Ehtesham Arif

Motor Protein Myo1c Is a Podocyte Protein That Facilitates the Transport of Slit Diaphragm Protein Neph1 to the Podocyte Membrane

ABSTRACT The podocyte proteins Neph1 and nephrin organize a signaling complex at the podocyte cell membrane that forms the structural framework for a functional glomerular filtration barrier. Mechanisms regulating the movement of these proteins to and from the membrane are currently unknown. This study identifies a novel interaction between Neph1 and the motor protein Myo1c, where Myo1c plays an active role in targeting Neph1 to the podocyte cell membrane. Using in vivo and in vitro experiments, we provide data supporting a direct interaction between Neph1 and Myo1c which is dynamic and actin dependent. Unlike wild-type Myo1c, the membrane localization of Neph1 was significantly reduced in podocytes expressing dominant negative Myo1c. In addition, Neph1 failed to localize at the podocyte cell membrane and cell junctions in Myo1c-depleted podocytes. We further demonstrate that similarly to Neph1, Myo1c also binds nephrin and reduces its localization at the podocyte cell membrane. A functional analysis of Myo1c knockdown cells showed defects in cell migration, as determined by a wound assay. In addition, the ability to form tight junctions was impaired in Myo1c knockdown cells, as determined by transepithelial electric resistance (TER) and bovine serum albumin (BSA) permeability assays. These results identify a novel Myo1c-dependent molecular mechanism that mediates the dynamic organization of Neph1 and nephrin at the slit diaphragm and is critical for podocyte function.

Slit Diaphragm Protein Neph1 and Its Signaling A NOVEL THERAPEUTIC TARGET FOR PROTECTION OF PODOCYTES AGAINST GLOMERULAR INJURY

Background: Neph1 is a podocyte protein that is critical for maintaining renal function. Results: Inhibiting Neph1 signaling preserves podocyte structure and function in response to glomerular injury-inducing agents. Conclusion: Maintaining a robust expression of Neph1 at the podocyte cell membrane protects podocytes from renal injury. Significance: This is the first report demonstrating that Neph1 signaling is a therapeutic target for preventing podocyte damage. Podocytes are specialized epithelial cells that are critical components of the glomerular filtration barrier, and their dysfunction leads to proteinuria and renal failure. Therefore, preserving podocyte function is therapeutically significant. In this study, we identified Neph1 signaling as a therapeutic target that upon inhibition prevented podocyte damage from a glomerular injury-inducing agent puromycin aminonucleoside (PAN). To specifically inhibit Neph1 signaling, we used a protein transduction approach, where the cytoplasmic domain of Neph1 (Neph1CD) tagged with a protein transduction domain trans-activator of transcription was transduced in cultured podocytes prior to treatment with PAN. The PAN-induced Neph1 phosphorylation was significantly reduced in Neph1CD-transduced cells; in addition, these cells were resistant to PAN-induced cytoskeletal damage. The biochemical analysis using subfractionation studies showed that unlike control cells Neph1 was retained in the lipid raft fractions in the transduced cells following treatment with PAN, indicating that transduction of Neph1CD in podocytes prevented PAN-induced mislocalization of Neph1. In accordance, the immunofluorescence analysis further suggested that Neph1CD-transduced cells had increased ability to retain endogenous Neph1 at the membrane in response to PAN-induced injury. Similar results were obtained when angiotensin was used as an injury-inducing agent. Consistent with these observations, maintaining high levels of Neph1 at the membrane using a podocyte cell line overexpressing chimeric Neph1 increased the ability of podocytes to resist PAN-induced injury and PAN-induced albumin leakage. Using a zebrafish in vivo PAN and adriamycin injury models, we further demonstrated the ability of transduced Neph1CD to preserve glomerular function. Collectively, these results support the conclusion that inhibiting Neph1 signaling is therapeutically significant in preventing podocyte damage from glomerular injury.

