Pathogenicity of a Human Laminin Β2 Mutation Revealed in Models Of

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Pathogenicity of a Human Laminin b2 Mutation Revealed in Models of Alport Syndrome

Steven D. Funk,1 Raymond H. Bayer,1 Andrew F. Malone,1 Karen K. McKee,2 Peter D. Yurchenco,2 and Jeffrey H. Miner1

1Division of Nephrology, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri and 2Department of Pathology and Laboratory Medicine, Robert Wood Johnson Medical School, Rutgers University, Piscataway, New Jersey

ABSTRACT Pierson syndrome is a congenital nephrotic syndrome with eye and neurologic defects caused by mutations in laminin b2(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 2 2 2 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 a3a4a5(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.

J Am Soc Nephrol 29: 949–960, 2018. doi: https://doi.org/10.1681/ASN.2017090997

Basement membranes are thin sheets of extracellu- Signiﬁcance Statement lar matrixconstructed from four major protein clas- ses: laminins (LMs), type IV collagens, nidogens, Pierson and Alport syndromes are genetic diseases that and sulfated proteoglycans.1,2 LMa,LMb,and affect the laminin and type IV collagen components, LMg chains assemble with a 1:1:1 stoichiometry respectively, of the glomerular basement membrane. Understanding the pathogenic mechanisms of muta- in the endoplasmic reticulum to form covalently tions found in patients may provide guidance for 3 linked heterotrimers. In the glomerulus, these choosing therapeutic approaches. Engineering a human LAMB2 mutation that causes a delayed nephrotic syndrome and impairs laminin polymerization into the Received September 17, 2017. Accepted November 19, 2017. mouse resulted in no detectable defect in glomerular permselectivity. However, breeding just one copy of Published online ahead of print. Publication date available at this mutation onto the Alport mouse background dra- www.jasn.org. matically increased the rate of progression to ESRD, Correspondence: Dr. Jeffrey H. Miner, Division of Nephrology, suggesting a genetic interaction. Thus, variants in a Washington University School of Medicine, 4523 Clayton Ave- noncollagen GBM protein can affect progression of nue, Campus Box 8126, St. Louis, MO 63110. Email: minerj@ Alport syndrome. This could explain in part the variation wustl.edu in disease presentation and progression observed in patients with Alport syndrome. Copyright © 2018 by the American Society of Nephrology

J Am Soc Nephrol 29: 949–960, 2018 ISSN : 1046-6673/2903-949 949 BASIC RESEARCH www.jasn.org are secreted into the space between podocytes and endothelial site mutations leading to absent or truncated LAMB2 protein. cells.4 The a-, b-, and g-chains’ laminin amino-terminal (LN) In contrast, LAMB2 missense mutations cause the less severe polymerization domains link LM heterotrimers together at nephrotic syndrome type 5 with or without ocular abnormal- a-b-g “trimeric nodes” to form a planar, sheet-like network ities (OMIM 614199).14,18 Most missense mutations cluster in in the matrix milieu, whereas the LMa chain’scarboxyl- or near the LN domain that mediates LM-521 polymeriza- terminal LM globular domain links the LM network to cell tion.14 Such LN domain mutations are logically predicted to surface receptors (e.g., integrins and dystroglycan).3,5 Simi- cause LM polymerization defects that should adversely affect larly, type IV collagens assemble intracellularly to form heter- GBM architecture and permselectivity, thereby causing ne- otrimeric “protomers” that are secreted into the extracellular phrotic syndrome. However, studies of two pathogenic mis- space, where distinct carboxyl-terminal and amino-terminal sense mutations in mice, LAMB2-R246Q and LAMB2-C321R, domain interactions promote collagen IV network formation.6 revealed a mechanism involving impaired protein folding and In the glomerular basement membrane (GBM), laminin a5b2g1 (LM-521) trimers form separate networks adjacent to podocytes and endothelial cells, and collagen a3a4a5(IV) (COL4A345) protomers form a network at the center of the GBM.7 A separate collagen a1a2a1(IV) network forms adjacent to the endothe- lium.7,8 Nidogen, a basement membrane protein with binding sites for LMg1and collagen IV, links the LM and collagen networks.9 The long heparan sulfate proteo- glycan 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 endo- thelium 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 b2[LAMB2]or COL4A345)causesvariousformsofhe- reditary kidney disease that are always ac- companied 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-a3, -a4, or -a5chains, which prevent or reduce production of the COL4A345 network, causes Alport Figure 1. LAMB2-S83R protein resulting from CRISPR/Cas9 mediated gene editing syndrome, a hereditary nephritis associ- 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 ated with hearing and eye defects.16,17 boxed sequence is the WT, and the lower sequence is the mutant. (B) SDS-PAGE 2/S83R The pathophysiologic mechanisms under- analysis of urine shows the lack of elevated albuminuria in Lamb2 mice versus lying these diseases are incompletely theWTatupto13months(n=3–5). (C) Immunoﬂuorescence analysis of LAMB2 in the +/+ +/S83R S83R/S83R understood. GBMs of Lamb2 (10 months), Lamb2 (10 months), Lamb2 (9 months), 2/S83R LAMB2 mutations that cause Pierson and Lamb2 (10 months) mice. LAMB2 levels in any S83R-containing mice were syndrome are typically nonsense or splice similar to the WT at any age up to 13 months; n=3–4 for each genotype.

