Genetic ablation of SLK exacerbates glomerular injury in adriamycin nephrosis in mice

2020 ◽  
Vol 318 (6) ◽  
pp. F1377-F1390 ◽  
Author(s):  
Boyan Woychyshyn ◽  
Joan Papillon ◽  
Julie Guillemette ◽  
José R. Navarro-Betancourt ◽  
Andrey V. Cybulsky
Keyword(s):  
Apical Membrane ◽  
Glomerular Injury ◽  
Podocyte Injury ◽  
Genetic Ablation ◽  
Apical Domain ◽  
Glomerular Epithelial Cells ◽  
The Matrix ◽  
Muscle Homeostasis ◽  
Specific Deletion

Ste20-like kinase SLK is critical for embryonic development and may play an important role in wound healing, muscle homeostasis, cell migration, and tumor growth. Mice with podocyte-specific deletion of SLK show albuminuria and damage to podocytes as they age. The present study addressed the role of SLK in glomerular injury. We induced adriamycin nephrosis in 3- to 4-mo-old control and podocyte SLK knockout (KO) mice. Compared with control, SLK deletion exacerbated albuminuria and loss of podocytes, synaptopodin, and podocalyxin. Glomeruli of adriamycin-treated SLK KO mice showed diffuse increases in the matrix and sclerosis as well as collapse of the actin cytoskeleton. SLK can phosphorylate ezrin. The complex of phospho-ezrin, Na+/H+ exchanger regulatory factor 2, and podocalyxin in the apical domain of the podocyte is a key determinant of normal podocyte architecture. Deletion of SLK reduced glomerular ezrin and ezrin phosphorylation in adriamycin nephrosis. Also, deletion of SLK reduced the colocalization of ezrin and podocalyxin in the glomerulus. Cultured glomerular epithelial cells with KO of SLK showed reduced ezrin phosphorylation and podocalyxin expression as well as reduced F-actin. Thus, SLK deletion leads to podocyte injury as mice age and exacerbates injury in adriamycin nephrosis. The mechanism may at least in part involve ezrin phosphorylation as well as disruption of the cytoskeleton and podocyte apical membrane structure.

scholarly journals Characterization of the composition and functioning of the Crumbs complex in C. elegans

2021 ◽  
Author(s):  
Victoria G Castiglioni ◽  
Joao J Ramalho ◽  
Jason R Kroll ◽  
Riccardo Stucchi ◽  
Hanna van Beuzekom ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Apical Membrane ◽  
Scaffolding Protein ◽  
Animal Development ◽  
C Elegans ◽  
Apical Domain ◽  
Junction Protein ◽  
Crumbs Complex

The apical domain of epithelial cells can acquire a diverse array of morphologies and functions, which is critical for the function of epithelial tissues. The Crumbs proteins are evolutionary conserved transmembrane proteins with essential roles in promoting apical domain formation in epithelial cells. The short intracellular tail of Crumbs proteins interacts with a variety of proteins, including the scaffolding protein Pals1 (protein associated with LIN7, Stardust in Drosophila). Pals1 in turn binds to a second scaffolding protein termed PATJ (Pals1-associated tight junction protein), to form the core Crumbs/ Pals1/PATJ Crumbs complex. While essential roles in epithelial organization have been shown for Crumbs proteins in Drosophila and mammalian systems, the three Caenorhabditis elegans crumbs genes are dispensable for epithelial polarization and animal development. Moreover, the presence and functioning of orthologs of Pals1 and PATJ has not been investigated. Here, we identify MAGU-2 and MPZ-1 as the C. elegans orthologs of Pals1 and PATJ, respectively. We show that MAGU-2 interacts with all three Crumbs proteins as well as MPZ-1, and localizes to the apical membrane domain in a Crumbs-dependent fashion. Similar to crumbs mutants, a magu-2 null mutant shows no developmental or epithelial polarity defects. Finally, we show that overexpression of the Crumbs proteins EAT-20 or CRB-3 in the C. elegans intestine can lead to apical membrane expansion. Our results shed light into the composition of the C. elegans Crumbs complex and indicate that the role of Crumbs proteins in promoting apical domain identity is conserved.