Myo1c is an unconventional myosin required for zebrafish glomerular development

The targeting and organization of podocyte slit diaphragm proteins nephrin and neph1 is critical for development and maintenance of a functional glomerular filtration barrier. Myo1c is a non-muscle myosin motor protein that interacts directly with nephrin and neph1 and mediates their intracellular transport to the podocyte intercellular junction. Here we investigated the necessity of Myo1c in podocyte development using zebrafish as a model system. Immunofluorescence microscopy and in situ RNA hybridization analysis of zebrafish embryos showed that Myo1c is widely expressed in various tissues including the zebrafish glomerulus. Knockdown of the Myo1c gene in zebrafish using antisense morpholino derivatives resulted in an abnormal developmental phenotype that included pericardial edema and dilated renal tubules. Ultra-structural analysis of the glomerulus in Myo1c depleted zebrafish showed abnormal podocyte morphology and absence of the slit diaphragm. Consistent with these observations, the glomerular filter permeability appeared altered in zebrafish in which Myo1c expression was attenuated. The specificity of Myo1c knockdown was confirmed by a rescue experiment in which co-injection of Myo1c morpholino derivatives with orthologous Myo1c mRNA prepared from mouse cDNA lessened phenotypic abnormalities including edema in Myo1c morphants. Thus, our results demonstrate that Myo1c is necessary for podocyte morphogenesis.

Multi-locus interactions of vascular homeostasis genes in essential hypertension: A gender-based study

BACKGROUND Studies on genes of endothelial and vascular homeostasis are inadequate in females. METHODS We investigated the role of 7 variants of ACE, AGT and NOS3 and their correlation with NO(x) levels and ACE activity in hypertension susceptibility in 910 case-controls of both genders. RESULTS Prevalence of alleles D of ACE I/D; -6A of AGT -6G/A; -786C, 894T and 4a of NOS3 -786T/C, 894G/T and 4b/4a polymorphisms was observed in patients (P< or =0.05). The 3 genotypes-combinations containing 6+5 wild-type alleles of AGT and NOS3 were significantly less prevalent in patients (P< or =0.0003). The haplotypes 235T/174T/-6A of AGT (P=4E-3) and -786T/894G/4a and -786C/894G/4a of NOS3 (P=2E-3, P=0.011, respectively) were significantly more prevalent in patients. The AGT and NOS3 findings were similar in males. Genotypes-combinations with 6+5 wild-type alleles of AGT correlated with higher NO(x) levels (P=0.03). The NOS3 genotypes-combinations having 6 and 6+5 wild-type alleles correlated with decreased ACE activity (P=0.025, P=0.0015, respectively) and increased NO(x) levels (P=0.001, P=0.0001, respectively) in patients. In gene-gene interactions, ACE D allele associated with < or =4 wild-type alleles containing genotypes-combinations of AGT and NOS3 in patients (P< or =0.04). CONCLUSION Within gene and between genes interactions of variants influence ACE activity and NO(x) levels and associate with EH.

Expression and functional activity of pro-oxidants and antioxidants in murine heart exposed to acute hypobaric hypoxia

Under hypobaric hypoxia, antioxidant defenses of the heart are stressed by the enhanced production of ROS. Mammalian heart acclimatizes to hypoxia through altered gene expression, which we studied in murine heart exposed to 10 h of acute hypobaric hypoxia (AHH), equivalent to 15 000 ft, using cDNA arrays. Functional classification of genes with a ⩾2‐fold change revealed a number of pro‐oxidants like Cyba, Xdh, Txnip, Ppp1r15b and antioxidants like Cat, Gpx1, Mt1, Mgst1. Interestingly, the protein level of Cyba, a subunit of NADPH oxidase, was markedly decreased in AHH exposed heart, suggesting the involvement of some stress response pathways. The AHH exposure also caused a significant reduction (50%) in the level of GSH (P < 0.05). The present study provides a retrospective insight on the cellular antioxidant defense mechanisms under AHH.

