IQGAP1 interacts with components of the slit diaphragm complex in podocytes and is involved in podocyte migration and permeability in vitro

IQGAP1 is a scaffold protein that interacts with proteins of the cytoskeleton and the intercellular adhesion complex. In podocytes, IQGAP1 is associated with nephrin in the glomerular slit diaphragm (SD) complex, but its role remains ill-defined. In this work, we investigated the interaction of IQGAP1 with the cytoskeleton and SD proteins in podocytes in culture, and its role in podocyte migration and permeability. Expression, localization, and interactions between IQGAP1 and SD or cytoskeletal proteins were determined in cultured human podocytes by Western blot (WB), immunocytolocalization (IC), immunoprecipitation (IP), and In situ Proximity Ligation assay (IsPL). Involvement of IQGAP1 in migration and permeability was also assessed. IQGAP1 expression in normal kidney biopsies was studied by immunohistochemistry. IQGAP1 expression by podocytes increased during their in vitro differentiation. IC, IP, and IsPL experiments showed colocalizations and/or interactions between IQGAP1 and SD proteins (nephrin, MAGI-1, CD2AP, NCK 1/2, podocin), podocalyxin, and cytoskeletal proteins (α-actinin-4). IQGAP1 silencing decreased podocyte migration and increased the permeability of a podocyte layer. Immunohistochemistry on normal human kidney confirmed IQGAP1 expression in podocytes and distal tubular epithelial cells and also showed an expression in glomerular parietal epithelial cells. In summary, our results suggest that IQGAP1, through its interaction with components of SD and cytoskeletal proteins, is involved in podocyte barrier properties.     

The Expression and Significance of Neuronal Iconic Proteins in Podocytes

Growing evidence suggests that there are many common cell biological features shared by neurons and podocytes; however, the mechanism of podocyte foot process formation remains unclear. Comparing the mechanisms of process formation between two cell types should provide useful guidance from the progress of neuron research. Studies have shown that some mature proteins of podocytes, such as podocin, nephrin, and synaptopodin, were also expressed in neurons. In this study, using cell biological experiments and immunohistochemical techniques, we showed that some neuronal iconic molecules, such as Neuron-specific enolase, nestin and Neuron-specific nuclear protein, were also expressed in podocytes. We further inhibited the expression of Neuron-specific enolase, nestin, synaptopodin and Ubiquitin carboxy terminal hydrolase-1 by Small interfering RNA in cultured mouse podocytes and observed the significant morphological changes in treated podocytes. When podocytes were treated with Adriamycin, the protein expression of Neuron-specific enolase, nestin, synaptopodin and Ubiquitin carboxy terminal hydrolase-1 decreased over time. Meanwhile, the morphological changes in the podocytes were consistent with results of the Small interfering RNA treatment of these proteins. The data demonstrated that neuronal iconic proteins play important roles in maintaining and regulating the formation and function of podocyte processes.     

Effects of insulin and high glucose on mobilization of slo1 BKCa channels in podocytes

Podocytes are dynamic polarized cells that lie on the surface of glomerular capillaries and comprise an essential component of the glomerular filtration barrier. Podocytes are affected in the earliest stages of diabetic nephropathy and insulin signaling to podocytes is essential for normal glomerular function. Large-conductance Ca(2+)-activated K(+) channels (BK(Ca) channels) encoded by the Slo1 gene are expressed in podocytes in a complex with multiple glomerular slit diaphragm proteins including nephrin, TRPC6 channels, and several different actin-binding proteins. Here we show that insulin increases cell surface expression of podocyte BK(Ca) channels, which is accompanied by a corresponding increase in the density of current flowing through these channels. Insulin stimulation of BK(Ca) channels was detectable in 15 min and required activation of both Erk and Akt signaling cascades. Exposure to high glucose (36.1 mM) for 24 h caused a marked reduction in the steady-state surface expression of BK(Ca) channels as well as of the slit diaphragm signaling molecule nephrin. High glucose treatment also abolished the stimulatory effects of insulin on BK(Ca) current density, although insulin continued to increase phosphorylation of Erk and Akt under those conditions. Therefore, in contrast to most other cell types, high glucose abrogates the effects of insulin in podocytes at relatively distal steps in its signaling pathway. Insulin stimulation of BK(Ca) channels in podocytes may prepare podocytes to adapt to changes in pressure gradients that occur during postprandial hyperfiltration.     