950 Journal of the American Society of Nephrology J Am Soc Nephrol 29: 949–960, 2018 www.jasn.org BASIC RESEARCH

secretion and induction of endoplasmic reticulum stress.19,20 Such knowledge about the pathogenic mechanisms of individ- ual 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 pre- sumed 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. LMb2 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 Trans- 2 2 genes 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

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

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“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 2 2 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 2 2 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 glomer- 2 2 uli, 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 2 2 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 overaccu- mulation 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 2 with Lamb2+/ (heterozygous null) mice to eventually pro- 2 duce Lamb2+/S83R, Lamb2S83R/S83R,andLamb2 /S83R mice. LAMB2 deposition in the GBM (Figure 1B) and colocalization with nidogen (shown for Lamb2S83R/S83R in Supplemental Fig- ure 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 2/2 knock-in mice expressing only LAMB2-S83R also lacked in- Figure 3. LAMB2-S83R worsens the Alport syndrome (Col4a3 ) +/+ 2/2 creased LAMB1 in the GBM (Supplemental Figure 2B). phenotype. (A) The age at ESRD of Lamb2 ;Col4a3 and +/S83R 2/2 Lamb2 ; Col4a3 mice was followed over the indicated time. Lamb2+/S83R Col4a32/2 fi Polymerization Properties of LAMB2-S83R In Vitro ; mice reached ESRD signi cantly faster Lamb2+/+;Col4a32/2 P, n – Previous biochemical studies of LMb1 short-arm (NH2- than mice; 0.01 by log rank test and =8 9. (B) Urinary ACRs were calculated at the indicated time points in terminal) fragments indicate that the LAMB1-S68R mutant, +/+ 2/2 +/S83R 2/2 control, Lamb2 ;Col4a3 ,andLamb2 ;Col4a3 mice; which is analogous to the LAMB2-S83R mutant, is polymer- +/+ 2/2 n=4–7. (C) BUN was measured in control, Lamb2 ;Col4a3 ,and 27 +/S83R 2/2 ization defective. In light of our genetic studies presented Lamb2 ;Col4a3 mice at the indicated time points; n=4–7. above, we tested the polymerization capacity of both the *P,0.05 by t test; #P,0.01 by t test. LAMB1-S68R and LAMB2-S83R mutants in vitro using a Schwann cell–based assay. Schwann cells do not express LMs

