Advancing Discovery Research in Nephrology in Canada: A Conference Report From the 2021 Molecules and Mechanisms Mediating Kidney Health and Disease (M3K) Scientific Meeting and Investigator Summit.

Purpose of conference: New discoveries arising from investigations into fundamental aspects of kidney development and function in health and disease are critical to advancing kidney care. Scientific meetings focused specifically on fundamental biology of the kidney can facilitate interactions, support the development of collaborative groups, and accelerate translation of key findings. The Canadian fundamental kidney researcher community has lacked such a forum. On December 3 to 4, 2021, the first Molecules and Mechanisms Mediating Kidney Health and Disease (M3K) Scientific Meeting and Investigator Summit was held to address this gap with the goal of advancing fundamental kidney research nationally. The meeting was held virtually and was supported by a planning and dissemination grant from the Canadian Institutes of Health Research. Attendees included PhD scientists, nephrology clinician scientists, engineers, industry representatives, graduate students, medical residents, and fellows. Sources of information: This report was prepared from the scientific program, registration numbers, and details obtained from the online platform WHOVA, and summaries written by organizers and participants of the 2021 meeting. Methods: A 21-person team, consisting of the organizing committee members and participants from the meeting, was assembled. Key highlights of the meeting and future directions were identified and the team jointly assembled this report. Key findings: Participation in the meeting was strong, with more than 140 attendees across a range of disciplines. The program featured state-of-the-art presentations on diabetic nephropathy, the immune system, kidney development, and fibrosis, and was heavily focused on trainee presentations. The moderated "Investigator Summit" identified key barriers to research advancement and discussed strategies for overcoming them. These included establishment of a pan-Canadian fundamental kidney research network, development of key resources, cross-pollination with clinical nephrology, better reintegration into the Canadian Society of Nephrology, and further establishment of identity and knowledge translation. Limitations and implications: The 2021 M3K meeting represented a key first step in uniting fundamental kidney researchers in Canada. However, it was universally agreed that regular meetings were necessary to sustain this momentum. The proceedings of this meeting and future actions to sustain the M3K Scientific Meeting and Investigator Summit are presented in this article.

Objectif de la conférence: De nouvelles découvertes découlant des enquêtes sur les aspects fondamentaux du développement et de la fonction des reins en santé ou malades sont essentielles pour faire progresser les soins rénaux. Les réunions scientifiques axées spécifiquement sur la biologie fondamentale du rein peuvent faciliter les interactions, appuyer le développement de groupes de collaboration et accélérer l'application des principaux résultats. La communauté canadienne des chercheurs fondamentaux en néphrologie a manqué d'un tel forum. Les 3 et 4 décembre 2021, le premier Sommet des chercheurs et la réunion scientifique M3K (Molecules and Mechanisms Mediating Kidney Health and Disease) sur les molécules et les médiateurs de la santé et des maladies rénales ont eu lieu pour combler cette lacune; l'objectif était de faire progresser la recherche fondamentale en néphrologie à l'échelle nationale. La réunion s'est tenue virtuellement et était financée par une subvention de planification et de diffusion des Instituts de recherche en santé du Canada. Des doctorants, cliniciens-chercheurs en néphrologie, ingénieurs, représentants de l'industrie, étudiants diplômés, résidents en médecine et en surspécialisation figuraient parmi les participants. Sources: Ce rapport a été préparé à partir du program scientifique, des informations et des numéros d'inscription tirés de la plateforme en ligne WHOVA, et des résumés rédigés par les organisateurs et les participants à la réunion de 2021. Méthodologie: Une équipe de 20 personnes composée de membres du comité organisateur et de participants à la réunion a été formée. Les principaux points saillants de la réunion et les orientations futures ont été déterminés, puis l'équipe a rédigé conjointement le présent rapport. Principaux résultats: La réunion s'est avérée un succès; plus de 140 personnes provenant d'un large éventail de disciplines y ont participé. Le program comprenait des présentations de pointe sur la néphropathie diabétique, le système immunitaire, le développement des reins et la fibrose, et était fortement axé sur des présentations par des stagiaires. Le « Sommet des chercheurs ¼, animé par un modérateur, a permis de déterminer les principaux obstacles à l'avancement de la recherche et de discuter des stratégies pour les surmonter. Ces dernières incluent notamment la création d'un réseau pancanadien de recherche fondamentale en néphrologie, le développement de ressources clés, la pollinisation croisée avec la néphrologie clinique, une « meilleure réintégration dans la Société canadienne de néphrologie ¼ et la poursuite de l'établissement de l'identité et de l'application des connaissances. Limites et implications: La réunion M3K de 2021 a constitué une première étape clé dans l'unification des chercheurs fondamentaux en néphrologie au Canada. On a cependant universellement convenu que des réunions régulières étaient nécessaires pour maintenir cet élan. Le compte rendu de cette réunion ainsi que les actions futures pour soutenir la réunion scientifique M3K et le Sommet des chercheurs sont présentés dans le présent article.

