Podocyte-associated talin1 is critical for glomerular filtration barrier maintenance
Xuefei TianJin‐Ju KimSusan J. MonkleyNanami GotohRamiro NándezK. SodaKazunori InoueDaniel M. BalkinHossam M. HassanSung Hyun SonYashang LeeGilbert MoeckelDavid CalderwoodLawrence B. HolzmanDavid R. CritchleyRoy ZentJochen ReiserShuta Ishibe
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Abstract:
Podocytes are specialized actin-rich epithelial cells that line the kidney glomerular filtration barrier. The interface between the podocyte and the glomerular basement membrane requires integrins, and defects in either α3 or β1 integrin, or the α3β1 ligand laminin result in nephrotic syndrome in murine models. The large cytoskeletal protein talin1 is not only pivotal for integrin activation, but also directly links integrins to the actin cytoskeleton. Here, we found that mice lacking talin1 specifically in podocytes display severe proteinuria, foot process effacement, and kidney failure. Loss of talin1 in podocytes caused only a modest reduction in β1 integrin activation, podocyte cell adhesion, and cell spreading; however, the actin cytoskeleton of podocytes was profoundly altered by the loss of talin1. Evaluation of murine models of glomerular injury and patients with nephrotic syndrome revealed that calpain-induced talin1 cleavage in podocytes might promote pathogenesis of nephrotic syndrome. Furthermore, pharmacologic inhibition of calpain activity following glomerular injury substantially reduced talin1 cleavage, albuminuria, and foot process effacement. Collectively, these findings indicate that podocyte talin1 is critical for maintaining the integrity of the glomerular filtration barrier and provide insight into the pathogenesis of nephrotic syndrome.Keywords:
Slit diaphragm
Synaptopodin
Glomerulosclerosis
Podocytes exhibit a unique cytoskeletal architecture that is fundamentally linked to their function in maintaining the kidney filtration barrier. The cytoskeleton regulates podocyte shape, structure, stability, slit diaphragm insertion, adhesion, plasticity, and dynamic response to environmental stimuli. Genetic mutations demonstrate that even slight impairment of the podocyte cytoskeletal apparatus results in proteinuria and glomerular disease. Moreover, mechanisms underpinning all acquired glomerular pathologies converge on disruption of the cytoskeleton, suggesting that this subcellular structure could be targeted for therapeutic purposes. This review summarizes our current understanding of the function of the cytoskeleton in podocytes and the associated implications for pathophysiology.
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Nephrin
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Podocytes are specialized actin-rich epithelial cells that line the kidney glomerular filtration barrier. The interface between the podocyte and the glomerular basement membrane requires integrins, and defects in either α3 or β1 integrin, or the α3β1 ligand laminin result in nephrotic syndrome in murine models. The large cytoskeletal protein talin1 is not only pivotal for integrin activation, but also directly links integrins to the actin cytoskeleton. Here, we found that mice lacking talin1 specifically in podocytes display severe proteinuria, foot process effacement, and kidney failure. Loss of talin1 in podocytes caused only a modest reduction in β1 integrin activation, podocyte cell adhesion, and cell spreading; however, the actin cytoskeleton of podocytes was profoundly altered by the loss of talin1. Evaluation of murine models of glomerular injury and patients with nephrotic syndrome revealed that calpain-induced talin1 cleavage in podocytes might promote pathogenesis of nephrotic syndrome. Furthermore, pharmacologic inhibition of calpain activity following glomerular injury substantially reduced talin1 cleavage, albuminuria, and foot process effacement. Collectively, these findings indicate that podocyte talin1 is critical for maintaining the integrity of the glomerular filtration barrier and provide insight into the pathogenesis of nephrotic syndrome.
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Synaptopodin
Glomerulosclerosis
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Podocytes are an integral part of the glomerular filtration barrier, a structure that prevents filtration of large proteins and macromolecules into the urine. Podocyte function is dependent on actin cytoskeleton regulation within the foot processes, structures that link podocytes to the glomerular basement membrane. Actin cytoskeleton dynamics in podocyte foot processes are complex and regulated by multiple proteins and other factors. There are two key signal integration and structural hubs within foot processes that regulate the actin cytoskeleton: the slit diaphragm and focal adhesions. Both modulate actin filament extension as well as foot process mobility. No matter what the initial cause, the final common pathway of podocyte damage is dysregulation of the actin cytoskeleton leading to foot process retraction and proteinuria. Disruption of the actin cytoskeleton can be due to acquired causes or to genetic mutations in key actin regulatory and signaling proteins. Here, we describe the major structural and signaling components that regulate the actin cytoskeleton in podocytes as well as acquired and genetic causes of actin dysregulation.