IL-1 receptor signaling in podocytes limits susceptibility to glomerular damage

2021 ◽  
Author(s):  
Jiafa Ren ◽  
Xiaohan Lu ◽  
Gentzon Hall ◽  
Jamie R Privratsky ◽  
Matthew J Robson ◽  
...  
Keyword(s):  
Kidney Injury ◽  
Protective Role ◽  
Glomerular Disease ◽  
Wild Type ◽  
Glomerular Injury ◽  
Podocyte Injury ◽  
Akt Activation ◽  
Injury Models ◽  
Dependent Signaling Pathway

IL-1 receptor (IL-1R1) activation triggers a proinflammatory signaling cascade that can exacerbate kidney injury. However, the functions of the podocyte IL-1R1 in glomerular disease remain unclear. To study the role of IL-1R1 signaling in podocytes, we selectively ablated the podocyte IL-1R1 in mice (PKO). We then subjected PKO mice and wild-type (WT) controls to 2 glomerular injury models: nephrotoxic serum (NTS)- and adriamycin (ADR)-induced nephropathy. Surprisingly, we found IL-1R1 activation in podocytes limited albuminuria and podocyte injury during NTS- and ADR-induced nephropathy. Moreover, deletion of IL-1R1 in podocytes drove podocyte apoptosis and glomerular injury through diminishing Akt activation. Activation of Akt signaling abrogated the differences in albuminuria and podocyte injury between WT and PKO mice during NTS. Thus, IL-1R1 signaling in podocytes limits susceptibility to glomerular injury via an Akt-dependent signaling pathway. These data identify an unexpected protective role for IL-1R1 signaling in podocytes in the pathogenesis of glomerular disease.

Membrane repolarization is delayed in proximal tubules after ischemia-reperfusion: possible role of microtubule-organizing centers

2003 ◽  
Vol 285 (2) ◽  
pp. F230-F240 ◽  
Author(s):  
Flavia A. Wald ◽  
Yolanda Figueroa ◽  
Andrea S. Oriolo ◽  
Pedro J. I. Salas
Keyword(s):  
Apical Membrane ◽  
Rat Kidney ◽  
Ischemia Reperfusion ◽  
Proximal Tubules ◽  
Microtubule Organizing Centers ◽  
Apical Domain ◽  
Brush Borders ◽  
Chemical Anoxia

We have previously shown that microtubule-organizing centers (MTOCs) attach to the apical network of intermediate filaments (IFs) in epithelial cells in culture and in epithelia in vivo. Because that attachment is important for the architecture of microtubules (MTs) in epithelia, we analyzed whether chemical anoxia in LLC-PK1 and CACO-2 cells or unilateral ischemia-reperfusion in rat kidney (performed under fluorane anesthesia) had an effect on the binding and distribution of MTOCs. In culture, we found that chemical anoxia induces MTOC detachment from IFs by morphological and biochemical criteria. In reperfused rat proximal tubules, noncentrosomal MTOCs were fully detached from the cytoskeleton and scattered throughout the cytoplasm at 3 days after reperfusion, when brush borders were mostly reassembled. At that time, MTs were also fully reassembled but, as expected, lacked their normal apicobasal orientation. Two apical membrane markers expressed in S2 and S3 segments were depolarized at the same stage. At 8 days after reperfusion, membrane polarity, MTOCs, and MTs were back to normal. Na+-K+-ATPase was also found redistributed, not to the apical domain but rather to an intracellular compartment, as described by others (Alejandro VS, Nelson W, Huie P, Sibley RK, Dafoe D, Kuo P, Scandling JD Jr., and Myers BD. Kidney Int 48: 1308–1315, 1995). The prolonged depolarization of the apical membrane may have implications in the pathophysiology of acute renal failure.

scholarly journals Genetic Ablation of Calcium-independent Phospholipase A2γ Exacerbates Glomerular Injury in Adriamycin Nephrosis in Mice

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Hanan Elimam ◽  
Joan Papillon ◽  
Julie Guillemette ◽  
José R. Navarro-Betancourt ◽  
Andrey V. Cybulsky
Keyword(s):  
Oxygen Consumption ◽  
Epithelial Cells ◽  
Mitochondrial Dysfunction ◽  
Oxygen Consumption Rate ◽  
Energy Homeostasis ◽  
Consumption Rate ◽  
Amp Kinase ◽  
Glomerular Injury ◽  
Genetic Ablation