Avian Podocytes,Which Lack Nephrin,Use Adherens Junction Proteins at Intercellular Junctions

Nephrin, a major intercellular junction (ICJ) molecule of mammalian podocytes in the renal glomerulus, is absent in the avian genome. We hypothesized that birds use ICJ molecules other than nephrin in their podocytes. Therefore, in the present study, we examined the possible involvement of adherens junction (AJ) proteins in the ICJs of avian podocytes. We found the AJ proteins N-cadherin and α- and β-catenins in podocytes of quail and chickens but not in those of rats, pigs or humans. The AJ proteins were prominent in avian glomerulus-rich fractions in immunoblot analyses, and in immunofluorescence microscopy analyses, they were localized along glomerular capillary walls appearing in at least two staining patterns: weakly diffuse and distinctly granular. Immunoelectron microscopy demonstrated that the significant accumulation of immunogold particles for the AJ proteins were especially evident in avian slit diaphragms and AJs. Furthermore, N-cadherin was found to be expressed in all nephron cells in the early developmental stage but became confined to podocytes during maturation. These results indicate that avian slit diaphragms clearly express AJ proteins as compared with that in the mammal—where AJ proteins are suppressed to an extremely low level—and that avian podocytes are interconnected by AJs per se in addition to slit diaphragms.     

Establishment of Protein Delivery Systems Targeting Podocytes

Background

Podocytes are uniquely structured cells that are critical to the kidney filtration barrier. Their anatomic location on the outer side of the glomerular capillaries expose podocytes to large quantities of both plasma and urinary components and thus are reachable for drug delivery. Recent years have made clear that interference with podocyte-specific disease pathways can modulate glomerular function and influence severity and progression of glomerular disease.

Methodology/Principal Findings

Here, we describe studies that show efficient transport of proteins into the mammalian cells mouse 3T3 fibroblasts and podocytes, utilizing an approach termed profection. We are using synthetic lipid structures that allow the safe packing of proteins or antibodies resulting in the subsequent delivery of protein into the cell. The uptake of lipid coated protein is facilitated by the intrinsic characteristic of cells such as podocytes to engulf particles that are physiologically retained in the extracellular matrix. Profection of the restriction enzyme MunI in 3T3 mouse fibroblasts caused an increase in DNA degradation. Moreover, purified proteins such as β-galactosidase and the large GTPase dynamin could be profected into podocytes using two different profection reagents with the success rate of 95–100%. The delivered β-galactosidase enzyme was properly folded and able to cleave its substrate X-gal in podocytes. Diseased podocytes are also potential recipients of protein cargo as we also delivered fluorophore labeled IgG into puromycin treated podocytes. We are currently optimizing our protocol for in vivo profection.

Conclusions

Protein transfer is developing as an exciting tool to study and target highly differentiated cells such as podocytes.     

