Loss of Epithelial Membrane Protein 2 Aggravates Podocyte Injury via Upregulation of Caveolin-1

Abstract

Nephrotic syndrome is a CKD defined by proteinuria with subsequent hypoalbuminemia, hyperlipidemia, and edema caused by impaired renal glomerular filtration barrier function. We previously identified mutations in epithelial membrane protein 2 (EMP2) as a monogenic cause of this disease. Here, we generated an emp2-knockout zebrafish model using transcription activator-like effector nuclease-based genome editing. We found that loss of emp2 in zebrafish upregulated caveolin-1 (cav1), a major component of caveolae, in embryos and adult mesonephric glomeruli and exacerbated podocyte injury. This phenotype was partially rescued by glucocorticoids. Furthermore, overexpression of cav1 in zebrafish podocytes was sufficient to induce the same phenotype observed in emp2 homozygous mutants, which was also treatable with glucocorticoids. Similarly, knockdown of EMP2 in cultured human podocytes resulted in increased CAV1 expression and decreased podocyte survival in the presence of puromycin aminonucleoside, whereas glucocorticoid treatment ameliorated this phenotype. Taken together, we have established excessive CAV1 as a mediator of the predisposition to podocyte injury because of loss of EMP2, suggesting CAV1 could be a novel therapeutic target in nephrotic syndrome and podocyte injury. Nephrotic syndrome (NS) is a CKD defined by nephrotic-range proteinuria caused by disruption of the renal glomerular filtration barrier, resulting in hypoalbuminemia, hyperlipidemia, and edema. Childhood NS, if untreated, is associated with increased risk of life-threatening infections, thromboembolism, lipid abnormalities, and malnutrition. 1 NS is classified by response or nonresponse to a standardized glucocorticosteroid therapy as steroid-sensitive nephrotic syndrome (SSNS) and steroid-resistant nephrotic syndrome (SRNS), respectively. Recent studies of SRNS have identified monogenic causes of SRNS to reside in podocytespecific genes, which if mutated, lead to podocyte dysfunction and defective glomerular filtration barrier. 2 Using the combined approach of homozygosity mapping and whole-exome sequencing, we have previously identified mutations in epithelial membrane protein 2 (EMP2) as a novel monogenic cause for NS. 3 EMP2 was initially identified by its homology with the growth-arrest-specific 3/peripheral myelin protein 22 family 4 and negatively regulates the mRNA and protein levels of CAV1, a major protein component of plasma membrane microdomain caveolae. 5,6 To explore the pathogenic mechanism underlying EMP2 mutations, we have now generated emp2

Advillin acts upstream of phospholipase C ϵ1 in steroid-resistant nephrotic syndrome

“…However, virtually the entire literature on zebrafish models for NS has been based on morpholino oligonucleotide (MO) knockdown in zebrafish larvae. This includes the genes PLCE1 (19), ADCK4 (28), ARHGDIA (8), and EMP2 (29). And it is of note that the approach of morpholino-based knockdown of genes in zebrafish disease models has been recently questioned in a major way, because these data were not reproducible in mutant stable lines (30).…”

Dosage-dependent role of Rac1 in podocyte injury

“…The podocyte-specific Gal4 driver lines Tg(pod:Gal4) were generated as previously reported (29). RAC1 Q61L and RAC1 T17N , the constitutively active form (CA-RAC1) and the dominant negative form (DN-RAC1) of the human RAC1 gene, were amplified from plasmids obtained from Addgene (12981, 12982) and cloned into pME-MCS.…”

“…The transgenic constructs Tol2-UAS:CA-RAC1/DN-RAC1/ EGFP-CA-rac1/EGFP-DN-rac1 were assembled using the multisite Gateway method (17). Tol2-pod:CreERT2 was created by replacing Gal4 with CreERT2 in the Tol2-pod:Gal4 construct (29). Each transgenic construct was coinjected with the in vitro synthesized capped RNA of the Tol2 transposase into one-cell-stage embryos.…”

“…Tg(pod: Gal4;UAS:NfsB-mCherry;UAS:RAC1,cmlc2:EGFP) were sorted out at 3 days postfertilization (dpf) based on fluorescence in the pronephros (red) and the heart (green). Thirty larvae were pooled as a cohort and subjected to metronidazole (MTZ) treatment for 48 h. Periorbital edema (POE) was characterized by the ratio of pupil spacing distance vs. body length as described previously (29). All experiments were repeated independently three times, and statistical analyses were performed based on Student's t-test.…”

“…To test whether Rac1 activation could lead to podocyte injury in zebrafish, we generated transgenic zebrafish that express either constitutively active (CA-RAC1) or dominant negative (DN-RAC1) human RAC1 (11) under the control of the UAS promoter. When combined with a podocyte-specific Gal4-driver transgenic line (29), these transgenic fish expressed either CA-RAC1 or DN-RAC1 specifically in the podocytes of pronephric glomeruli (Fig. 2, A and B).…”