Steroid-resistant nephrotic syndrome associated MYO1E mutations have differential effects on myosin 1e localization, dynamics, and activity

Myo1e is a non-muscle motor protein enriched in the podocyte foot processes. Mutations in MYO1E are associated with steroid-resistant nephrotic syndrome (SRNS). Here, we set out to differentiate between the pathogenic and neutral MYO1E variants identified in SRNS patients by exome sequencing. Based on protein sequence conservation and structural predictions, two mutations in the motor domain, T119I and D388H, were selected for this study. EGFP-tagged Myo1e constructs were delivered into the Myo1e-KO podocytes via adenoviral infection to analyze Myo1e protein stability, Myo1e localization, and clathrin-dependent endocytosis, which is known to involve Myo1e activity. Furthermore, truncated Myo1e constructs were expressed using the baculoviral expression system and used to measure Myo1e ATPase and motor activity in vitro. Both mutants were expressed as full-length proteins in the Myo1e-KO podocytes. However, unlike wild-type (WT) Myo1e, the T119I variant was not enriched at the cell junctions or clathrin-coated vesicles (CCVs) in podocytes. In contrast, the D388H variant localization was similar to the WT. Surprisingly, the dissociation of the D388H variant from cell-cell junctions and CCVs was decreased, suggesting that this mutation also affects Myo1e activity. The ATPase activity and the ability to translocate actin filaments were drastically reduced for the D388H mutant, supporting the findings from cell-based experiments. The experimental pipeline developed in this study allowed us to determine that the T119I and D388H mutations appear to be pathogenic and gain additional knowledge in the Myo1e role in podocytes. This workflow can be applied to the future characterization of novel MYO1E variants associated with SRNS.

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GAPVD1 and ANKFY1 Mutations Implicate RAB5 Regulation in Nephrotic Syndrome

Journal of the American Society of Nephrology ◽

10.1681/asn.2017121312 ◽

2018 ◽

Vol 29 (8) ◽

pp. 2123-2138 ◽

Cited By ~ 13

Author(s):

Tobias Hermle ◽

Ronen Schneider ◽

David Schapiro ◽

Daniela A. Braun ◽

Amelie T. van der Ven ◽

...

Keyword(s):

Nephrotic Syndrome ◽

Binding Affinity ◽

Ectopic Expression ◽

Physical Interaction ◽

Missense Mutations ◽

Wild Type ◽

Steroid Resistant ◽

Steroid Resistant Nephrotic Syndrome ◽

Whole Exome

BackgroundSteroid-resistant nephrotic syndrome (SRNS) is a frequent cause of CKD. The discovery of monogenic causes of SRNS has revealed specific pathogenetic pathways, but these monogenic causes do not explain all cases of SRNS.MethodsTo identify novel monogenic causes of SRNS, we screened 665 patients by whole-exome sequencing. We then evaluated the in vitro functional significance of two genes and the mutations therein that we discovered through this sequencing and conducted complementary studies in podocyte-like Drosophila nephrocytes.ResultsWe identified conserved, homozygous missense mutations of GAPVD1 in two families with early-onset NS and a homozygous missense mutation of ANKFY1 in two siblings with SRNS. GAPVD1 and ANKFY1 interact with the endosomal regulator RAB5. Coimmunoprecipitation assays indicated interaction between GAPVD1 and ANKFY1 proteins, which also colocalized when expressed in HEK293T cells. Silencing either protein diminished the podocyte migration rate. Compared with wild-type GAPVD1 and ANKFY1, the mutated proteins produced upon ectopic expression of GAPVD1 or ANKFY1 bearing the patient-derived mutations exhibited altered binding affinity for active RAB5 and reduced ability to rescue the knockout-induced defect in podocyte migration. Coimmunoprecipitation assays further demonstrated a physical interaction between nephrin and GAPVD1, and immunofluorescence revealed partial colocalization of these proteins in rat glomeruli. The patient-derived GAPVD1 mutations reduced nephrin-GAPVD1 binding affinity. In Drosophila, silencing Gapvd1 impaired endocytosis and caused mistrafficking of the nephrin ortholog.ConclusionsMutations in GAPVD1 and probably in ANKFY1 are novel monogenic causes of NS. The discovery of these genes implicates RAB5 regulation in the pathogenesis of human NS.

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ADCK4 Deficiency Destabilizes the Coenzyme Q Complex, Which Is Rescued by 2,4-Dihydroxybenzoic Acid Treatment

Journal of the American Society of Nephrology ◽

10.1681/asn.2019070756 ◽

2020 ◽

Vol 31 (6) ◽

pp. 1191-1211 ◽

Cited By ~ 5

Author(s):

Eugen Widmeier ◽

Seyoung Yu ◽

Anish Nag ◽

Youn Wook Chung ◽

Makiko Nakayama ◽

...