952 Journal of the American Society of Nephrology J Am Soc Nephrol 29: 949–960, 2018 www.jasn.org BASIC RESEARCH 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). Reten- tion of all heterotrimers tested was further decreased by addition of netrin-4 (Figure Figure 4. LAMB2-S83R incorporation into the GBM is unimpaired in Alport mice. +/S83R 2/2 2B), a matrix protein that competitively Immunofluorescence assay for LAMB2 in Lamb2 ; Col4a3 mice was compared +/+ 2/2 inhibits LM LN domain ternary node with control and Lamb2 ; Col4a3 littermates at P7–P11. Similar levels of LAMB2 formation. These data indicate that the were observed in each genotype as well as clear colocalization with the ubiquitous pathogenic mechanism of the human basement membrane protein nidogen in the GBM; n=3–4. Scale bar, 50 mm. LAMB2-S80R allele should involve a polymerization defect. However, the apparently normal LAMB2-S83R accumulation in the mouse GBM (Fig- Urinalysis showed a significant increase in the urinary 2 2 ure 1, Supplemental Figures 1 and 2) does not reflect the in albumin-to-creatinine ratio (ACR) in Lamb2+/S83R; Col4a3 / 2 2 vitro findings and does not support a polymerization defect. mice versus Lamb2+/+; Col4a3 / and control (non-Alport) Nevertheless, we note that some potentially polymerization- littermates as early as P7–P11 (Figure 3B). Although ACRs defective LAMA2 missense mutations causing a relatively remained high through 7 weeks, ACRs decreased as BUN levels mild muscular dystrophy in patients result in nearly normal increased at 5–7 weeks (Figure 3C) and renal failure occurred levels of LM-211 in muscle and Schwann cell basement (Figure 3A). Statistically insignificant differences in ACR and 2 2 membranes.5,30–32 BUN between Lamb2+/+; Col4a3 / and control littermates at these ages further showed the dramatic effect of the Lamb2S83R Effect of LAMB2-S83R on Disease Progression in allele on the Alport phenotype. Alport Mice The Alport phenotype presents histologically with a mix Wenext hypothesized that the murine model lacks a contextual offocalsegmentalandglobalglomerulosclerosis,some- factor(s) necessary to elicit LAMB2-S83R’s pathogenicity and times with crescents. In contrast, diffuse mesangial sclerosis that Lamb2S83R might act as a modifier allele in mice. To test is the histologic hallmark of Pierson syndrome. Periodic 2 2 2 2 this, we crossed the Lamb2S83R allele onto the Col4a3 / Al- acid–Schiff (PAS) staining of Lamb2+/S83R; Col4a3 / kid- 2 2 port background.33 Strikingly, Lamb2+/S83R; Col4a3 / mice neys revealed normal features at P7–P11 (Figure 5A) but (harboring just one Lamb2S83R allele) showed a significant re- pronounced histopathologic features in 6- to 8-week-old 2 2 2 2 duction in survival compared with Lamb2+/+; Col4a3 / lit- mice (Figure 5B). In comparison, Lamb2+/+; Col4a3 / termates, indicating a dominant pathogenic effect of the kidneys exhibited less severe pathology at 6–8weeksof LAMB2-S83R protein (Figure 3A). Notably, a Lamb2 null allele age (Figure 5B). Furthermore, PAS analysis of P7 homozy- 2 2 2 2 2 did not affect the Alport phenotype in Lamb2+/ ; Col4a3 / mice gous Lamb2S83R/S83R; Col4a3 / kidneys revealed a pheno- (Supplemental Figure 3). LAMB2 expression and colocaliza- type more consistent with 6- to 8-week-old Lamb2+/S83R; 2 2 2 2 tion with nidogen in the Lamb2+/S83R; Col4a3 / GBM at post- Col4a3 / kidneys (Figure 5C). Blinded quantification of natal days 7–11 (P7–P11) (Figure 4) were similar to those glomerular lesions showed significantly accelerated pathol- 2 2 2 2 observed in Lamb2+/+; Col4a3 / littermates. Thus, the re- ogy in Lamb2+/S83R; Col4a3 / mice versus Lamb2+/+; 2 2 2 2 duced survivability of Lamb2+/S83R; Col4a3 / mice cannot Col4a3 / and control mice (Figure 5D). Thus, histologic be attributed to reduced LAMB2 levels but is likely due to de- analysis also indicates that a single Lamb2S83R allele is suf- fective function of LAMB2-S83R within the GBM. ficient to worsen the Alport phenotype.

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+/+ Figure 5. The LAMB2-S83R allele dramatically worsened Alport phenotype histopathology. (A) PAS staining of P7–P11 control, Lamb2 ; 2/2 +/S83R 2/2 Col4a3 ,andLamb2 ; Col4a3 kidney sections revealed no pathology; n=3–5. (B) PAS staining of kidneys at 6 weeks revealed +/S83R 2/2 signiﬁcant pathologic features in Lamb2 ; Col4a3 mice, including glomerulosclerosis, crescents, and tubular protein casts, but +/+ 2/2 S83R/S83R 2/2 no severe lesions in Lamb2 ;Col4a3 littermates; n=3–4. (C) Widespread and severe pathology in a P7 Lamb2 ;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; +/S83R S83R/S83R +/+, WT; +/S, Lamb2 ;S/S,Lamb2 .