The Glomerular Endothelium Restricts Albumin Filtration.

Inflammatory activation and/or dysfunction of the glomerular endothelium triggers proteinuria in many systemic and localized vascular disorders. Among them are the thrombotic microangiopathies, many forms of glomerulonephritis, and acute inflammatory episodes like sepsis and COVID-19 illness. Another example is the chronic endothelial dysfunction that develops in cardiovascular disease and in metabolic disorders like diabetes. While the glomerular endothelium is a porous sieve that filters prodigious amounts of water and small solutes, it also bars the bulk of albumin and large plasma proteins from passing into the glomerular filtrate. This endothelial barrier function is ascribed predominantly to the endothelial glycocalyx with its endothelial surface layer, that together form a relatively thick, mucinous coat composed of glycosaminoglycans, proteoglycans, glycolipids, sialomucins and other glycoproteins, as well as secreted and circulating proteins. The glycocalyx/endothelial surface layer not only covers the glomerular endothelium; it extends into the endothelial fenestrae. Some glycocalyx components span or are attached to the apical endothelial cell plasma membrane and form the formal glycocalyx. Other components, including small proteoglycans and circulating proteins like albumin and orosomucoid, form the endothelial surface layer and are bound to the glycocalyx due to weak intermolecular interactions. Indeed, bound plasma albumin is a major constituent of the endothelial surface layer and contributes to its barrier function. A role for glomerular endothelial cells in the barrier of the glomerular capillary wall to protein filtration has been demonstrated by many elegant studies. However, it can only be fully understood in the context of other components, including the glomerular basement membrane, the podocytes and reabsorption of proteins by tubule epithelial cells. Discovery of the precise mechanisms that lead to glycocalyx/endothelial surface layer disruption within glomerular capillaries will hopefully lead to pharmacological interventions that specifically target this important structure.

TIMAP inhibits endothelial myosin light chain phosphatase by competing with MYPT1 for the catalytic protein phosphatase 1 subunit PP1cß.

Transforming growth factor-ß membrane associated protein (TIMAP) is an endothelial cell (EC)-predominant PP1 regulatory subunit and a member of the myosin phosphatase target (MYPT) protein family. The MYPTs preferentially bind the catalytic protein phosphatase 1 subunit PP1cß, forming myosin phosphatase holoenzymes. We investigated whether TIMAP/PP1cß could also function as a myosin phosphatase. Endogenous PP1cß, myosin light chain 2 (MLC2), and myosin IIA heavy chain coimmunoprecipitated from EC lysates with endogenous TIMAP, and endogenous MLC2 colocalized with TIMAP in EC projections. Purified recombinant GST-TIMAP interacted directly with purified recombinant His-MLC2. However, TIMAP overexpression in EC enhanced MLC2 phosphorylation, an effect not observed with a TIMAP mutant that does not bind PP1cß. Conversely, MLC2 phosphorylation was reduced in lung lysates from TIMAP-deficient mice and upon silencing of endogenous TIMAP expression in ECs. Ectopically expressed TIMAP slowed the rate of MLC2 dephosphorylation, an effect requiring TIMAP-PP1cß interaction. The association of MYPT1 with PP1cß was profoundly reduced in the presence of excess TIMAP, leading to proteasomal MYPT1 degradation. In the absence of TIMAP, MYPT1-associated PP1cß readily bound immobilized microcystin-LR, an active-site inhibitor of PP1c. By contrast, TIMAP-associated PP1cß did not interact with microcystin-LR, indicating that the active site of PP1cß is blocked when it is bound to TIMAP. Thus, TIMAP inhibits myosin phosphatase activity in ECs by competing with MYPT1 for PP1cß and blocking the PP1cß active site.

An In Vitro Kinase Assay to Assess Rac1 Phosphorylation by ERK.

Recent findings suggest that phosphorylation might further contribute to the tight regulation of Rho GTPases. Interestingly, sequence analysis of Rac1 shows that T108 within the 106PNTP109 motif of Rac1 is likely an ERK phosphorylation site and Rac1 also has an ERK docking site 183KKRKRKCLLL192 (D-site) at the C-terminus. Protein phosphorylation could be assayed by many different methods. Here, we describe an in vitro kinase assay we used to assess Rac1 phosphorylation by ERK. Rac1 phosphorylation is detected based on the transfer of a radiolabeled phosphate from ATP to Rac1 by the phosphotransferase activity of the kinase EKR. This in vitro kinase assay uses commercially available purified active ERK. Substrate Rac1 was generated and purified as a glutathione S-transferase (GST) fusion protein. [γ-32P]ATP is used to radiolabel Rac1. Phosphorylation of Rac1 is viewed by autoradiography.

