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. 2020 Jul;98(1):133-146.
doi: 10.1016/j.kint.2020.01.033. Epub 2020 Feb 20.

A deletion in the N-terminal polymerizing domain of laminin β2 is a new mouse model of chronic nephrotic syndrome

Steven D Funk  1 , Raymond H Bayer  1 , Karen K McKee  2 , Kazushi Okada  3 , Hiroshi Nishimune  3 , Peter D Yurchenco  2 , Jeffrey H Miner  4
Affiliations

A deletion in the N-terminal polymerizing domain of laminin β2 is a new mouse model of chronic nephrotic syndrome

Steven D Funk et al. Kidney Int. 2020 Jul.

Abstract

The importance of the glomerular basement membrane (GBM) in glomerular filtration is underscored by the manifestations of Alport and Pierson syndromes, caused by defects in type IV collagen α3α4α5 and the laminin β2 chain, respectively. Lamb2 null mice, which model the most severe form of Pierson syndrome, exhibit proteinuria prior to podocyte foot process effacement and are therefore useful for studying GBM permselectivity. We hypothesize that some LAMB2 missense mutations that cause mild forms of Pierson syndrome induce GBM destabilization with delayed effects on podocytes. While generating a CRISPR/Cas9-mediated analogue of a human LAMB2 missense mutation in mice, we identified a 44-amino acid deletion (LAMB2-Del44) within the laminin N-terminal domain, a domain mediating laminin polymerization. Laminin heterotrimers containing LAMB2-Del44 exhibited a 90% reduction in polymerization in vitro that was partially rescued by type IV collagen and nidogen. Del44 mice showed albuminuria at 1.8-6.0 g/g creatinine (ACR) at one to two months, plateauing at an average 200 g/g ACR at 3.7 months, when GBM thickening and hallmarks of nephrotic syndrome were first observed. Despite the massive albuminuria, some Del44 mice survived for up to 15 months. Blood urea nitrogen was modestly elevated at seven-nine months. Eight to nine-month-old Del44 mice exhibited glomerulosclerosis and interstitial fibrosis. Similar to Lamb2-/- mice, proteinuria preceded foot process effacement. Foot processes were widened but not effaced at one-two months despite the high ACRs. At three months some individual foot processes were still observed amid widespread effacement. Thus, our chronic model of nephrotic syndrome may prove useful to study filtration mechanisms, long-term proteinuria with preserved kidney function, and to test therapeutics.

Keywords: Pierson syndrome; glomerular basement membrane; laminin; nephrotic syndrome.