Nephrin and CD2AP associate with phosphoinositide 3-OH kinase and stimulate AKT-dependent signaling

Mutations of NPHS1 or NPHS2, the genes encoding nephrin and podocin, as well as the targeted disruption of CD2-associated protein (CD2AP), lead to heavy proteinuria, suggesting that all three proteins are essential for the integrity of glomerular podocytes, the visceral glomerular epithelial cells of the kidney. It has been speculated that these proteins participate in common signaling pathways; however, it has remained unclear which signaling proteins are actually recruited by the slit diaphragm protein complex in vivo. We demonstrate that both nephrin and CD2AP interact with the p85 regulatory subunit of phosphoinositide 3-OH kinase (PI3K) in vivo, recruit PI3K to the plasma membrane, and, together with podocin, stimulate PI3K-dependent AKT signaling in podocytes. Using two-dimensional gel analysis in combination with a phosphoserine-specific antiserum, we demonstrate that the nephrin-induced AKT mediates phosphorylation of several target proteins in podocytes. One such target is Bad; its phosphorylation and inactivation by 14-3-3 protects podocytes against detachment-induced cell death, suggesting that the nephrin-CD2AP-mediated AKT activity can regulate complex biological programs. Our findings reveal a novel role for the slit diaphragm proteins nephrin, CD2AP, and podocin and demonstrate that these three proteins, in addition to their structural functions, initiate PI3K/AKT-dependent signal transduction in glomerular podocytes.     

A proteomic investigation of glomerular podocytes from a Denys-Drash syndrome patient with a mutation in the Wilms tumour suppressor gene WT1

Glomerular podocytes are essential for blood filtration in the kidney underpinned by their unique cytoskeletal morphology. An increasing number of kidney diseases are being associated with key podocyte abnormalities. The Wilms tumour suppressor gene (WT1) encodes a zinc finger protein with a crucial role in normal kidney development; and in the adult, WT1 is required for normal podocyte function. Denys-Drash Syndrome (DDS) results from mutations affecting the zinc finger domain of WT1. The aim of this study was to undertake, for the first time, a proteomic analysis of cultured human podocytes; and to analyse the molecular changes in DDS podocytes. The morphology of DDS podocytes was highly irregular, reminiscent of a fibroblastic appearance. A reference 2-D gel was generated, and 75 proteins were identified of which 43% involved in cytoskeletal architecture. The DDS and wild-type proteomes were compared by 2-D DIGE. The level of 95.6% of proteins was unaltered; but 4.4% were altered more than two-fold. A sample of proteins involved in cytoskeletal architecture appeared to be misexpressed in DDS podocytes. Consistent with this finding, overall levels of filamentous actin also appeared reduced in DDS podocytes. We conclude that one of WT1 functions in podocytes is to regulate the expression of key components and regulators of the cytoskeleton.     

The struggle for energy in podocytes leads to nephrotic syndrome

Podocytes are terminally differentiated post-mitotic cells similar to neurons, and their damage leads to nephrotic syndrome, which is characterized by massive proteinuria associated with generalized edema. A recent functional genetic approach has identified the pathological relevance of several mutated proteins in glomerular podocytes to the mechanism of proteinuria in hereditary nephrotic syndrome. In contrast, the pathophysiology of acquired primary nephrotic syndrome, including minimal change disease, is still largely unknown. We recently demonstrated the possible linkage of an energy-consuming process in glomerular podocytes to the mechanism of proteinuria. Puromycin aminonucleoside nephrosis, a rat model of minimal change disease, revealed the activation of the unfolded protein response (UPR) in glomerular podocytes to be a cause of proteinuria. The pretreatment of puromycin aminonucleoside rat podocytes and cultured podocytes with the mammalian target of rapamycin (mTOR) inhibitor everolimus further revealed that mTOR complex 1 consumed energy, which was followed by UPR activation. In this paper, we will review nutritional transporters to summarize the energy uptake process in podocytes and review the involvement of the UPR in the pathogenesis of glomerular diseases. We will also present additional data that reveal how mTOR complex 1 acts upstream of the UPR. Finally, we will discuss the potential significance of targeting the energy metabolism of podocytes to develop new therapeutic interventions for acquired nephrotic syndrome.     