Abstract Genetic ablation of calcium-independent phospholipase A2γ (iPLA2γ) in mice results in marked damage of mitochondria and enhanced autophagy in glomerular visceral epithelial cells (GECs) or podocytes. The present study addresses the role of iPLA2γ in glomerular injury. In adriamycin nephrosis, deletion of iPLA2γ exacerbated albuminuria and reduced podocyte number. Glomerular LC3-II increased and p62 decreased in adriamycin-treated iPLA2γ knockout (KO) mice, compared with treated control, in keeping with increased autophagy in KO. iPLA2γ KO GECs in culture also demonstrated increased autophagy, compared with control GECs. iPLA2γ KO GECs showed a reduced oxygen consumption rate and increased phosphorylation of AMP kinase (pAMPK), consistent with mitochondrial dysfunction. Adriamycin further stimulated pAMPK and autophagy. After co-transfection of GECs with mito-YFP (to label mitochondria) and RFP-LC3 (to label autophagosomes), or RFP-LAMP1 (to label lysosomes), there was greater colocalization of mito-YFP with RFP-LC3-II and with RFP-LAMP1 in iPLA2γ KO GECs, compared with WT, indicating enhanced mitophagy in KO. Adriamycin increased mitophagy in WT cells. Thus, iPLA2γ has a cytoprotective function in the normal glomerulus and in glomerulopathy, as deletion of iPLA2γ leads to mitochondrial damage and impaired energy homeostasis, as well as autophagy and mitophagy.

scholarly journals Role of IRE1α in podocyte proteostasis and mitochondrial health

2020 ◽  
Vol 6 (1) ◽  
Author(s):  
José R. Navarro-Betancourt ◽  
Joan Papillon ◽  
Julie Guillemette ◽  
Takao Iwawaki ◽  
Chen-Fang Chung ◽  
...  
Keyword(s):  
Mechanistic Studies ◽  
Mitochondrial Ultrastructure ◽  
Glomerular Diseases ◽  
Podocyte Injury ◽  
Glomerular Epithelial Cell ◽  
Unfolded Protein ◽  
The Unfolded Protein Response ◽  
Protein Response ◽  
Specific Deletion

AbstractGlomerular epithelial cell (GEC)/podocyte proteostasis is dysregulated in glomerular diseases. The unfolded protein response (UPR) is an adaptive pathway in the endoplasmic reticulum (ER) that upregulates proteostasis resources. This study characterizes mechanisms by which inositol requiring enzyme-1α (IRE1α), a UPR transducer, regulates proteostasis in GECs. Mice with podocyte-specific deletion of IRE1α (IRE1α KO) were produced and nephrosis was induced with adriamycin. Compared with control, IRE1α KO mice had greater albuminuria. Adriamycin increased glomerular ER chaperones in control mice, but this upregulation was impaired in IRE1α KO mice. Likewise, autophagy was blunted in adriamycin-treated IRE1α KO animals, evidenced by reduced LC3-II and increased p62. Mitochondrial ultrastructure was markedly disrupted in podocytes of adriamycin-treated IRE1α KO mice. To pursue mechanistic studies, GECs were cultured from glomeruli of IRE1α flox/flox mice and IRE1α was deleted by Cre–lox recombination. In GECs incubated with tunicamycin, deletion of IRE1α attenuated upregulation of ER chaperones, LC3 lipidation, and LC3 transcription, compared with control GECs. Deletion of IRE1α decreased maximal and ATP-linked oxygen consumption, as well as mitochondrial membrane potential. In summary, stress-induced chaperone production, autophagy, and mitochondrial health are compromised by deletion of IRE1α. The IRE1α pathway is cytoprotective in glomerular disease associated with podocyte injury and ER stress.

scholarly journals Proteostasis as a therapeutic target in glomerular injury associated with mutant α-actinin-4

2018 ◽  
Vol 315 (4) ◽  
pp. F954-F966 ◽  
Author(s):  
Albert Yee ◽  
Joan Papillon ◽  
Julie Guillemette ◽  
Daniel R. Kaufman ◽  
Chris R. J. Kennedy ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Er Stress ◽  
Glomerular Injury ◽  
Glomerular Diseases ◽  
Podocyte Injury ◽  
Chemical Chaperone ◽  
Accelerated Degradation ◽  
Glomerular Epithelial Cells ◽  
Enhanced Expression ◽  
Tight Association