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Figures

Figure 1.
Figure 1.. CRISPR/Cas9 gene editing induced a 44-amino acid deletion in the LAMB2 protein.
(a) PCR of genomic DNA using primers flanking the gRNA target/Cas9 cleavage site revealed a 219 bp shift in the product. (b) The deduced partial LAMB2 LN domain mutant protein sequence (top and middle rows) shows a 44-amino acid loss (hyphens) due to deletion of parts of exons 3 and 4, and intron 3. This 44-amino acid sequence includes S83 (CIVSHLQ). (c) A ribbon diagram of LAMB2 LN and LEa1 domains was rendered in PyMOL after a homology fit to LAMB1 in HHPred software. The Del44 sequence is shown in red, and S83 is shown as a space-filling molecule in purple. (d) A zoomed image of the LAMB2-Del44 ribbon diagram in (c) rotated 90° clockwise. In addition to a space-filling representation of S83 (purple), cysteines C80, C91, C94, and C71 are shown as space filling models in yellow. Red text for C80-C94 indicates their inclusion in the region deleted in Del44.
Figure 2.
Figure 2.. Defective polymerization of laminin containing LAMB2-S83R or -Del44 is modulated by multiple molecular interactions.
(a) Schwann cell cultures were incubated with wild-type, S83R, or Del44 laminin-121 (Lm-121) with or without the presence of collagen IV α1α1α2 (ColIV) and nidogen (Nd + ColIV), or only with collagen IV and nidogen. Laminin was visualized with anti-laminin E3 antibody and cells were stained with DAPI; n = 9-10 for each concentration and every condition. (b) Anti-laminin E3 immunofluorescence was normalized to DAPI for each condition. 2-way ANOVA followed by Holm-Sidak pairwise comparisons indicated significant concentration-dependent increases in polymerization (28nM vs 14nM and 7nM, P < 0.05) for wild-type and S83R laminins with and without nidogen and collagen IV, excepting Del44 LM-121 by itself (--Δ--). All laminin conditions at 28nM were significantly different (P < 0.003), except for S83R LM-121 vs. Del44 LM-121 with nidogen and collagen IV, indicating polymerization defects in LAMB2 mutants and a stabilizing effect on LAMB2 mutant heterotrimers by nidogen + collagen IV.
Figure 3.
Figure 3.. Urinalysis reveals a long-term, chronic proteinuria in Del44 mice.
(a) Urinary albumin was quantified by ELISA and normalized to urinary creatinine at the indicated time points; n = 3-5 for controls and 3-9 for Del44 mice. 2-way ANOVA revealed significant differences between Del44 and controls (P < 0.05). Tukey multiple pairwise comparison revealed an adjusted P < 0.05 between week 15 and weeks 4-7. (b) Representative creatinine-normalized urines collected at the indicated ages were run on an SDS-PAGE gel and stained with Coomassie to reveal urinary albumin. Cntrl = Control. (c) 24-hour urine collection from 3-month old control and Del44 mice indicated development of polyuria in Del44 mice. n = 4-6; P < 0.004 by Student’s t-test.
Figure 4.
Figure 4.. Survival and plasma analyses reveal features of nephrotic syndrome in Del44 mice.
(a) A log-rank survival analysis comparing control and Del44 littermates revealed significantly reduced viability in Del44 mice. n = 11 control and 15 Del44 mice. P < 1.0 x10−5. (b) BUN levels in control and Del44 mice at the indicated ages revealed the onset of renal function decline at 7 months, which is delayed relative to proteinuria onset and plateau shown in Figure 3. n = 4-6; #, P = 0.0505; *, P < 0.05; $, P < 0.01. (c) Plasma albumin levels in control and Del44 mice revealed onset of hypoalbuminemia at 2-3 months of age. n = 3-5; *, P < 0.001. (d) Levels of plasma cholesterol indicated onset of hyperlipidemia in Del44 mice at 2-3 months. n = 3-5; *, P < 0.05 by Student’s t-test.
Figure 5.
Figure 5.. Periodic acid Schiff staining revealed delayed glomerular and interstitial fibrosis in Del44 mice.
Throughout the 2nd month of life (5-8 weeks of age) almost no pathology was observed in Del44 mice. Tubular dilations, protein casts, and reduced proximal tubule brush border staining were evident at 2 months of age (9 weeks). After 3.5 months (15 weeks) tubular dilations and protein casts were more frequent, and GBM thickening was pronounced. Between 8-9 months (37 weeks is shown) glomerulosclerosis was consistently observed, including global sclerosis and FSGS, with immune infiltrates and fibrosis evident throughout the interstitium. n = 3-5 per age group. Scale bars as indicated.
Figure 6.
Figure 6.. Transmission electron microscopy (TEM) reveals prolonged foot process maintenance in Del44 mice.
(a-c) TEM of Del44 glomerular capillary loops at 1-2 months of age (5, 7, 9 weeks) revealed widening of foot processes with maintenance of architecture and slit diaphragms, occasional GBM out-pocketing (notable in the 7-week tissue shown here), and very sparse foot process effacement concomitant with heavy and progressive albuminuria. (d) After 3 months of age (15 weeks) most foot processes were dramatically dysmorphic or effaced, but occasional slit diaphragms were still observable. (e) TEM of a glomerular capillary loop from a 15-week old control mouse. (f) Foot process number per μm GBM length were quantified in at least 5 micrographs taken from at least 4 different glomeruli per mouse. *, P < 0.05; #, P < 0.005; $, P < 1 x10−9 by Student’s t-test. Scale bars = 500 nm
Figure 7.
Figure 7.. Immunofluorescence analysis of Del44 podocytes reveals early desmin expression but late foot process marker abnormalities.
(a) Expression of desmin, a marker of podocyte injury, in Del44 podocytes preceded proteinuria as early as 1 month of age, but expression varied among podocytes and glomeruli; n = 3-5. (b) Podocin and nephrin co-localized in 3 month-old Del44 glomeruli with intensities similar to controls, but with a moderately punctate appearance. Some Del44 glomerular capillary tufts appeared dilated at this age. n = 3-4 mice. (c) Synaptopodin expression remained robust in Del44 mice until 8 months of age, at which point the staining became punctate but exhibited intensity similar to controls; n = 3-4. Scale bars = 50 μm.
Figure 8.
Figure 8.. LAMB2-Del44 exhibits reduced expression in the GBM.
(a) Sections of kidneys aged 1 and 3 months from control and Del44 mice were stained for LAMB2 by immunofluorescence and imaged with confocal microscopy. (b) Anti-LAMB2 signal was ~30% reduced at 1 month and 3 months of age in Del44 glomeruli versus controls. n = 3 at each age. Statistical significance was determined with the Student’s t-test for each age. Scale bars = 50 μm.
Figure 9.
Figure 9.. Aberrant expression of laminin isoforms reveals a unique molecular pathology.
(a) Del44 capillary loop GBMs exhibited accumulation of the embryonic LAMB1 isoform that co-localized with nidogen at 1 month of age preceding heavy proteinuria, suggesting a defective laminin network in Del44 mice; n = 3-4. (b) Accumulation of the embryonic LAMA1 isoform was also enhanced GBMs of Del44 mice at 1 month of age; n = 3-4. (c) LAMA2 was scarcely observed in Del44 capillary loops through 8 months of age, in contrast to Lamb2−/−, Alport, and Cd151−/− mice. n=3-4. (d) COL4A1 was modestly increased segmentally in the Del44 GBM at 8 months of age; n = 3-5. Scale bars = 50 μm.
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References

    1. Miner JH. The glomerular basement membrane. Exp Cell Res. 2012;318:973–978. - PMC - PubMed
    1. Abrahamson DR. Origin of the glomerular basement membrane visualized after in vivo labeling of laminin in newborn rat kidneys. J Cell Biol. 1985;100:1988–2000. - PMC - PubMed
    1. St John PL, Abrahamson DR. Glomerular endothelial cells and podocytes jointly synthesize laminin-1 and -11 chains. Kidney Int. 2001;60:1037–1046. - PubMed
    1. Reiser J, Kriz W, Kretzler M et al. The glomerular slit diaphragm is a modified adherens junction. J Am Soc Nephrol. 2000;11:1–8. - PubMed
    1. Simic I, Tabatabaeifar M, Schaefer F. Animal models of nephrotic syndrome. Pediatr Nephrol. 2013;28:2079–2088. - PubMed

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