Shp2 Associates with and Enhances Nephrin Tyrosine Phosphorylation and Is Necessary for Foot Process Spreading in Mouse Models of Podocyte Injury

In most forms of glomerular diseases, loss of size selectivity by the kidney filtration barrier is associated with changes in the morphology of podocytes. The kidney filtration barrier is comprised of the endothelial lining, the glomerular basement membrane, and the podocyte intercellular junction, or slit diaphragm. The cell adhesion proteins nephrin and neph1 localize to the slit diaphragm and transduce signals in a Src family kinase Fyn-mediated tyrosine phosphorylation-dependent manner. Studies in cell culture suggest nephrin phosphorylation-dependent signaling events are primarily involved in regulation of actin dynamics and lamellipodium formation. Nephrin phosphorylation is a proximal event that occurs both during development and following podocyte injury. We hypothesized that abrogation of nephrin phosphorylation following injury would prevent nephrin-dependent actin remodeling and foot process morphological changes. Utilizing a biased screening approach, we found nonreceptor Src homology 2 (sh2) domain-containing phosphatase Shp2 to be associated with phosphorylated nephrin. We observed an increase in nephrin tyrosine phosphorylation in the presence of Shp2 in cell culture studies. In the human glomerulopathies minimal-change nephrosis and membranous nephropathy, there is an increase in Shp2 phosphorylation, a marker of increased Shp2 activity. Mouse podocytes lacking Shp2 do not develop foot process spreading when subjected to podocyte injury in vivo using protamine sulfate or nephrotoxic serum (NTS). In the NTS model, we observed a lack of foot process spreading in mouse podocytes with Shp2 deleted and smaller amounts of proteinuria. Taken together, these results suggest that Shp2-dependent signaling events are necessary for changes in foot process structure and function following injury.     

Actin up: regulation of podocyte structure and function by components of the actin cytoskeleton

Podocytes of the renal glomerulus are unique cells with a complex cellular organization consisting of a cell body, major processes and foot processes. Podocyte foot processes form a characteristic interdigitating pattern with foot processes of neighboring podocytes, leaving in between the filtration slits that are bridged by the glomerular slit diaphragm. The highly dynamic foot processes contain an actin-based contractile apparatus comparable to that of smooth muscle cells or pericytes. Mutations affecting several podocyte proteins lead to rearrangement of the actin 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.     

Glomerular podocytes: a study of mechanical properties and mechano-chemical signaling

Kidney glomeruli function as filters, allowing the passage of small solutes and waste products into the urinary tract, while retaining essential proteins and macromolecules in the blood stream. These structures are under constant mechanical stress due to fluid pressure, driving filtration across the barrier. We mechanically stimulated adherent wildtype podocytes using the methods of magnetic tweezer and twisting as well as cell stretching. Attaching collagen IV-coated or poly-l-lysine-coated magnetic beads to cell receptors allowed for the determination of cellular stiffness. Angiotensin II-treated podocytes showed slightly higher stiffness than untreated cells, the cell fluidity (i.e. internal dynamics) remained similar, and showed an increase with force. The bead detachment (a measure of the binding strength) was higher in angiotensin II-treated compared to untreated podocytes. Magnetic twisting confirmed that angiotensin II treatment of podocytes increases and CDTA treatment decreases cell stiffness. However, treatment with both angiotensin II and CDTA increased the cell stiffness only slightly compared to solely CDTA-treated cells. Exposing podocytes to cyclic, uniaxial stretch showed an earlier onset of ERK^1/2 phosphorylation compared to MEF (control) cells. These results indicate that angiotensin II might free intracellularly stored calcium and affects actomyosin contraction, and that mechanical stimulation influences cell signaling.     

Contractile proteins in podocytes: immunocytochemical localization of actin and alpha-actinin in normal and nephrotic rat kidneys.