Mutations in α-actinin-4 (actinin-4) result in hereditary focal segmental glomerulosclerosis (FSGS) in humans. Actinin-4 mutants induce podocyte injury because of dysregulation of the cytoskeleton and proteotoxicity. Injury may be associated with endoplasmic reticulum (ER) stress and polyubiquitination of proteins. We assessed if the chemical chaperone 4-phenylbutyrate (4-PBA) can ameliorate the proteotoxicity of an actinin-4 mutant. Actinin-4 K255E, which causes FSGS in humans (K256E in the mouse), showed enhanced ubiquitination, accelerated degradation, aggregate formation, and enhanced association with filamentous (F)-actin in glomerular epithelial cells (GECs). The mutant disrupted ER function and stimulated autophagy. 4-PBA reduced actinin-4 K256E aggregation and its tight association with F-actin. Transgenic mice that express actinin-4 K256E in podocytes develop podocyte injury, proteinuria, and FSGS in association with glomerular ER stress. Treatment of these mice with 4-PBA in the drinking water over a 10-wk period significantly reduced albuminuria and ER stress. Another drug, celastrol, which enhanced expression of ER and cytosolic chaperones in GECs, tended to reduce actinin-4 aggregation but did not decrease the tight association of actinin-4 K256E with F-actin and did not reduce albuminuria in actinin-4 K256E transgenic mice. Thus, chemical chaperones, such as 4-PBA, may represent a novel therapeutic approach to certain hereditary glomerular diseases.

scholarly journals AMP-Kinase mediates regulation of glomerular volume and podocyte survival

2021 ◽  
Author(s):  
Khadija Banu ◽  
Qisheng Lin ◽  
John M Basgen ◽  
Marina Planoutene ◽  
Chengguo Wei ◽  
...  
Keyword(s):  
Protective Role ◽  
Negative Regulator ◽  
Amp Kinase ◽  
Glomerular Injury ◽  
Podocyte Injury ◽  
Kinase Activation ◽  
Glomerular Volume ◽  
Foot Process ◽  
Injury Models

We reported that Shroom3 knockdown, via Fyn inhibition, induced albuminuria with foot process effacement (FPE) without glomerulosclerosis (FSGS) or podocytopenia. Interestingly, knockdown mice had reduced podocyte volumes. Human minimal change disease, where podocyte Fyn inactivation was reported, also showed lower glomerular volumes than FSGS. We hypothesized that lower glomerular volume prevented the progression to podocytopenia. To test this hypothesis, we utilized unilateral- and 5/6th nephrectomy models in Shroom3 knockdown mice. Knockdown mice exhibited lower glomerular volume, and less glomerular and podocyte hypertrophy after nephrectomy. FYN-knockdown podocytes had similar reductions in podocyte volume, implying Fyn was downstream of Shroom3. Using SHROOM3- or FYN-knockdown, we confirmed reduced podocyte protein content, along with significantly increased phosphorylated AMP-kinase, a negative regulator of anabolism. AMP-Kinase activation resulted from increased cytoplasmic redistribution of LKB1 in podocytes. Inhibition of AMP-Kinase abolished the reduction in glomerular volume and induced podocytopenia in mice with FPE, suggesting a protective role for AMP-Kinase activation. In agreement with this, treatment of glomerular injury models with AMP-Kinase activators restricted glomerular volume, podocytopenia and progression to FSGS. In summary, we demonstrate the important role of AMP-Kinase in glomerular volume regulation and podocyte survival. Our data suggest that AMP-Kinase activation adaptively regulates glomerular volume to prevent podocytopenia in the context of podocyte injury.

scholarly journals Role of miRNA-671-5p in Mediating Wnt/β-Catenin-Triggered Podocyte Injury

2022 ◽  
Vol 12 ◽  
Author(s):  
Chunhong Wang ◽  
Jiafeng Liu ◽  
Xiaoyao Zhang ◽  
Qiyan Chen ◽  
Xiaoyan Bai ◽  
...  
Keyword(s):  
Wilms Tumor ◽  
Luciferase Reporter ◽  
Glomerular Injury ◽  
Bioinformatics Analyses ◽  
Podocyte Injury ◽  
Specific Proteins ◽  
Underlying Mechanisms ◽  
End Stage

