Mutations in six nephrosis genes delineate a pathogenic pathway amenable to treatment

Abstract

No efficient treatment exists for nephrotic syndrome (NS), a frequent cause of chronic kidney disease. Here we show mutations in six different genes (MAGI2, TNS2, DLC1, CDK20, ITSN1, ITSN2) as causing NS in 17 families with partially treatment-sensitive NS (pTSNS). These proteins interact and we delineate their roles in Rho-like small GTPase (RLSG) activity, and demonstrate deficiency for mutants of pTSNS patients. We find that CDK20 regulates DLC1. Knockdown of MAGI2, DLC1, or CDK20 in cultured podocytes reduces migration rate. Treatment with dexamethasone abolishes RhoA activation by knockdown of DLC1 or CDK20 indicating that steroid treatment in patients with pTSNS and mutations in these genes is mediated by this RLSG module. Furthermore, we discover ITSN1 and ITSN2 as podocytic guanine nucleotide exchange factors for Cdc42. We generate Itsn2-L knockout mice that recapitulate the mild NS phenotype. We, thus, define a functional network of RhoA regulation, thereby revealing potential therapeutic targets.

Fig. 1

High-throughput sequencing reveals recessive mutations of MAGI2, TNS2, DLC1, CDK20, ITSN1, or ITSN2 as causing NS in humans. a Renal histology of individual A5146-21 with focal segmental glomerulosclerosis (FSGS) and MAGI2 mutation (scale bar = 50 μm). b Exon structure of human MAGI2 cDNA and mutations. Below is the protein domain structure of MAGI2, showing GuK, WW1, WW2, and six different PDZ domains. Two different homozygous truncating mutations of MAGI2 were detected in two families with NS and neurological impairment. c Homozygosity mapping identifies ten recessive candidate loci (red circles) in family A1358 with NS, and WES identifies a homozygous mutation of TNS2 (p.Arg292Gln). Non-parametric lod scores (NPL) were calculated and plotted across the human genome. The TNS2 locus (arrowhead) is positioned within one of the maximum NPL peaks on chromosome 12q. d Exon and protein domain structure of human TNS2. Six different TNS2 mutations were detected in five families with NS. Family numbers and amino acid changes are given (Supplementary Table 1). Arrow heads denote altered nucleotides. Lines and arrows indicate positions of mutations in relation to exons and protein domains. Family numbers with compound heterozygous mutations (het) are highlighted in gray. e Renal histology of individual A4967-21 with FSGS and DLC1 mutations. TEM reveals podocyte foot process effacement (arrow heads, magnification 8000x). f Exon and protein domain structure of human DLC1. The SAM, RhoGAP, and START domains are depicted by colored bars, in relation to encoding exon position. Six different DLC1 mutations were detected in four families with NS. Positions of amino acid changes (Supplementary Table 1) are marked with arrowheads. g Renal histology of individual A5013-21 with membranoproliferative glomerulonephritis (MPGN) and mutation in CDK20. TEM reveals podocyte foot process effacement and thickening of glomerular basement membrane (arrow heads, magnification 7000x). h Homozygosity mapping identifies twelve recessive candidate loci (red circles) in family A5013 with NS, and WES identifies a homozygous mutation of CDK20 (p.Phe204Leu), positioned within one of the maximum NPL peaks on chromosome 9q. i Exon and protein domain structure of human CDK20. The serine threonine kinase (S_TKc) domain is depicted by a colored bar, in relation to encoding exon position. One homozygous mutation in CDK20 was detected in family A5013 with NS. j Homozygosity mapping in family A3706 with NS identifies 17 recessive candidate loci (red circles), and WES identifies a homozygous mutation of ITSN1 (p.Pro180Ser), positioned within one of the maximum NPL peaks on chromosome 21q. k Exon and protein domain structure of human ITSN1. Five different ITSN1 mutations were detected in three families with NS. l Renal histology of Patient-1 with mutations in ITSN2 revealed minimal change disease (scale bar = 50 μm). EM showed foot process effacement (scale bar = 2 μm). m Exon and protein domain structure of human ITSN2. Three different ITSN2 mutations were detected in two families with NS

