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  • BASIC RESEARCH www.jasn.org

    HANAC Syndrome Col4a1 Mutation Causes NeonateGlomerular Hyperpermeability and AdultGlomerulocystic Kidney Disease

    Zhiyong Chen,* Tiffany Migeon,* Marie-Christine Verpont,* Mohamad Zaidan,*Yoshikazu Sado, Dontscho Kerjaschki, Pierre Ronco,*| and Emmanuelle Plaisier*|

    *Institut National de la Sant et de la Recherche Mdicale, Unit Mixte de Recherche (UMR)S 1155, Paris, France;Sorbonne University, Universit Pierre et Marie Curie, Paris 06, UMR_S 1155, Paris, France; Division of Immunology,Shigei Medical Research Institute, Okayama, Japan; Clinical Institute of Pathology, Medical University of Vienna,Vienna, Austria; and |Assistance Publique-Hpitaux de Paris, Department of Nephrology and Dialysis, Tenon Hospital,Paris, France

    ABSTRACTHereditary angiopathy, nephropathy, aneurysms, and muscle cramps (HANAC) syndrome is an autosomaldominant syndromecausedbymutations inCOL4A1 that encodes thea1chainof collagen IV, amajor componentof basement membranes. Patients present with cerebral small vessel disease, retinal tortuosity, muscle cramps,and kidney disease consisting of multiple renal cysts, chronic kidney failure, and sometimes hematuria. MutationsproducingHANAC syndrome localizewithin the integrin binding site containingCB3[IV] fragment of theCOL4A1protein. To investigate the pathophysiology of HANAC syndrome, we generated mice harboring the Col4a1p.Gly498Val mutation identified in a family with the syndrome. Col4a1 G498V mutation resulted in delayed glo-merulogenesis and podocyte differentiation without reduction of nephron number, causing albuminuria and he-maturia in newborns. The glomerular defects resolved within the first month, but glomerular cysts developed in3-month-old mutant mice. Abnormal structure of Bowmans capsule was associated with metalloproteinase in-duction and activation of the glomerular parietal epithelial cells that abnormally expressedCD44,a-SMA, ILK, andDDR1. Inflammatory infiltrates were observed around glomeruli and arterioles. HomozygousCol4a1G498Vmu-tant mice additionally showed dysmorphic papillae and urinary concentration defects. These results reveal a de-velopmental role for thea1a1a2collagen IVmolecule in theembryonicglomerularbasementmembrane,affectingpodocyte differentiation. The observed association between molecular alteration of the collagenous network inBowmans capsule of the mature kidney and activation of parietal epithelial cells, matrix remodeling, and inflam-mation may account for glomerular cyst development and CKD in patients with COL4A1-related disorders.

    J Am Soc Nephrol 27: 10421054, 2016. doi: 10.1681/ASN.2014121217

    Type IV collagen is a major component of basementmembranes (BMs). It consists of six homologous achains designated a1(IV)a6(IV) encoded byCOL4A1COL4A6 genes.1 Each a-chain contains alarge collagenous domain composed of Gly-Xaa-Yaarepeats, a C-terminal noncollagenous region, and asmall N-terminal domain. Three a-chains assembleto form three distinct triple-helical molecules.2 Thea1a1a2(IV) trimer is ubiquitously expressed in em-bryonic BMs, including embryonic glomerular base-mentmembranes (GBMs), and persists inmost adultorgans. In the mature kidney, it represents the maincollagen IV molecule of the tubular, vascular, and

    Bowmans capsule BM, whereas it remains onlyweakly expressed in the GBM.3 The a3a4a5(IV)and a5a5a6(IV) trimers display a more restricted

    Received December 12, 2014. Accepted June 16, 2015.

    Published online ahead of print. Publication date available atwww.jasn.org.

    Correspondence: Prof. Emmanuelle Plaisier, Department ofNephrology and Dialysis, INSERM UMR_S 1155, Hpital Tenon, 4rue de la Chine, 75020 Paris, France. Email: emmanuelle.plaisier@tnn.aphp.fr

    Copyright 2016 by the American Society of Nephrology

    1042 ISSN : 1046-6673/2704-1042 J Am Soc Nephrol 27: 10421054, 2016

    http://www.jasn.orgmailto:emmanuelle.plaisier@tnn.aphp.frmailto:emmanuelle.plaisier@tnn.aphp.fr

  • distribution, including in the kidney, wherethe former is expressed in the mature GBMand distal tubule BM, and the latter is selec-tively found in the adult Bowmans capsule.3

    Collagen IV networks play a crucial struc-tural role inBMs, but they are also involved inembryogenesis, cell migration and differenti-ation, angiogenesis, tumor growth, and me-tastasis.These functionsaremediated throughinteraction with cell receptors, mainly in-tegrins and discoidin domain receptors 1(DDR1).46

    Alport syndrome was the first collagenIVinherited disease characterized withmutations affecting COL4A3, COL4A4, orCOL4A5 genes.3 COL4A1 mutations weremore recently identified in families affectedby autosomal dominant porencephaly andcerebral small vessel disease.711 Eye abnor-malities, such as cataract, anterior segmentdysgenesis, and retinal tortuosity, were occa-sionally reported together with the brain de-fects.912 Concomitantly, severalCol4a1 ENUmutant mice were analyzed, showing severebrain disease and eye alterations similar to theones observed in patients.7,1315