Actin and alpha-actinin immunoreactive sites have been localized at the electron microscope level by the protein A-gold immunocytochemical technique in podocytes of normal and nephrotic rat renal tissues. In normal renal glomeruli, fibrillar networks located in the core of foot processes or bundles of microfilaments interconnecting them were found to be labelled for these two cytoskeletal proteins. On the other hand, in nephrotic renal glomeruli, concomitant with the loss of podocytic foot processes a reorganization of the podocytic cytoskeleton and a concentration of some of its elements into thick uniform bands was observed. Actin and alpha-actinin were revealed in these bands. Control experiments confirmed the specificity of the labelling obtained. Our results suggest that normal podocytes contain an actin-based contractile system that might contribute to the maintenance of the particular cell shape of these cells and that the rearrangement of the podocytic cytoskeleton occurring in the nephrotic syndrome might account for the changes in the foot processes and contribute to the alteration in glomerular function.     

Cyclin I-Cdk5 governs survival in post-mitotic cells

Cdk5 has long been recognized to play an important role in development, maturation and apoptosis of post-mitotic and terminally differentiated cells. Activation of Cdk5 is tightly regulated by specific activators. Cyclin I was recently characterized as the first cyclin protein that binds to and activates Cdk5. Cyclin I-Cdk5 activates the MEK-ERK pathway and results in increased Bcl-2 and Bcl-XL mRNA and protein levels. Lack of cyclin I renders podocytes more susceptible to apoptosis. Interestingly, activation of Cdk5 by p35 is also involved in the podocytes’ response to injury. In the absence of p35, podocytes are more prone to undergo apoptosis. Here, we propose a new model where Cdk5 plays a central role in the cellular response machinery against injury-induced apoptosis of post-mitotic cells. While cyclin I-Cdk5 regulates Bcl-2 family proteins through activation of the MEK-ERK pathway, p35-Cdk5 directly phosphorylates and stabilizes Bcl-2.     

The glomerulus – a view from the outside – the podocyte

In the past decade, podocyte research has been greatly aided by the development of powerful new molecular, cellular and animal tools, leading to elucidation of an increasing number of proteins involved in podocyte function and identification of mutated genes in hereditary glomerulopathies. Accumulating evidence indicates that podocyte disorders may not only underlie these hereditary glomerulopathies but also play crucial role in a broad spectrum of acquired glomerular diseases. Genetic susceptibility, environmental influence and systemic responses are all involved in the mediation of the pathogenesis of podocytopathies. Injured podocytes may predisopose to further injury of other podocytes and other adjacent/distant renal cells in a vicious cycle, leading to inexorable progression of glomerular injury. The classic view is that podocytes have a limited ability to proliferate in the normal mature kidney. However, recent research in rodents has provided suggestive evidence for podocyte regeneration resulting from differentiation of progenitor cells within Bowman's capsule.     

“Treasure your exceptions”: recent advances in molecular genetics of glomerular disease

The glomerular filtration barrier consists of endothelial cells, the glomerular basement membrane, and podocytes. The membrane is a highly crosslinked macromolecular meshwork composed of specific extracellular matrix proteins. The adjacent foot processes of podocytes are bridged along their basolateral surfaces by a slit diaphragm (a porous filter structure of nephrin molecules). Recent discoveries of mutations in the range of genes encoding proteins involved in the structure or function of the glomerular filtration barrier have provided new insights into mechanisms of glomerular diseases. In this review, we summarize recent progress in the elucidation of the genetic basis of some glomerulopathies in humans.     

(Pro)renin receptor and autophagy in podocytes

Within the kidneys, podocytes are highly specialized postmitotic cells. Podocytes, together with endothelial cells and the glomerular basement membrane (GBM), maintain the filtration barrier and the normal structure of the glomerular capillary, are involved in the remodeling of the GBM and the endocytosis of filtered proteins, and counteract intracapillary hydrostatic pressure. The (pro)renin receptor [(P)RR], as ATP6AP2, is an accessory subunit of the vacuolar H⁺-ATPase, implying more fundamental developmental functions for the (pro)renin receptor in addition to its role in activating the local renin-angiotensin system, and is also expressed in podocytes, where it is involved in both tissue angiotensin II production and (P)RR-mediated intracellular signaling. Overexpression of human (P)RR in rats caused slowly progressive proteinuria and glomerular sclerosis, which suggests that (P)RR-mediated signaling is involved in the development of glomerular diseases. However, the physiological role of the (P)RR in podocytes has not yet been fully understood.     