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ABSTRACT
Background: Nephrotic syndrome is a disorder of the
glomerular filtration barrier, a highly specialized trilayer
structure with unique functional properties.
Recent advances emanating from the field of
molecular genetics have revealed the podocyte as
probably the central player in the control of glomerular
filtration. More specifically, the cell–cell junction
between adjacent podocyte foot processes, namely, the
slit diaphragm, has been revealed to be made up of a
sophisticated multi-protein complex which
dynamically controls foot process architecture via
signaling to the actin cytoskeleton. Nephrotic
Syndrome (NS) is one of the most common idiopathic
primary diseases in childhood. Which is defined as
presence of four main symptoms: proteinuria,
hyperlipidemia, hypoalbuminemia and edema.
According to the patient’s response to the steroid
therapy the disease divided into: resistant and sensitive
groups. About 90% of patients are responsive to
steroid therapy during four weeks who called steroid
sensitive nephrotic syndrome (SSNS). Patients in
whom proteinuria does not stop after about one month
are classified as resistant which describe as steroid
resistant nephrotic syndrome (SRNS).SRNS is
considered as a poor prognostic disease, in which 30-
40% of it progresses to end stage renal disease
(ESRD), requiring dialysis and transplantation. The
most frequent renal histological feature associated with
SRNS is focal segmental glomerulosclerosis (FSGS).
Moreover minimal change nephrotic syndrome
(MCNS), and diffuse mesangial sclerosis (DMS) have
been identified. Genetic forms of SRNS are classified
as isolated kidney disease or syndromic disorder. The
fenestrated endothelium, the glomerular basement
membrane (GBM) and the podocytes form three layers
of glomerular filtration barrier (GFB) which is
impaired in NS and cause proteinuria.
2major proteins of podocytes including nephrin and
podocin, coded by NPHS1 and NPHS2, are considered
to play an important role in GFB. Mutations in these
genes result in altering conformation and stability of
podocytes and causing proteinuria and SRNS. Most
cases of SRNS are considered as sporadic representing
both AR and AD inheritance. NPHS1 and NPHS2
genes are the most common identified genes in AR
form.
This study was aimed to screen mutations causing
disease within NPHS1 and NPHS2, figuring out the
most common mutations in Iranian children and
comprising the prevalence of such mutations among
different nations. Due to heterogeneity of this disease,
This work is partly presented at 5th World Heart and Brain Conference September 24-26, 2018 Abu Dhabi, UAE
Extended Abstract
Vol. 5, Iss.3
2019
Insights in Blood Pressure
WES was performed for 10 patients in pilot study to
evaluate other related genes and exploring new
potential mutations. Indeed, preventing of ineffective
treatment with steroids and helping proper clinician
prediction in post transplantation outcome may be
facilitated via indicating the specific mutations.
Nephrotic syndrome is one of the most common
kidney diseases in childhood. About 20% of children
are steroid-resistant NS (SRNS) which progress to
end-stage renal disease (ESRD). More than 53 genes
are associated with SRNS which represent the genetic
heterogeneity of SRNS. This study was aimed to
screen disease causing mutations within NPHS1 and
NPHS2 and evaluate new potential variants in other
genes.
Method: In first phase of study, 25 patients with
SRNS were analyzed for NPHS1 (exon 2, 26) and all
exons of NPHS2 genes by Sanger sequencing. In the
second phase, whole exome sequencing was performed
on 10 patients with no mutations in NPHS1 and
NPHS2.
Result: WES analysis revealed a novel mutation in
FAT1 (c.10570C>A; Q3524K). We identified 4
pathogenic mutations, located in exon 4 and 5 of
NPHS2 gene in 20% of patients (V180M, P118L,
R168C and Leu156Phe). Also our study has
contributed to the descriptions of previously known
pathogenic mutations across WT1 (R205C) and
SMARCAL1 (R764Q) and a novel polymorphism in
CRB2.
Conclusion:In summary, this is the first and largest
study among Iranian population with different ethnic
origins that investigates causative variants associated
with SRNS through screening both common genes
(NPHS1 and NPHS2) and whole exome study. Among
25 patients who underwent for PCR sequencing for all
exons of NPHS2, 5 patients carried a mutation causing
disease, suggesting that NPHS2 especially exons 4 and
5 of this gene should be considered as the first step
genetic approach in children with SRNS. For the first
time in this country, 3 known variant were detected in
WT1, SMARCAL1 and CRB2, significantly a novel
variant were identified in FAT1 gene.Our study
concludes that mutations of exon 4 and 5 NPHS2 gene
are common in Iranian and some other ethnic groups.