Both CLIC4 and CLIC5A activate ERM proteins in glomerular endothelium.

The chloride intracellular channel (CLIC) 5A is expressed at very high levels in renal glomeruli, in both endothelial cells (EC) and podocytes. CLIC5A stimulates Rac1- and phosphatidylinositol (4,5)-bisphosphate-dependent ERM (ezrin, radixin, moesin) activation. ERM proteins, in turn, function in lumen formation and in the development of actin-based cellular projections. In mice lacking CLIC5A, ERM phosphorylation is profoundly reduced in podocytes, but preserved in glomerular EC. Since glomerular EC also express CLIC4, we reasoned that, if CLIC4 activates ERM proteins like CLIC5A, then CLIC4 could compensate for the CLIC5A loss in glomerular EC. In glomeruli of CLIC5-deficient mice, CLIC4 expression was upregulated and colocalized with moesin and ezrin in glomerular EC, but not in podocytes. In cultured glomerular EC, CLIC4 silencing reduced ERM phosphorylation and cytoskeletal association, and expression of exogenous CLIC4 or CLIC5A rescued ERM de-phosphorylation due to CLIC4 silencing. In mice lacking either CLIC4 or CLIC5, ERM phosphorylation was retained in glomerular EC, but, in mice lacking both CLIC4 and CLIC5, glomerular EC ERM phosphorylation was profoundly reduced. Although glomerular EC fenestrae developed normally in dual CLIC4/CLIC5-deficient mice, the density of fenestrae declined substantially by 8 mo of age, along with the deposition of subendothelial electron-lucent material. The dual CLIC4/CLIC5-deficient mice developed spontaneous proteinuria, glomerular cell proliferation, and matrix deposition. Thus CLIC4 stimulates ERM activation and can compensate for CLIC5A in glomerular EC. The findings indicate that CLIC4/CLIC5A-mediated ERM activation is required for maintenance of the glomerular capillary architecture.

The chloride intracellular channel 5A stimulates podocyte Rac1, protecting against hypertension-induced glomerular injury.

Glomerular capillary hypertension elicits podocyte remodeling and is a risk factor for the progression of glomerular disease. Ezrin, which links podocalyxin to actin in podocytes, is activated through the chloride intracellular channel 5A (CLIC5A)-dependent phosphatidylinositol 4,5 bisphosphate (PI[4,5]P2) accumulation. Because Rac1 is involved in podocyte actin remodeling and can promote PI[4,5]P2 production we determined whether CLIC5A-dependent PI[4,5]P2 generation and ezrin activation are mediated by Rac1. In COS7 cells, CLIC5A expression stimulated Rac1 but not Cdc42 or Rho activity. CLIC5A also stimulated phosphorylation of the Rac1 effector Pak1 in COS7 cells and in cultured mouse podocytes. CLIC5A-induced PI[4,5]P2 accumulation and Pak1 and ezrin phosphorylation were all Rac1 dependent. In DOCA/Salt hypertension, phosphorylated Pak increased in podocytes of wild-type, but not CLIC5-deficient mice. In DOCA/salt hypertensive mice lacking CLIC5, glomerular capillary microaneurysms were more frequent and albuminuria was greater than in wild-type mice. Thus, augmented hypertension-induced glomerular capillary injury in mice lacking CLIC5 results from abrogation of Rac1-dependent Pak and ezrin activation, perhaps reducing the tensile strength of the podocyte actin cytoskeleton.

Phosphorylation and Activation of RhoA by ERK in Response to Epidermal Growth Factor Stimulation.

The small GTPase RhoA has been implicated in various cellular activities, including the formation of stress fibers, cell motility, and cytokinesis. In addition to the canonical GTPase cycle, recent findings have suggested that phosphorylation further contributes to the tight regulation of Rho GTPases. Indeed, RhoA is phosphorylated on serine 188 (188S) by a number of protein kinases. We have recently reported that Rac1 is phosphorylated on threonine 108 (108T) by extracellular signal-regulated kinases (ERK) in response to epidermal growth factor (EGF) stimulation. Here, we provide evidence that RhoA is phosphorylated by ERK on 88S and 100T in response to EGF stimulation. We show that ERK interacts with RhoA and that this interaction is dependent on the ERK docking site (D-site) at the C-terminus of RhoA. EGF stimulation enhanced the activation of the endogenous RhoA. The phosphomimetic mutant, GFP-RhoA S88E/T100E, when transiently expressed in COS-7 cells, displayed higher GTP-binding than wild type RhoA. Moreover, the expression of GFP-RhoA S88E/T100E increased actin stress fiber formation in COS-7 cells, which is consistent with its higher activity. In contrast to Rac1, phosphorylation of RhoA by ERK does not target RhoA to the nucleus. Finally, we show that regardless of the phosphorylation status of RhoA and Rac1, substitution of the RhoA PBR with the Rac1 PBR targets RhoA to the nucleus and substitution of Rac1 PBR with RhoA PBR significantly reduces the nuclear localization of Rac1. In conclusion, ERK phosphorylates RhoA on 88S and 100T in response to EGF, which upregulates RhoA activity.