Fig. 2

Gene products of MAGI2, TNS2, DLC1, CDK20, and CAV1 physically and functionally interact to regulate RhoA/Rac1/Cdc42 activation. a Identification of six novel monogenic causes of NS reveals a regulatory network of RhoA activation. The large rounded square symbolizes a podocyte. All six proteins MAGI2, TNS2, DLC1, CDK20, ITSN1, and ITSN2, in which recessive defects were detected herein as novel causes of pTSNS, interact physically or functionally to regulate RhoA/Rac1/Cdc42. Yellow labels highlight proteins encoded by genes, which if mutated give rise to monogenic nephrosis as shown in this study (MAGI2, TNS2, DLC1, CDK20, ITSN1, and ITSN2) or as published (EMP2, ARHGDIA). Blue frame around yellow labels indicate that there is also a monogenic mouse model of NS or a zebrafish model known, as shown in this study for ITSN2 or as published for MAGI2, TNS2, EMP2, and RhoGDI-α. For each of the proteins, MAGI2, TNS2, DLC1, CDK20, TLN1, or ITSN1, the protein domains are shown. Red circles denote positions of mutations that we found in patients. Truncation mutations are represented by “x”. By identifying novel monogenic causes of NS we discovered a cluster proteins that regulate Rho/Rac/Cdc42 activation as being central for the pathogenesis of these patients. b MAGI2 interacts with TNS2 upon co-overexpression and coimmunoprecipitation (coIP) in HEK293T cells. Both mutant MAGI2 clones, Gly39* and Tyr746* (underlined) that reflect alleles of NS patients A5146-21 and B91 respectively, abrogate this interaction. c MAGI2 interacts with DLC1 upon co-overexpression and coIP in HEK293T cells. One mutant MAGI2 clone Gly39* (underlined) reflecting a mutation of NS patient A5146-21 abrogates this interaction. d DLC1 interacts with CDK20 upon co-overexpression in HEK293T cells. e DLC1 interacts with CAV1. Two mutant DLC1 c-DNA clones, reflecting Trp10* and Lys1358Thr alleles of NS patients A548-21 and A4967-21 respectively, lack this interaction. f In IMCD3 cells, migration rate is induced in the presence of serum as compared to scrambled control. Knockdown of Dlc1 in IMCD3 cells using mouse Dlc1 siRNA #1 impairs cell migration rate (red vs. black curve with serum). The decrease in migration is rescued by transfection with full-length human DLC1 cDNA (green curve). Transfection with four out ot six mutants (Trp10*, Ala456Val, Ala1352Val, and Lys1358Thr) failed to rescue this migratory phenotype