We suggest conducting WES after NPHS2 screening
and further comprehensive studies to identify the most
common genes in the development of SRNS, which
might help in clinical impact on management in
patients with SRNS.
Because of the heterogeneous clinical and pathological
spectrum, a molecular diagnosis based on sequencing
is required. Identification of mutations causing SRNS
is of importance, not only for therapeutic
considerations but also for genetic counseling.
Glomerulosclerosis
Minimal change disease
Slit diaphragm
Hypoalbuminemia
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Glomerular podocytes are highly specialized cells with a complex cytoarchitecture. Their most prominent features are interdigitated foot processes with filtration slits in between. These are bridged by the slit diaphragm, which plays a major role in establishing the selective permeability of the glomerular filtration barrier. We searched Medline and Pubmed using the combination of keywords "NPHS2", "podocin", "steroid-resistant nephrotic syndrome," and "genetics" to identify studies describing an association between NPHS2 gene and renal disease. The highly dynamic foot processes contain an actin-based contractile apparatus comparable to that of smooth muscle cells. Mutations affecting several podocyte proteins lead to rearrangement of the cytoskeleton, disruption of the filtration barrier, and subsequent renal disease. The fact that the dynamic regulation of the podocyte cytoskeleton is vital to kidney function has led to podocytes emerging as an excellent model system for studying actin cytoskeleton dynamics in a physiological context. Injury to podocytes leads to proteinuria, a hallmark of most glomerular diseases. Recent studies have led to a considerable increase in our understanding of podocyte biology including composition and arrangement of the cytoskeleton involved in the control of ultrafiltration. Moreover, disturbances of podocyte architecture resulting in the retraction of foot processes and proteinuria appear to be a common theme in the progression of an acquired glomerular disease. In hereditary nephrotic syndromes identified over the last few years, all mutated gene products were localized in podocytes. This review integrates our recent physiological and molecular understanding of the role of podocytes during the maintenance and failure of the glomerular filtration barrier.
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Glomerulus
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Nephrotic syndrome is a disorder of the glomerular filtration barrier, and central to the filtration mechanism of the glomerular filtration barrier is the podocyte. We are starting to better understand how this cell, with its unique architectural features, fulfils its exact filtration properties. The multiprotein complex between adjacent podocyte foot processes, the slit diaphragm, is essential to the control of the actin cytoskeleton and cell morphology. Many of the proteins within the slit diaphragm, including nephrin, podocin, transient receptor potential-6 channel, and α-actinin-4, have been identified via genetic studies of inherited nephrotic syndromes. Signaling from slit diaphragm proteins to the actin cytoskeleton is mediated via the Rho GTPases. These are thought to be involved in the control of podocyte motility, which has been postulated as a focus of proteinuric pathways. Nephrotic syndrome is currently treated with immunosuppressive therapy, with significant adverse effects. These therapies may work in nephrotic syndrome due to specific effects on the podocytes. This review aims to describe our current understanding of the cellular pathways and molecules within the podocyte relevant to nephrotic syndrome and its treatment. With our current knowledge of the cellular biology of the podocyte, there is much hope for targeted therapies for nephrotic syndromes.
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Nephrin
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Aging nephropathy is characterized by podocyte depletion accompanied by progressive glomerulosclerosis. Replacement of terminally differentiated podocytes by local stem/progenitor cells is likely a critical mechanism for their regeneration. Recent studies have shown that cells of renin lineage (CoRL), normally restricted to the kidney's extraglomerular compartment, might serve this role after an abrupt depletion in podocyte number. To determine the effects of aging on the CoRL reserve and if CoRL moved from an extra- to the intraglomerular compartment during aging, genetic cell fate mapping was performed in aging Ren1cCre × Rs-ZsGreen reporter mice. Podocyte number decreased and glomerular scarring increased with advanced age. CoRL number decreased in the juxtaglomerular compartment with age. There was a paradoxical increase in CoRL in the intraglomerular compartment at 52 and 64 wk of age, where a subset coexpressed the podocyte proteins nephrin, podocin, and synaptopodin. Transmission electron microscopy studies showed that a subset of labeled CoRL in the glomerulus displayed foot processes, which attached to the glomerular basement membrane. No CoRL in the glomerular compartment stained for renin. These results suggest that, despite a decrease in the reserve, a subpopulation of CoRL moves to the glomerulus after chronic podocyte depletion in aging nephropathy, where they acquire a podocyte-like phenotype. This suggests that they might serve as adult podocyte stem/progenitor cells under these conditions, albeit in insufficient numbers to fully replace podocytes depleted with age.