Solution Structure Analysis of Cytoplasmic Domain of Podocyte Protein Neph1 Using Small/Wide Angle X-ray Scattering (SWAXS)

Background: Solution structure of the cytoplasmic domain of a podocyte protein Neph1 will provide functional insight into the Neph1 molecule. Results: A structural model of the Neph1-CD and its complex with ZO-1-PDZ1 was generated, and the interacting sites were mapped. Conclusion: Neph1-CD adopts a global shape in solution, and its interaction with ZO-1 involves multiple sites. Significance: This study advances our understanding of the molecular network of podocyte proteins in three dimensions. Neph1 is present in podocytes, where it plays a critical role in maintaining the filtration function of the glomerulus, in part through signaling events mediated by its cytoplasmic domain that are involved in actin cytoskeleton organization. To understand the function of this protein, a detailed knowledge of the structure of the Neph1 cytoplasmic domain (Neph1-CD) is required. In this study, the solution structure of this domain was determined by small/wide angle x-ray scattering (SWAXS). Analysis of Neph1-CD by SWAXS suggested that this protein adopts a global shape with a radius of gyration and a maximum linear dimension of 21.3 and 70 Å, respectively. These parameters and the data from circular dichroism experiments were used to construct a structural model of this protein. The His-ZO-1-PDZ1 (first PDZ domain of zonula occludens) domain that binds Neph1-CD was also analyzed by SWAXS, to confirm that it adopts a global structure similar to its crystal structure. We used the SWAXS intensity profile, the structural model of Neph1-CD, and the crystal structure of ZO-1-PDZ1 to construct a structural model of the Neph1-CD·ZO-1-PDZ1 complex. Mapping of the intermolecular interactions suggested that in addition to the C-terminal residues Thr-His-Val, residues Lys-761 and Tyr-762 in Neph1 are also critical for stabilizing the complex. Estimated intensity values from the SWAXS data and in vivo and in vitro pull-down experiments demonstrated loss of binding to ZO-1 when these residues were individually mutated to alanines. Our findings present a structural model that provides novel insights into the molecular structure and function of Neph1-CD.

Structural Analysis of the Myo1c and Neph1 Complex Provides Insight into the Intracellular Movement of Neph1

ABSTRACT The Myo1c motor functions as a cargo transporter supporting various cellular events, including vesicular trafficking, cell migration, and stereociliary movements of hair cells. Although its partial crystal structures were recently described, the structural details of its interaction with cargo proteins remain unknown. This study presents the first structural demonstration of a cargo protein, Neph1, attached to Myo1c, providing novel insights into the role of Myo1c in intracellular movements of this critical slit diaphragm protein. Using small angle X-ray scattering studies, models of predominant solution conformation of unliganded full-length Myo1c and Myo1c bound to Neph1 were constructed. The resulting structures show an extended S-shaped Myo1c with Neph1 attached to its C-terminal tail. Importantly, binding of Neph1 did not induce a significant shape change in Myo1c, indicating this as a spontaneous process or event. Analysis of interaction surfaces led to the identification of a critical residue in Neph1 involved in binding to Myo1c. Indeed, a point mutant from this site abolished interaction between Neph1 and Myo1c when tested in the in vitro and in live-cell binding assays. Live-cell imaging, including fluorescence recovery after photobleaching, provided further support for the role of Myo1c in intracellular vesicular movement of Neph1 and its turnover at the membrane.

A Novel CLCN5 Mutation Associated With Focal Segmental Glomerulosclerosis and Podocyte Injury