2 2 Alport syndrome features ectopic deposition of LAMA2, at early stages in Lamb2+/S83R; Col4a3 / mice, which even- LAMB1, and COL4A112 in the GBM, which could be com- tually worsened to become extremely thick and electron lucent 2 2 2 2 pensatory and/or pathogenic.8,34,35 Lamb2+/S83R; Col4a3 / compared with those in Lamb2+/+; Col4a3 / mice (Figure 7). 2 2 mice exhibited these matrix constituents in the GBM at levels At P7–P11, Lamb2+/+; Col4a3 / mice exhibited mild foot pro- 2 2 2 2 higher than age-matched Lamb2+/+; Col4a3 / mice (Figure cess effacement, and by comparison, Lamb2+/S83R; Col4a3 / 6, Supplemental Figures 4 and 5). Elevated expression of mice showed a statistically insigniﬁcant trend of increased ef- LAMB2-S83R between 6–10 weeks of age (Figure 6, Supple- facement (Figure 7B). By 5 weeks, foot process effacement in 2 2 mental Figure 4) indicates that accelerated disease progres- Lamb2+/S83R; Col4a3 / mice was signiﬁcantly worse than that 2 2 2 2 sion in Lamb2+/S83R;Col4a3 / mice is not due to loss of in Lamb2+/+; Col4a3 / littermates. Consistent with the histo- 2 2 LAMB2 expression. Transmission electron microscopy re- logic data, P7–P11 Lamb2S83R/S83R; Col4a3 / mice exhibited vealed GBM splitting and bulging typical of Alport syndrome effacement that was more consistent with that of 5- and 7-

954 Journal of the American Society of Nephrology J Am Soc Nephrol 29: 949–960, 2018 www.jasn.org BASIC RESEARCH

Curiously, the average ACR of Lamb2+/+; 2 Col4a5+/ mice was not different from 2 that of Lamb2+/S83R; Col4a5+/ mice at 8 weeks, but it was signiﬁcantly higher in 2 Lamb2+/S83R; Col4a5+/ mice at 12 weeks, indicating accelerated disease progression. Histologic analysis revealed enhanced glomerulosclerosis and protein casts in 2 Lamb2+/S83R; Col4a5+/ kidneys compared 2 with Lamb2+/+; Col4a5+/ kidneys as early as 8 weeks of age (Figure 8C). Collectively, urinalysis and histology both showed accelerated disease progression in female 2 Col4a5+/ Alport mice harboring the Lamb2S83R allele, similar to the effects of 2 2 Lamb2S83R in Col4a3 / mice.