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The understanding of the unique molecular apparatus of the podocyte has increased dramatically in recent years. This new knowledge has improved the diagnosis and classification of the diseases that have been termed podocytopathies. Podocyte injury frequently leads to reorganization of the slit diaphragm and reorganization of the foot process structure. Four major causes of foot process effacement can be identified, with some due to genetic mutations and others due to acquired conditions: (1) impaired formation of the slit diaphragm complex; (2) abnormalities of the glomerular basement membrane or the adhesion of podocytes to the glomerular basement membrane; (3) abnormalities of the actin cytoskeleton and associated proteins, and (4) alterations in the apical membrane domain of the podocyte. The major podocytopathies can also be organized into four categories, including those with a normal glomerular histology, diffuse mesangial sclerosis, focal segmental glomerulosclerosis, and collapsing glomerulopathy.
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Glomerulopathy
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Increased glomerular permeability of the glomerular capillary wall for macromolecules caused by the changes of the structure of the glomerular basement membrane, or podocytes and slit diaphragm between foot processes of podocytes is the main cause of nephrotic syndrome. Recently new information about podocyte proteins emerged. Mutation of the basic structural protein of slit diaphragm, nephrin, results in the Finnish type of the congenital nephrotic syndrome, mutations of other podocyte proteins, e.g. podocin, or alpha-actinin-4 result in congenital focal segmental glomerulosclerosis. Primary focal segmental glomerulosclerosis is a clinical syndrome, caused either by the mutation of podocyte proteins, or by circulating permeability factors, or by the deficiency of their circulating inhibitors. New information about the role of cubilin and megalin in the reabsorption of filtered albumin in the proximal tubule may contribute to the elucidation of the mechanisms of the tubulotoxicity of proteinuria; inhibition of albumin reabsorption in nephrotic subjects could lower the risk of interstitial fibrosis and progressive renal insufficiency.
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Slit diaphragm
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Congenital nephrotic syndrome
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Synaptopodin (Synpo) is an actin-associated protein in podocyte foot processes. By generating mice that completely lack Synpo, we previously showed that Synpo is dispensable for normal kidney function. However, lack of Synpo worsened adriamycin-induced nephropathy, indicating a protective role for Synpo in injured podocytes. Here, we investigated whether lack of Synpo directly impacts a genetic disease, Alport syndrome (AS), because Synpo is reduced in podocytes of affected humans and mice; whether this is merely an association or pathogenic is unknown. We used collagen type IV-α5 (Col4a5) mutant mice, which model X-linked AS, showing glomerular basement membrane (GBM) abnormalities, eventual foot process effacement, and progression to end-stage kidney disease. We intercrossed mice carrying mutations in Synpo and Col4a5 to produce double-mutant mice. Urine and tissue were taken at select time points to evaluate albuminuria, histopathology, and glomerular capillary wall composition and ultrastructure. Lack of Synpo in Col4a5-/Y, Col4a5-/-, or Col4a5+/- Alport mice led to the acceleration of disease progression, including more severe proteinuria and glomerulosclerosis. Absence of Synpo attenuated the shift of myosin IIA from the podocyte cell body and major processes to actin cables near the GBM in the areas of effacement. We speculate that this is mechanistically associated with enhanced loss of podocytes due to easier detachment from the GBM. We conclude that Synpo deletion exacerbates the disease phenotype in Alport mice, revealing the podocyte actin cytoskeleton as a target for therapy in patients with AS.NEW & NOTEWORTHY Alport syndrome (AS) is a hereditary disease of the glomerular basement with hematuria and proteinuria. Podocytes eventually exhibit foot process effacement, indicating actin cytoskeletal changes. To investigate how cytoskeletal changes impact podocytes, we generated Alport mice lacking synaptopodin, an actin-binding protein in foot processes. Analysis showed a more rapid disease progression, demonstrating that synaptopodin is protective. This suggests that the actin cytoskeleton is a target for therapy in AS and perhaps other glomerular diseases.
Alport syndrome
Synaptopodin
Glomerulosclerosis
Albuminuria
Podocin
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