Keyword(s):

Nephrotic Syndrome ◽

Knockout Mice ◽

Molecular Mechanisms ◽

Coenzyme Q ◽

Mass Spectrometry Analysis ◽

Dihydroxybenzoic Acid ◽

Mitochondrial Potential ◽

Steroid Resistant ◽

Steroid Resistant Nephrotic Syndrome

BackgroundMutations in ADCK4 (aarF domain containing kinase 4) generally manifest as steroid-resistant nephrotic syndrome and induce coenzyme Q10 (CoQ10) deficiency. However, the molecular mechanisms underlying steroid-resistant nephrotic syndrome resulting from ADCK4 mutations are not well understood, largely because the function of ADCK4 remains unknown.MethodsTo elucidate the ADCK4’s function in podocytes, we generated a podocyte-specific, Adck4-knockout mouse model and a human podocyte cell line featuring knockout of ADCK4. These knockout mice and podocytes were then treated with 2,4-dihydroxybenzoic acid (2,4-diHB), a CoQ10 precursor analogue, or with a vehicle only. We also performed proteomic mass spectrometry analysis to further elucidate ADCK4’s function.ResultsAbsence of Adck4 in mouse podocytes caused FSGS and albuminuria, recapitulating features of nephrotic syndrome caused by ADCK4 mutations. In vitro studies revealed that ADCK4-knockout podocytes had significantly reduced CoQ10 concentration, respiratory chain activity, and mitochondrial potential, and subsequently displayed an increase in the number of dysmorphic mitochondria. However, treatment of 3-month-old knockout mice or ADCK4-knockout cells with 2,4-diHB prevented the development of renal dysfunction and reversed mitochondrial dysfunction in podocytes. Moreover, ADCK4 interacted with mitochondrial proteins such as COQ5, as well as cytoplasmic proteins such as myosin and heat shock proteins. Thus, ADCK4 knockout decreased the COQ complex level, but overexpression of ADCK4 in ADCK4-knockout podocytes transfected with wild-type ADCK4 rescued the COQ5 level.ConclusionsOur study shows that ADCK4 is required for CoQ10 biosynthesis and mitochondrial function in podocytes, and suggests that ADCK4 in podocytes stabilizes proteins in complex Q in podocytes. Our study also suggests a potential treatment strategy for nephrotic syndrome resulting from ADCK4 mutations.

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TBC1D8B Mutations Implicate RAB11-Dependent Vesicular Trafficking in the Pathogenesis of Nephrotic Syndrome

Journal of the American Society of Nephrology ◽

10.1681/asn.2019040414 ◽

2019 ◽

Vol 30 (12) ◽

pp. 2338-2353 ◽

Cited By ~ 3

Author(s):

Lina L. Kampf ◽

Ronen Schneider ◽

Lea Gerstner ◽

Roland Thünauer ◽

Mengmeng Chen ◽

...

Keyword(s):

Nephrotic Syndrome ◽

Slit Diaphragm ◽

Missense Mutations ◽

Loss Of Function ◽

Cellular Functions ◽

Steroid Resistant ◽

Steroid Resistant Nephrotic Syndrome ◽

Whole Exome ◽

Immortalized Cell

BackgroundMutations in about 50 genes have been identified as monogenic causes of nephrotic syndrome, a frequent cause of CKD. These genes delineated the pathogenetic pathways and rendered significant insight into podocyte biology.MethodsWe used whole-exome sequencing to identify novel monogenic causes of steroid-resistant nephrotic syndrome (SRNS). We analyzed the functional significance of an SRNS-associated gene in vitro and in podocyte-like Drosophila nephrocytes.ResultsWe identified hemizygous missense mutations in the gene TBC1D8B in five families with nephrotic syndrome. Coimmunoprecipitation assays indicated interactions between TBC1D8B and active forms of RAB11. Silencing TBC1D8B in HEK293T cells increased basal autophagy and exocytosis, two cellular functions that are independently regulated by RAB11. This suggests that TBC1D8B plays a regulatory role by inhibiting endogenous RAB11. Coimmunoprecipitation assays showed TBC1D8B also interacts with the slit diaphragm protein nephrin, and colocalizes with it in immortalized cell lines. Overexpressed murine Tbc1d8b with patient-derived mutations had lower affinity for endogenous RAB11 and nephrin compared with wild-type Tbc1d8b protein. Knockdown of Tbc1d8b in Drosophila impaired function of the podocyte-like nephrocytes, and caused mistrafficking of Sns, the Drosophila ortholog of nephrin. Expression of Rab11 RNAi in nephrocytes entailed defective delivery of slit diaphragm protein to the membrane, whereas RAB11 overexpression revealed a partial phenotypic overlap to Tbc1d8b loss of function.ConclusionsNovel mutations in TBC1D8B are monogenic causes of SRNS. This gene inhibits RAB11. Our findings suggest that RAB11-dependent vesicular nephrin trafficking plays a role in the pathogenesis of nephrotic syndrome.