Fig. 3

The novel RhoA regulatory network of MAGI2, TNS2, DLC1, CDK20, and CAV1 is a central part of the pTSNS pathogenesis. a Overexpression of GFP-tagged MAGI2 wild type (WT) but not the two MAGI2 mutants from pTSNS patients resulted in a significant increase in active RhoA. Active RhoA was measured by G-LISA assay in HEK293T cells. b Myc-tagged TNS2-WT increased active RhoA levels upon overexpression in HEK293T cells as compared to Mock. However, all six TNS2 mutant clones from pTSNS patients failed to increase active RhoA. c GFP-tagged CDK20-WT showed a significant increase in active RhoA level upon overexpression in HEK293T cells, while the mutant clone Phe240Leu, from a pTSNS patient, failed to do so. d Myc-tagged DLC1-WT showed a significant decrease in active RhoA level upon overexpression in HEK293T cells as compared to Mock. However, none of the six DLC1 mutant clones from pTSNS patients showed this effect. e siRNA mediated knockdown of MAGI2 decreased active RhoA. The overexpression of Magi2-WT rescued this effect while both the mutants from pTSNS patients did not. f siRNA mediated knockdown of TNS2 decreased active RhoA. The overexpression of TNS2-WT rescued this effect, while four out of six TNS2 mutant clones from pTSNS patients did not. g siRNA mediated knockdown of CDK20 decreased active RhoA. This effect was rescued by the overexpression of CDK20-WT, but not by the overexpression of mutant clone Phe240Leu. h siRNA mediated knockdown of DLC1 showed a significant increase in active RhoA. i siRNA mediated knockdown of TNS2 or DLC1 in HEK293T cells decreased or increased active RhoA levels, respectively, as compared to scrambled control. The parallel knockdown of both TNS2 and DLC1 rescued this effect on active RhoA. j siRNA mediated knockdown of DLC1 increased active RhoA and this effect was unaltered upon overexpression of TNS2 in DLC1 knockdown cells, suggesting that TNS2 modulates RhoA activity indirectly via DLC1. k siRNA mediated knockdown of DLC1 or CDK20 in HEK293T cells increased and decreased active RhoA levels, respectively, as compared to scrambled control. The combined knockdown of both DLC1 and CDK20 rescued this effect on active RhoA. l siRNA mediated knockdown of DLC1 or MAGI2 in HEK293T cells increased and decreased active RhoA levels, respectively, as compared to scrambled control. The combined knockdown of both DLC1 and MAGI2 failed to rescue this effect on active RhoA. m Treatment with dexamethasone (75 and 100 µM) inhibited in a dose-dependent manner the increase of active RhoA elicited by DLC1 knockdown. n The change in active RhoA upon knockdown of CDK20, but not MAGI2, TNS2, or CAV1 was found reversible by treatment of HEK293T cells with 100 µM dexamethasone. Error bars are defined as the standard error of at least three independent experiments. One-way ANOVA with Dunnet’s post hoc test was performed vs. control. Data are presented as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. ns, non significant

Fig. 4

ITSN1 and ITSN2 are GEFs for Cdc42, regulating NS-related podocyte function, and Itsn2^−/− mice develop incompletely penetrant NS upon LPS injection. a Myc-tagged ITSN1-WT increased active Cdc42 levels upon overexpression in HEK293T cells as compared to mock, whereas three out of five ITSN1 mutants from pTSNS patients failed to show any significant increase in active Cdc42 upon overexpression. b Active Cdc42 level was measured in COS7 cells. Myc-tagged ITSN2-WT overexpression increased active Cdc42 levels as compared to Mock. However, all three ITSN2 mutants from pTSNS patients failed to show any significant increase in active Cdc42 upon overexpression. Error bars are defined as the standard error of at least four independent experiments. One-way ANOVA with Dunnet’s post hoc test vs. wild-type (WT) ITSN2 expressing control. c Filopodia induction in cultured human podocytes was quantified by counting the transfected cells with filopodia and showed as a percentage of cells. For each construct, 50 transfected cells from three independent experiments were analyzed. One-way ANOVA with Dunnet’s post hoc test vs. WT ITSN1 expressing control. Data are presented as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. d Filopodia induction in cultured human podocytes was quantified by counting the transfected cells with filopodia and showed as a percentage of cells. For each construct, 50 transfected cells from three independent experiments were analyzed. One-way ANOVA with Dunnet’s post hoc test vs. WT ITSN2 expressing control. Data are presented as the mean ± SEM. * P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. e Kidneys of WT and Itsn2 ^L−/L− mice were stained with periodic acid Schiff. The glomeruli of Itsn2 ^L−/L− mice showed normal findings, similar to WT. f Urine albumin of Itsn2 ^L−/L−, Itsn2 ^+/L−, and WT mice was measured before and after LPS injection. Albuminuria level was increased in Itsn2 ^L−/L− mice from 12 to 48 h after LPS injection compared with WT and Itsn2 ^+/L− mice. Two-tailed Student’s t-tests. Data are presented as the mean ± SEM. *P < 0.05. g Representative electron microscopy images. LPS injection-induced foot process (FP) effacement in both WT and Itsn2 ^L−/L− mice within 24 h. At 48 h after LPS injection, FP effacement was still observed in Itsn2 ^L−/L− mice, although WT mice recovered from FP effacement