Podocyte injury and proteinuria are the most common features of glomerular disease, which is the leading cause of end-stage renal failure. Hyperactivated Wnt/β-catenin signaling is closely associated with podocyte injury, but the underlying mechanisms are incompletely understood. Here we show that miRNA-671-5p (miR-671-5p) plays a crucial role in mediating β-catenin-triggered podocyte injury by targeting Wilms tumor 1 (WT1). Microarray-based expression profiling revealed that miR-671-5p was the most upregulated miRNA in podocytes after β-catenin activation. MiR-671-5p was colocalized with β-catenin in the glomeruli of proteinuric CKD in vivo. Bioinformatics analyses and luciferase reporter assays confirmed that miR-671-5p targeted WT1 mRNA. Overexpression of miR-671-5p mimics inhibited WT1 and impaired podocyte integrity, whereas miR-671-5p antagomir preserved the expression of WT1 and other podocyte-specific proteins under basal conditions or after β-catenin activation. In mouse remnant kidney model, overexpression of miR-671-5p aggravated podocyte injury, worsened kidney dysfunction and exacerbated renal fibrosis after 5/6 nephrectomy. In contrast, miR-671-5p antagomir alleviated podocyte injury and attenuated proteinuria and renal fibrotic lesions after glomerular injury in vivo. These studies underscore a pivotal role of miR-671-5p in mediating WT1 depletion and podocyte injury induced by β-catenin. Targeting miR-671-5p may serve as a new approach to prevent podocyte injury and proteinuria in proteinuric CKD.

scholarly journals Cdc42 negatively regulates endocytosis during apical membrane maintenance in live animals

2019 ◽  
Vol 30 (3) ◽  
pp. 324-332 ◽  
Author(s):  
Akiko Shitara ◽  
Lenka Malec ◽  
Seham Ebrahim ◽  
Desu Chen ◽  
Christopher Bleck ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Apical Membrane ◽  
Fluid Secretion ◽  
Small Gtpase ◽  
Model Systems ◽  
Adult Mice ◽  
Apical Domain ◽  
Endocytic Pathways

Lumen establishment and maintenance are fundamental for tubular organs physiological functions. Most of the studies investigating the mechanisms regulating this process have been carried out in cell cultures or in smaller organisms, whereas little has been done in mammalian model systems in vivo. Here we used the salivary glands of live mice to examine the role of the small GTPase Cdc42 in the regulation of the homeostasis of the intercellular canaliculi, a specialized apical domain of the acinar cells, where protein and fluid secretion occur. Depletion of Cdc42 in adult mice induced a significant expansion of the apical canaliculi, whereas depletion at late embryonic stages resulted in a complete inhibition of their postnatal formation. In addition, intravital subcellular microscopy revealed that reduced levels of Cdc42 affected membrane trafficking from and toward the plasma membrane, highlighting a novel role for Cdc42 in membrane remodeling through the negative regulation of selected endocytic pathways.

Role of PKC isozymes in water transport in toad urinary bladder

1991 ◽  
Vol 49 ◽  
pp. 302-303
Author(s):  
A.J. Mia ◽  
L.X. Oakford ◽  
T. Yorio
Keyword(s):  
Urinary Bladder ◽  
Apical Membrane ◽  
Membrane Surface ◽  
Protein A ◽  
Cell Types ◽  
Leukemic Cells ◽  
Toad Urinary Bladder ◽  
Pkc Isozymes ◽  
Antibody Labeling

Protein kinase C (PKC) isozymes, when activated, are translocated to particulate membrane fractions for transport to the apical membrane surface in a variety of cell types. Evidence of PKC translocation was demonstrated in human megakaryoblastic leukemic cells, and in cardiac myocytes and fibroblasts, using FTTC immunofluorescent antibody labeling techniques. Recently, we reported immunogold localizations of PKC subtypes I and II in toad urinary bladder epithelia, following 60 min stimulation with Mezerein (MZ), a PKC activator, or antidiuretic hormone (ADH). Localization of isozyme subtypes I and n was carried out in separate grids using specific monoclonal antibodies with subsequent labeling with 20nm protein A-gold probes. Each PKC subtype was found to be distributed singularly and in discrete isolated patches in the cytosol as well as in the apical membrane domains. To determine if the PKC isozymes co-localized within the cell, a double immunogold labeling technique using single grids was utilized.

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