed intensity per cell of the signal detected on Schwann cells cultured with the indicated basement membrane constituents; n=3–5. P values were determined by one-way ANOVA followed by Holm–Sidak pairwise comparisons. *Denotes the representation of 2 independent comparisons by the adjacent bar, whereas every other bar indicates single comparison between 2 experimental groups.

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 Lamb2^S83R might act as a modifier allele in mice. To test this, we crossed the Lamb2^S83R allele onto the Col4a3^−/− Alport background.³³ Strikingly, Lamb2^+/S83R; Col4a3^−/− mice (harboring just one Lamb2^S83R 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.

Figure 3.: LAMB2-S83R worsens the Alport syndrome (Col4a3 ^−/−) phenotype. (A) The age at ESRD of Lamb2 ^+/+ ; Col4a3 ^−/− and Lamb2 ^+/S83R; Col4a3 ^−/− mice was followed over the indicated time. Lamb2 ^+/S83R; Col4a3 ^−/− mice reached ESRD significantly faster than Lamb2 ^+/+ ; Col4a3 ^−/− mice; P<0.01 by log rank test and n=8–9. (B) Urinary ACRs were calculated at the indicated time points in control, Lamb2 ^+/+ ; Col4a3 ^−/−, and Lamb2 ^+/S83R ; Col4a3 ^−/− mice; n=4–7. (C) BUN was measured in control, Lamb2 ^+/+ ; Col4a3 ^−/−, and Lamb2 ^+/S83R ; Col4a3 ^−/− mice at the indicated time points; n=4–7. *P<0.05 by t test; ^# P<0.01 by t test.

Figure 4.: LAMB2-S83R incorporation into the GBM is unimpaired in Alport mice. Immunofluorescence assay for LAMB2 in Lamb2 ^+/S83R; Col4a3 ^−/− mice was compared with control and Lamb2 ^+/+; Col4a3 ^−/− littermates at P7–P11. Similar levels of LAMB2 were observed in each genotype as well as clear colocalization with the ubiquitous basement membrane protein nidogen in the GBM; n=3–4. Scale bar, 50 μm.

Figure 5.: The LAMB2-S83R allele dramatically worsened Alport phenotype histopathology. (A) PAS staining of P7–P11 control, Lamb2 ^+/+ ; Col4a3 ^−/−, and Lamb2 ^+/S83R ; Col4a3 ^−/− kidney sections revealed no pathology; n=3–5. (B) PAS staining of kidneys at 6 weeks revealed significant pathologic features in Lamb2 ^+/S83R ; Col4a3 ^−/− mice, including glomerulosclerosis, crescents, and tubular protein casts, but no severe lesions in Lamb2 ^+/+ ; Col4a3 ^−/− littermates; n=3–4. (C) Widespread and severe pathology in a P7 Lamb2 ^S83R/S83R ; Col4a3 ^−/− double-homozygous kidney shows a very early effect on the Alport phenotype; n=3. (D) Quantification of glomerulosclerosis in multiple kidneys representative of those shown in A–C. The indicated P values were calculated by t test. C, control; Col, Col4a3; LM, Lamb2; +/+, WT; +/S, Lamb2 ^+/S83R; S/S, Lamb2 ^S83R/S83R.

Figure 6.: LAMB2-S83R increases Alport-associated deposition of ectopic laminins into the GBM. Frozen sections of kidneys from 6- to 10-week-old mice of the indicated genotypes were stained for LAMB2 (6 weeks shown), LAMB1 (10 weeks shown), and LAMA2 (10 weeks shown for control and Lamb2 ^+/+; Col4a3 ^−/− and 8 weeks shown for Lamb2 ^+/S83R; Col4a3 ^−/−). LAMB2 was detected in the GBM in all cases. LAMB1 and LAMA2 are normally in the mesangial matrix but were detected ectopically in the Alport GBM regardless of Lamb2 genotype, although the levels appeared slightly increased in some Lamb2 ^+/S80R ; Col4a3 ^−/− versus Lamb2 ^+/+ ; Col4a3 ^−/− glomeruli. Colocalization with GBM agrin is shown in Supplemental Figures 3 and 4; n=4–5.

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 Lamb2^S83R 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 Lamb2^S83R/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 Lamb2^S83R 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 Lamb2^S83R/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 Lamb2^S83R/S83R; Col4a3^−/− GBM’s ultrastructure resembled that of 2.5-day-old Lamb2^−/−; Col4a3^−/− mice (Supplemental Figure 3).

Figure 7.: The Lamb2-S83R allele enhances Alport-associated GBM abnormalities and foot process effacement. (A) Transmission electron micrographs of glomeruli from mice of the indicated genotypes at the indicated ages reveal GBM splitting by P7–P11 and depict the progression of foot process effacement and sclerosis; n=3–5. Note the unusually thick and electron-lucent GBM in the Lamb2 ^+/S80R ; Col4a3 ^−/− kidney at 7 weeks. (B) The number of foot processes per 1 μm of GBM was counted for each genotype at each time point; n=3–5. *P<5×10⁻⁴ by t test; **P<6×10⁻⁵ by t test; ***P<2×10⁻⁷ by t test.

We next tested the Lamb2^S83R 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 Lamb2^S83R 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 Lamb2^S83R allele, similar to the effects of Lamb2^S83R in Col4a3^−/− mice.

Figure 8.: LAMB2-S83R accelerates disease in Col4a5 ^+/− females. (A) LAMB2 and COL4Α345 were detected by immunofluorescence in 8-week-old tissues. Anti-COL4A345 NC1 domain staining revealed the expected mosaic expression pattern mediated by naturally random inactivation of either the WT or the Col4a5 mutant X chromosome in each podocyte. LAMB2 was detected in both COL4Α345-negative and -positive regions of the GBM but exhibited increased intensity in COL4Α345-negative capillary loops (red in the merged images); n=4. (B) Urinalysis revealed significantly higher ACRs in Lamb2 ^+/S83R; Col4a5 ^+/− mice versus Col4a5 ^+/− littermates between 2 and 6 weeks of age and at 12 weeks of age but with similar ratios at week 8; n=4–7 at each time point. Urine volumes normalized to 20 mg/dl creatinine are shown on a representative SDS-PAGE gel for Lamb2 ^+/+; Col4a5 ^+/− and Lamb2 ^+/S83R; Col4a5 ^+/− littermates at 12 weeks. (C) PAS staining indicated accelerated glomerulosclerosis, inflammation, and protein casts in 8-week-old Lamb2 ^+/S83R; Col4a5 ^+/− females versus Col4a5 ^+/− females; n=3–5. *P<0.05 by t-test.

Discussion

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 LAMB2^S80R 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 Lamb2^S83R 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 Lamb2^S83R/S83R; Col4a3^+/− mice, the severe detrimental effect of Lamb2^S83R 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 Lamb2^S83R/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 Lamb2^S83R 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

Concise Methods

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 mice^25,51 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 Lamb2^S83R 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 Lamb2^S83R/S83R homozygotes or Lamb2^−/S83R compound heterozygotes. For Alport studies, Lamb2^S83R 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.

Microscopy

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.

Disclosures

None.