Introduction Tubular dysfunction is characteristic of Dent’s disease; however, focal segmental glomerulosclerosis (FSGS) can also be present. Glomerulosclerosis could be secondary to tubular injury, but it remains uncertain whether the CLCN5 gene, which encodes an endosomal chloride and/or hydrogen exchanger, plays a role in podocyte biology. Here, we implicate a role for CLCN5 in podocyte function and pathophysiology. Methods Whole exome capture and sequencing of the proband and 5 maternally-related family members was conducted to identify X-linked mutations associated with biopsy-proven FSGS. Human podocyte cultures were used to characterize the mutant phenotype on podocyte function. Results We identified a novel mutation (L521F) in CLCN5 in 2 members of a Hispanic family who presented with a histologic diagnosis of FSGS and low-molecular-weight proteinuria without hypercalciuria. Presence of CLCN5 was confirmed in cultured human podocytes. Podocytes transfected with the wild-type or the mutant (L521F) CLCN5 constructs showed differential localization. CLCN5 knockdown in podocytes resulted in defective transferrin endocytosis and was associated with decreased cell proliferation and increased cell migration, which are hallmarks of podocyte injury. Conclusions The CLCN5 mutation, which causes Dent’s disease, may be associated with FSGS without hyercalcuria and nepthrolithiasis. The present findings supported the hypothesis that CLCN5 participates in protein trafficking in podocytes and plays a critical role in organizing the components of the podocyte slit diaphragm to help maintain normal cell physiology and a functional filtration barrier. In addition to tubular dysfunction, mutations in CLCN5 may also lead to podocyte dysfunction, which results in a histologic picture of FSGS that may be a primary event and not a consequence of tubular damage.

High-content screening assay-based discovery of paullones as novel podocyte-protective agents

Podocyte dysfunction and loss is an early event and a hallmark of proteinuric kidney diseases. A podocytes normal function is maintained via its unique cellular architecture that relies on an intracellular network of filaments, including filamentous actin (F-actin) and microtubules, that provides mechanical support. Damage to this filamentous network leads to changes in cellular morphology and results in podocyte injury, dysfunction, and death. Conversely, stabilization of this network protects podocytes and ameliorates proteinuria. This suggests that stabilization of podocyte architecture via its filamentous network could be a key therapeutic strategy for proteinuric kidney diseases. However, development of podocyte-directed therapeutics, especially those that target the cells filamentous network, is still lacking, partly because of unavailability of appropriate cellular assays for use in a drug discovery environment. Here, we describe a new high-content screening-based methodology and its implementation on podocytes to identify paullone derivatives as a novel group of podocyte-protective compounds. We find that three compounds, i.e., kenpaullone, 1-azakenpaullone, and alsterpaullone, dose dependently protect podocytes from puromycin aminonucleoside (PAN)-mediated injury in vitro by reducing PAN-induced changes in both the filamentous actin and microtubules, with alsterpaullone providing maximal protection. Mechanistic studies further show that alsterpaullone suppressed PAN-induced activation of signaling downstream of GSK3β and p38 mitogen-activated protein kinase. In vivo it reduced ADR-induced glomerular injury in a zebrafish model. Together, these results identify paullone derivatives as novel podocyte-protective agents for future therapeutic development.

Targeting Neph1 and ZO-1 protein-protein interaction in podocytes prevents podocyte injury and preserves glomerular filtration function

Targeting protein-protein interaction (PPI) is rapidly becoming an attractive alternative for drug development. While drug development commonly involves inhibiting a PPI, in this study, we show that stabilizing PPI may also be therapeutically beneficial. Junctional proteins Neph1 and ZO-1 and their interaction is an important determinant of the structural integrity of slit diaphragm, which is a critical component of kidney’s filtration system. Since injury induces loss of this interaction, we hypothesized that strengthening this interaction may protect kidney’s filtration barrier and preserve kidney function. In this study, Neph1-ZO-1 structural complex was screened for the presence of small druggable pockets formed from contributions from both proteins. One such pocket was identified and screened using a small molecule library. Isodesmosine (ISD) a rare naturally occurring amino acid and a biomarker for pulmonary arterial hypertension was selected as the best candidate and to establish the proof of concept, its ability to enhance Neph1-CD and ZO-1 binding was tested. Results from biochemical binding analysis showed that ISD enhanced Neph1 and ZO-1 interaction under in vitro and in vivo conditions. Importantly, ISD treated podocytes were resistant to injury-induced loss of transepithelial permeability. Finally, mouse and zebrafish studies show that ISD protects from injury-induced renal damage.