(Pro)renin receptor and autophagy in podocytes

Within the kidneys, podocytes are highly specialized postmitotic cells. Podocytes, together with endothelial cells and the glomerular basement membrane (GBM), maintain the filtration barrier and the normal structure of the glomerular capillary, are involved in the remodeling of the GBM and the endocytosis of filtered proteins, and counteract intracapillary hydrostatic pressure. The (pro)renin receptor [(P)RR], as ATP6AP2, is an accessory subunit of the vacuolar H (+) -ATPase, implying more fundamental developmental functions for the (pro)renin receptor in addition to its role in activating the local renin-angiotensin system, and is also expressed in podocytes, where it is involved in both tissue angiotensin II production and (P)RR-mediated intracellular signaling. Overexpression of human (P)RR in rats caused slowly progressive proteinuria and glomerular sclerosis, which suggests that (P)RR-mediated signaling is involved in the development of glomerular diseases. However, the physiological role of the (P)RR in podocytes has not yet been fully understood.     

Contractile proteins in podocytes: immunocytochemical localization of actin and alpha-actinin in normal and nephrotic rat kidneys

Actin and alpha-actinin immunoreactive sites have been localized at the electron microscope level by the protein A-gold immunocytochemical technique in podocytes of normal and nephrotic rat renal tissues. In normal renal glomeruli, fibrillar networks located in the core of foot processes or bundles of micro filaments interconnecting them were found to be labelled for these two cytoskeletal proteins. On the other hand, in nephrotic renal glomeruli, concomitant with the loss of podocytic foot processes a reorganization of the podocytic cytoskeleton and a concentration of some of its elements into thick uniform bands was observed. Actin and alpha-actinin were revealed in these bands. Control experiments confirmed the specificity of the labelling obtained. Our results suggest that normal podocytes contain an actin-based contractile system that might contribute to the maintenance of the particular cell shape of these cells and that the rearrangement of the podocytic cyto-skeleton occurring in the nephrotic syndrome might account for the changes in the foot processes and contribute to the alteration in glomerular function. This work was supported by grants from the Medical Research Council of Canada     

The Directed Differentiation of Human iPS Cells into Kidney Podocytes

The loss of glomerular podocytes is a key event in the progression of chronic kidney disease resulting in proteinuria and declining function. Podocytes are slow cycling cells that are considered terminally differentiated. Here we provide the first report of the directed differentiation of induced pluripotent stem (iPS) cells to generate kidney cells with podocyte features. The iPS-derived podocytes share a morphological phenotype analogous with cultured human podocytes. Following 10 days of directed differentiation, iPS podocytes had an up-regulated expression of mRNA and protein localization for podocyte markers including synaptopodin, nephrin and Wilm’s tumour protein (WT1), combined with a down-regulation of the stem cell marker OCT3/4. In contrast to human podocytes that become quiescent in culture, iPS-derived cells maintain a proliferative capacity suggestive of a more immature phenotype. The transduction of iPS podocytes with fluorescent labeled-talin that were immunostained with podocin showed a cytoplasmic contractile response to angiotensin II (AII). A permeability assay provided functional evidence of albumin uptake in the cytoplasm of iPS podocytes comparable to human podocytes. Moreover, labeled iPS-derived podocytes were found to integrate into reaggregated metanephric kidney explants where they incorporated into developing glomeruli and co-expressed WT1. This study establishes the differentiation of iPS cells to kidney podocytes that will be useful for screening new treatments, understanding podocyte pathogenesis, and offering possibilities for regenerative medicine.