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Polypoid Change of the Glomerular Basement Membrane in a Child with Steroid Resistant Nephrotic Syndrome and ARHGAP24 Mutation: A Case Report

The Open Urology & Nephrology Journal ◽

10.2174/1874303x01609010088 ◽

2016 ◽

Vol 9 (1) ◽

pp. 88-93 ◽

Cited By ~ 1

Author(s):

Anna Francis ◽

John Burke ◽

Leo Francis ◽

Steven McTaggart ◽

Andrew Mallett

Keyword(s):

Nephrotic Syndrome ◽

Basement Membrane ◽

Gene Product ◽

Glomerular Basement Membrane ◽

Steroid Resistant ◽

Steroid Resistant Nephrotic Syndrome ◽

Membrane Ruffling ◽

Genes Encoding ◽

Important Player

Background: Steroid resistant nephrotic syndrome (SRNS) is increasingly recognised to have a genetic basis following the identification of a number of mutations within genes encoding podocyte and basement membrane proteins. The ARHGAP24 gene product is a recently recognised important player in podocyte interaction with the glomerular basement membrane. The ARHGAP24 gene encodes a protein involved in regulating cell motility, membrane structure and polarity. Mutations in the gene have been shown in vitro to cause cell membrane ruffling. Case Presentation: We report a novel missense mutation in exon 4 (c.[284G>A]; p.[Arg95Gln]) of the ARHGAP24 gene in a child that presented with SRNS at four years of age. Renal biopsy demonstrated unusual polypoid changes of the glomerular basement membrane (GBM). Conclusion: We propose this novel ARHGAP24 mutation as causative for SRNS associated with unusual polypoid basement membrane changes. These biopsy findings, in association with ARHGAP24 mutation and clinical nephrotic syndrome are a novel finding. This finding may advance the understanding of ARHGAP24 gene product function.

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Faculty Opinions recommendation of Tacrolimus: a new therapy for steroid-resistant nephrotic syndrome in children.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1104725.560794 ◽

2008 ◽

Author(s):

Neil Turner

Keyword(s):

Nephrotic Syndrome ◽

Steroid Resistant ◽

Steroid Resistant Nephrotic Syndrome ◽

New Therapy

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A rare cause of steroid-resistant nephrotic syndrome in a child: Answers

Pediatric Nephrology ◽

10.1007/s00467-019-04385-6 ◽

2019 ◽

Vol 35 (4) ◽

pp. 621-623

Author(s):

Lale Guliyeva ◽

Yılmaz Tabel ◽

Ali Düzova ◽

Nusret Akpolat ◽

Seza Özen ◽

...

Keyword(s):

Nephrotic Syndrome ◽

Steroid Resistant ◽

Steroid Resistant Nephrotic Syndrome

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Whole-Exome Sequencing Solved over 2-Decade Kidney Disease Enigma

Nephron ◽

10.1159/000514293 ◽

2021 ◽

pp. 1-6

Author(s):

Suramath Isaranuwatchai ◽

Ankanee Chanakul ◽

Chupong Ittiwut ◽

Chalurmpon Srichomthong ◽

Vorasuk Shotelersuk ◽

...

Keyword(s):

Nephrotic Syndrome ◽

Kidney Disease ◽

Exome Sequencing ◽

Whole Exome Sequencing ◽

Treatment Plan ◽

Unknown Etiology ◽

Pediatric Case ◽

Steroid Resistant ◽

Steroid Resistant Nephrotic Syndrome ◽

Whole Exome

Chronic kidney disease of unknown etiology (CKDu) has been a problem in renal practice as indefinite diagnosis may lead to inappropriate management. Here, we report a 54-year-old father diagnosed with CKDu at 33 years old and his 8-year-old son with steroid-resistant nephrotic syndrome. Using whole-exome sequencing, both were found to be heterozygous for c.737G>A (p.Arg246Gln) in LMX1B. The diagnosis of LMX1B-associated nephropathy has led to changes in the treatment plan with appropriate genetic counseling. The previously reported cases with this particular mutation were also reviewed. Most children with LMX1B-associated nephropathy had nonnephrotic proteinuria with normal renal function. Interestingly, our pediatric case presented with steroid-resistant nephrotic syndrome at 8 years old and progressed to ESRD requiring peritoneal dialysis at the age of 15 years. Our report emphasized the need of genetic testing in CKDu for definite diagnosis leading to precise management.

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