DISCUSSION

Loss of LAMB2 causes Pierson syndrome, a rare but catastrophic congenital disease Figure 6. LAMB2-S83R increases Alport-associated deposition of ectopic laminins with both renal and extrarenal phenotypes into the GBM. Frozen sections of kidneys from 6- to 10-week-old mice of the indicated that leads to death, usually within the first genotypes were stained for LAMB2 (6 weeks shown), LAMB1 (10 weeks shown), and year of life.14 Missense mutations clustered +/+ 2/2 LAMA2 (10 weeks shown for control and Lamb2 ; Col4a3 and 8 weeks shown for in or near the LAMB2 LN domain have Lamb2+/S83R Col4a32/2 ; ). LAMB2 was detected in the GBM in all cases. LAMB1 and been discovered in patients who exhibit LAMA2 are normally in the mesangial matrix but were detected ectopically in the Lamb2 phenotypes that are, in general, less severe Alport GBM regardless of genotype, although the levels appeared slightly 14 +/S80R 2/2 +/+ 2/2 than Pierson syndrome, suggesting that a increased in some Lamb2 ; Col4a3 versus Lamb2 ; Col4a3 glomeruli. Colocalization with GBM agrin is shown in Supplemental Figures 3 and 4; n=4–5. 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 2 2 week-old Lamb2+/S83R; Col4a3 / mice, and the P7–P11 missense mutation, which was discovered as homozygous in a girl 2 2 Lamb2S83R/S83R; Col4a3 / GBM’s ultrastructure resembled with an unusually late-onset nephrotic-range proteinuria at age 6 2 2 2 2 that of 2.5-day-old Lamb2 / ;Col4a3 / mice (Supple- years old.14,21 The failure of the analogous murine Lamb2S83R mental Figure 3). mutation to cause proteinuria when either homozygous or oppo- We next tested the Lamb2S83R allele in a less severe Alport site a Lamb2 null allele was surprising, because the patient also syndrome model. The Col4a5 gene resides on the X chromo- exhibited a retinal phenotype consistent with the Pierson syn- some, accounting for the increased frequency of Alport syndrome spectrum.21 This discrepancy may have resulted from 2 drome in males. Because of random X inactivation, Col4a5+/ either differences in mouse and human glomerular physiology females show mosaic, discontinuous deposition of COL4A345 or an unknown second genetic or environmental hit in the in the GBM and a mild Alport phenotype. Wecrossed Lamb2S83R patient. Previous analysis of the patient ruled out potential mice with Col4a5 mutant mice36 to generate Lamb2+/S83R; contributions by mutations in NPHS1, NPHS2, and WT1,21 2 Col4a5+/ females and compared COL4A345 and LAMB2 ex- but it is possible that the patient harbors mutations in other pression, albuminuria levels, and histology with those of con- genes that affect the phenotype. 2 trol littermates at 2–12 weeks of age. Lamb2+/+; Col4a5+/ and Strong candidates for a potential second hit that also asso- 2 Lamb2+/S83R; Col4a5+/ GBMs exhibited the expected mosaic ciate with GBM defects are COL4A3 and COL4A4, because pattern of COL4A345 staining (Figure 8A). LAMB2 deposi- approximately 1% of the population carries mutations that 2 tion in COL4A345-negative segments of the Col4a5+/ GBM can cause Alport syndrome when homozygous or compound was enhanced (Figure 8A), similar to the homogeneous in- heterozygous. Moreover, COL4A3 and COL4A4 heterozygos- 2 2 crease in LAMB2 observed in the Col4a3 / GBM (Figure 6, ity can present either with no clinical phenotype or with fea- Supplemental Figure 4). Urinalysis revealed significantly in- tures as variable as hematuria, thin basement membrane 2 creased albuminuria in Lamb2+/S83R; Col4a5+/ versus Lamb2+/+; nephropathy, proteinuria, focal segmental glomerulosclero- 2 Col4a5+/ mice between 4 and 6 weeks of age (Figure 8B). sis, and even renal failure.37–42 Although we did not find a

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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 +/S80R 2/2 Lamb2 ; Col4a3 kidney at 7 weeks. (B) The number of foot processes per 1 mm of GBM was counted for each genotype at each 2 2 2 time point; n=3–5. *P,5310 4 by t test; **P,6310 5 by t test; ***P,2310 7 by t test.

2 phenotype in Lamb2S83R/S83R; Col4a3+/ mice, the severe det- is noteworthy. These ﬁndings provide support for the exis- rimental effect of Lamb2S83R heterozygosity on the Alport tence of modiﬁer genes that encode GBM proteins and could 2 2 2 phenotype in both Col4a3 / and Col4a5+/ mutant mice contribute to the well known variability in phenotypes of