    We have characterized a distinct systemicphenotype associated with COL4A1 muta-tions in six families, which we named hered-itary angiopathy, nephropathy, aneurysms,and muscle cramps (HANAC).16 Affectedpatients usually show muscle cramps, mildcerebral small vessel disease, retinal arteriolartortuosity, intracranial aneurysms, and a

    Figure 1. Col4a1 G498V mutant newborns show albuminuria and hematuria associ-ated with podocyte and GBM defects at P0. (A) Urine dipstick tests revealed heavyproteinuria and hematuria in heterozygous (+/G498V) and homozygous (G498V/G498V) Col4a1 mutant mice at P0. (B) Phase-contrast microscopy analysis of spoturine sample of Col4a1 homozygous mutant P0 mice showed numerous dimorphicerythrocytes (arrow). (C) Quantification of albuminuria in wild-type (+/+), heterozygousand homozygous mutant newborns at P0. Values represent mean6SEM ***P,0.0001compared with +/+. (D and E) Compared with wild-type animals (D), sagittal sectionsof newborn kidneys in Col4a1 homozygous mutant mice at P0 (E) showed enlargedBowmans spaces with retracted capillary tufts in a subset of glomeruli (white arrow)and dilated proximal tubular sections with flattened epithelial cells. Note also thewidening of the collecting duct lumen in the medulla (periodic acidSchiff). (F and G)Semi-thin sections of immature glomeruli after the formation of the first capillary loopsin wild-type (F) and homozygous mutant (G) P0 kidneys showing podocyte disorga-nization in mutant glomeruli (toluidine blue stain). (H and I) Mature glomeruli in thedeep cortex of wild-type (H) and homozygous mutant (I) P0 kidneys. Mutant miceshowed dilated glomerular capillaries (toluidine blue stain). (J) Erythrocytes in dilatedtubule sections with epithelial cell flattening, adjacent to a glomerulus with retractedcapillary tuft and enlarged Bowmans space in a homozygous mutant kidney (Massons

    trichrome). (K) Intracytoplasmic eosinophilicvacuoles in proximal tubular cells of homozy-gous mice (hematoxylin and eosin). (L and M)Collectingduct sections. Flattenedepitheliumand lumen widening of collecting ducts inhomozygous kidneys (M) compared with wild-type animals (L). Note the expansion of thestroma component between ducts (*) (PAS).(MO) EM analysis of mature glomeruli. Po-docyte foot process effacement (arrowheads)and segmental duplication of GBM (arrows) inhomozygous mutant newborn P0 kidneys (N)contrasting with normal architecture of po-docytes and GBM in wild-type littermates.Note the presence of large intercellular junc-tions between podocytes in homozygousmutant kidneys (white arrow) (P). Scalebars: (DandE):500mm; (FI,K): 50mm; (LandM):20 mm; (J) 100 mm; (N and O): 1 mm; and (O):25 nm.

    J Am Soc Nephrol 27: 10421054, 2016 Renal Defects in HANAC Col4a1 Mice 1043

    www.jasn.org BASIC RESEARCH

  • renal disease that associates multicystic kidneys, sometimes he-maturia, and decreased GFR in older patients.1719 HANACCOL4A1 mutations all substitute glycine residues between

    Gly498 and Gly528 within the cyanogenbromidederived fragment CB3[IV] ofthe protein that contains integrin-bindingsites.1618 These data suggest that thepathogenic effects of HANAC mutationsmay occur through defective cellBM in-teractions. Because COL4A1mutations as-sociated with the brain and eye phenotypeare usually localized in the C-terminal halfof the protein, phenotype-genotype correla-tions may occur among COL4A1-relateddiseases.

    Because kidney disease, including cystsand hematuria, has not been reported inpatients with brain-eye restricted dis-ease and in ENU Col4a1 mice,7,14,15 wegenerated mice harboring the Col4a1p.Gly498Val mutation previously identifiedin a family with HANAC to specifically in-vestigate the pathophysiology of renaldefects.

    RESULTS

    Generation of HANAC Col4a1 G498VMutant MiceA targeting vector was designed to intro-duce the Col4a1 c.G1617T mutation inexon 25 leading to the p.Gly498Val substi-tution (G498V) (Supplemental Figure 1A).Electroporation of the construct intomouse embryonic stem (ES) cells resultedin 372 neomycin-resistant clones of whichfive were targeted (Supplemental Figure1B). Three positive clones were injectedinto mouse blastocysts, and four chimericmice were obtained. The targeted allele wastransmitted through the germline (Supple-mental Figure 1, C and D). Crossbreedingof Col4a1+/G498V mice generated 32% ofCol4a1+/+, 51% of Col4a1+/G498V, and17% of Col4a1G498V/G498V, indicatingincreased embryonic lethality of the ho-mozygous mutants (expected Mendeliandistribution 25%, 50%, and 25%, respec-tively). After birth however, heterozygousand homozygous mice showed normal vi-ability. Brain hemorrhages were observedin Col4a1+/G498V and Col4a1G498V/G498V

    newborns that did not impair life span(data not shown), and retinal tortuosity

    and muscular dystrophy were pr