956 Journal of the American Society of Nephrology J Am Soc Nephrol 29: 949–960, 2018 www.jasn.org BASIC RESEARCH

including LAMA2, LAMB1, and COL4A1, to the GBM was similar in Lamb2+/+; 2 2 2 2 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 2 2 effacement in Lamb2+/S83R; Col4a3 / 2 2 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, 2 2 by 7 weeks, the Lamb2+/S83R; Col4a3 / GBM exhibited a severe thickening and unusual podocyte disorganization that re- 2 2 sembled the Lamb2S83R/S83R; Col4a3 / 2 2 2 2 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 wors- ening GBM ultrastructure, eventually leading to lesions resembling those observed in the total absence of LAMB2 2 shortly after birth. Figure 8. LAMB2-S83R accelerates disease in Col4a5+/ females. (A) LAMB2 and Why is the LAMB2-S83R mutant pro- COL4Α345 were detected by immunofluorescence in 8-week-old tissues. Anti- COL4A345 NC1 domain staining revealed the expected mosaic expression pattern tein innocuous on its own in mice but mediated by naturally random inactivation of either the WT or the Col4a5 mutant X detrimentalontheAlportbackground?Ex- chromosome in each podocyte. LAMB2 was detected in both COL4Α345-negative isting biochemical studies27 and our cell and -positive regions of the GBM but exhibited increased intensity in COL4Α345- biologic studies (Figure 2) of mutant negative capillary loops (red in the merged images); n=4. (B) Urinalysis revealed sig- LAMB1 and LAMB2 chains show that the 2 2 nificantly higher ACRs in Lamb2+/S83R; Col4a5+/ mice versus Col4a5+/ littermates conserved Ser in the LN domain is neces- between 2 and 6 weeks of age and at 12 weeks of age but with similar ratios at week 8; saryforproperternarynodeformation n=4–7 at each time point. Urine volumes normalized to 20 mg/dl creatinine are shown with LMa and LMg LN domains and Lamb2+/+ Col4a5+/2 Lamb2+/S83R on a representative SDS-PAGE gel for ; and ; efficient LM trimer polymerization and Col4a5+/2 littermates at 12 weeks. (C) PAS staining indicated accelerated glomer- 29 2 accumulation at cell surfaces. Our data ulosclerosis, inflammation, and protein casts in 8-week-old Lamb2+/S83R; Col4a5+/ +/2 showing that LAMB2-S83R incorporates females versus Col4a5 females; n=3–5. *P,0.05 by t-test. into the GBM (Figures 1, 3, and 5, Supple- mental Figures 1 and 3) together with the presence of LAMB2-S80R in the patient’s patients with Alport syndrome and identical COL4 muta- GBM21 indicate that the mutant LM-521 (LM-52*1) is some- tions. This variability could result from second hits in mod- how stabilized in vivo via a mechanism not active in vitro.We ifier genes that might not otherwise be overtly pathogenic. To propose that the macromolecular complex that forms by our knowledge, this is the first report describing a genetic integrin-LM-nidogen-COL4 interactions in the confined modifier of Alport syndrome that involves a GBM compo- space between podocytes and endothelial cells facilitates re- nent; previous studies have shown roles for Itga1, Itga2, Ddr1, tention of LM-52*1 within the GBM, despite the proven de- Trp53,andAlb in modulating kidney disease progression in fect in LM polymerization. In contrast, in Alport GBM, the Alport mice.43–47 In addition, a variant in NPHS2 (podocin) LM-52*1’s defective polymerization apparently synergizes has been associated with proteinuria in patients with thin with the COL4 defect to destabilize the GBM and hasten basement membrane nephropathy caused by a heterozygous its demise and the resulting decrease in kidney function. mutation in COL4A3 or COL4A4.48 The fact that LAMB2-S83R is pathogenic, despite the pres- Histology revealed features consistent with Alport syn- ence of WT LAMB2 presumably expressed at a 1:1 ratio in 2 2 2 2 drome that were accelerated in Lamb2+/S83R; Col4a3 / Lamb2+/S80R; Col4a3 / mice, suggests that even modestly mice. The localization of ectopic matrix protein isoforms, modulating the efficiency of LM polymerization can

J Am Soc Nephrol 29: 949–960, 2018 LAMB2 Mutation Modifies Alport Syndrome 957 BASIC RESEARCH www.jasn.org drastically affect progression of Alport syndrome. Finally, we 7 mm. Antibodies, including rabbit anti-mouse LAMB1 and speculate that methods of improving WT LM-521 polymer- LAMB2,52 mouse anti-rat LAMB2,24 rat anti-mouse LAMA2 (clone ization and strengthening its interactions with other 4H8–2; Axxora),53 rat anti-mouse nidogen (Clone ELM1), mouse GBM components in the Alport context exist, and they anti-bovine COL4A345,54 and rabbit anti-mouse agrin,55 were di- can be exploited to slow damage to the GBM and attenuate luted into PBS containing 1% BSA. the resulting decline in kidney function. In this regard, var- ious designer basement membrane proteins have been LM Polymerization Assay used to slow or prevent muscular dystrophy due to Lama2 Schwann cells were grown as described.29 Cells were incubated at 10,30,49,50 mutation. 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 netrin4DC for 1 hour followed by CONCISE METHODS washing with PBS and fixation with 3% paraformaldehyde for 15 minutes at room temperature. After blocking with 5% goat serum Transgenes and Mice and 0.5% BSA in PBS, wells were incubated with rat anti-LMg1 All animal experiments conformed to the National Institutes of Health (MAB1920; Millipore), washed, and then incubated with secondary Guide for the Care and Use of Laboratory Animals and were approved antibody. Schwann cells were viewed by indirect immunofluores- by the Washington University Institutional Animal Care and Use Com- cence, and digital images were recorded (six to seven fields mittee. For transgenic expression, the S80R mutation (S83R in rat and per condition, each 130031030 pixels) using a 310 microscope 24 mouse) wasengineered into the previously described rat Lamb2 cDNA objective with controlled exposure times. Fluorescence levels were by site-directed mutagenesis (QuickChange XL; Stratagene, La Jolla, estimated with ImageJ.29 Nuclei were stained with 49,6-diamidino- CA) and placed under control of the mouse 4.2-kb nephrin pro- 2-phenylindole to determine the number of cells in each field. The 22 fi moter for podocyte-speci c expression. Founders were mated with average background per pixel was determined using untreated cul- 25,51 Lamb2 mutant mice for functional analysis of the nephrin-LAMB2- tures. The summed intensities from each field were divided by the 2/2 2/2 S83R transgene on the Lamb2 background. Lamb2 mice also area followed by background subtraction and division by the carried the Muscle Creatine Kinase-rat LAMB2 transgene to prevent number of counted nuclei. Data were expressed as the mean6 24 lethal neuromuscular junction defects. SD of normalized net summed intensities per cell, with plots S83R To generate the mouse Lamb2 knock-in allele via CRISPR/ and statistical calculations prepared in SigmaPlot 12.5 (Systat). 9 9 Cas9,aguideRNA(5-aggtgacagacaatacagta-3 )wasdesignedby Significance was determined by one-way ANOVA followed by the Washington University Transgenic Vectors Core and microinjec- Holm–Sidak pairwise comparisons. ted 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 2 were mated with Lamb2+/ mice on a mixed B6CBA background51 to ACKNOWLEDGMENTS 2 produce Lamb2S83R/S83R homozygotes or Lamb2 /S83R compound heterozygotes. For Alport studies, Lamb2S83R mice were mated with We thank Gloriosa Go and Jennifer Richardson for technical as- Col4a3 and Col4a5 mutant mice on mixed B6CBA backgrounds.33,36 sistance, the Transgenic Vectors Core for design and validation of 2 2 Mice with Lamb2+/+, Lamb2+/ , Col4a3+/+,andCol4a3+/ genotypes the Clustered Regularly Interspaced Short Palindromic Repeats were used as controls. (CRISPR)/Cas9 guideA, the Mouse Genetics Core for nucleic acid microinjections and mouse husbandry, the Pulmonary Urinalyses and BUN Assays Morphology Core and Advanced Imaging and Tissue Analysis Urine creatinine levels were determined with the Quantichrome Core (supported by National Institutes of Health [NIH] grant creatinine detection kit. Blood was collected from cheek punc- P30DK052574)forhistology,andtheWashingtonUniversityCenter ture–inducedbleedsandcentrifugedat5500rpmfor5minutesto for Cellular Imaging for electron microscopy. We are grateful to collect serum. BUN was assayed with the Quantichrome Urea Takako Sasaki, Yoshikazu Sado, and Dorin-Bogdan Borza for gifts of detection kit. To determine albumin levels, creatinine-normal- antibodies. ized urines were subjected to SDS-PAGE and Coomassie blue S.D.F. was supported by NIH grant T32DK007126. This work was staining with BSA standards, quantified by densitometry with funded by NIH grants R01DK036425 (to P.D.Y.) and R01DK078314 ImageJ software, and extrapolated from a best fitcurveofthe (to J.H.M.). Production of transgenic and knock-in mice was sup- BSA standards. ported by Diabetes Research Center Transgenic and Embryonic Stem Cell Core grant P30DK020579 and Digestive Diseases Research Core Microscopy Center Murine Models Core grant P30DK052574. PAS staining of paraffin kidney sections and transmission electron microscopy were performed by standard methods. Glomerulosclero- sis was quantified by an observer blinded to genotypes. For immu- DISCLOSURES nofluorescence, kidneys were frozen in OCT and cryosectioned at None.

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