Clustered PI(4,5)P2 accumulation and ezrin phosphorylation in response to CLIC5A.

CLIC5A (encoded by CLIC5) is a component of the ezrin-NHERF2-podocalyxin complex in renal glomerular podocyte foot processes. We explored the mechanism(s) by which CLIC5A regulates ezrin function. In COS-7 cells, CLIC5A augmented ezrin phosphorylation without changing ezrin abundance, increased the association of ezrin with the cytoskeletal fraction and enhanced actin polymerization and the formation of cell surface projections. CLIC5A caused the phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] reporter RFP-PH-PLC to translocate from the cytosol to discrete plasma membrane clusters at the cell surface, where it colocalized with CLIC5A. Transiently expressed HA-PIP5Kα colocalized with GFP-CLIC5A and was pulled from cell lysates by GST-CLIC5A, and silencing of endogenous PIP5Kα abrogated CLIC5A-dependent ERM phosphorylation. N- and C-terminal deletion mutants of CLIC5A, which failed to associate with the plasma membrane, failed to colocalize with PIP5Kα, did not alter the abundance of PI(4,5)P2 plasma membrane clusters and failed to enhance ezrin phosphorylation. Relative to wild-type mice, in CLIC5-deficient mice, the phosphorylation of glomerular ezrin was diminished and the cytoskeletal association of both ezrin and NHERF2 was reduced. Therefore, the mechanism of CLIC5A action involves clustered plasma membrane PI(4,5)P2 accumulation through an interaction of CLIC5A with PI(4,5)P2-generating kinases, in turn facilitating ezrin activation and actin-dependent cell surface remodeling.

TIMAP promotes angiogenesis by suppressing PTEN-mediated Akt inhibition in human glomerular endothelial cells.

The function of TIMAP, an endothelial cell (EC)-predominant protein phosphatase 1-regulatory subunit, is poorly understood. We explored the potential role of TIMAP in the Akt-dependent regulation of glomerular EC proliferation, survival, and in vitro angiogenesis. To deplete TIMAP, the EC were transfected with TIMAP-specific or nonspecific small interfering (si) RNA. The rate of electrical impedance development across subconfluent EC monolayers, a measure of the time-dependent increase in EC number, was 93 ± 2% lower in TIMAP-depleted than in control EC. This effect on cell proliferation was associated with reduced DNA synthesis and increased apoptosis: TIMAP silencing reduced 5-ethynyl-2'-deoxyuridine incorporation by 38 ± 2% during the exponential phase of EC proliferation, and cleaved caspase 3 as well as caspase 3 activity increased in TIMAP-depleted relative to control cells. Furthermore, TIMAP depletion inhibited the formation of angiogenic sprouts by glomerular EC in three-dimensional culture. TIMAP depletion strongly diminished growth factor-stimulated Akt phosphorylation without altering ERK1/2 phosphorylation, suggesting a specific effect on the PI3K/Akt/PTEN pathway. Endogenous TIMAP and PTEN colocalized in EC and coimmunoprecipitated from EC lysates. The inhibitory PTEN phosphorylation on S370 was significantly reduced in TIMAP-depleted compared with control EC, while phosphorylation of PTEN on the S380/T382/T383 cluster remained unchanged. Finally, the PTEN inhibitor bpV(phen) fully reversed the suppressive effect of TIMAP depletion on Akt phosphorylation. The data indicate that in growing EC, TIMAP is necessary for Akt-dependent EC proliferation, survival, and angiogenic sprout formation and that this effect of TIMAP is mediated by inhibition of the tumor suppressor PTEN.

Tipping the balance from angiogenesis to fibrosis in CKD.

Chronic progressive renal fibrosis leads to end-stage renal failure many patients with chronic kidney disease (CKD). Loss of the rich peritubular capillary network is a prominent feature, and seems independent of the specific underlying disease. The mechanisms that contribute to peritubular capillary regression include the loss of glomerular perfusion, as flow-dependent shear forces are required to provide the survival signal for endothelial cells. Also, reduced endothelial cell survival signals from sclerotic glomeruli and atrophic or injured tubule epithelial cells contribute to peritubular capillary regression. In response to direct tubular epithelial cell injury, and the inflammatory reaction that ensues, capillary pericytes dissociate from their blood vessels, also reducing endothelial cell survival. In addition, direct inflammatory injury of capillary endothelial cells, for instance in chronic allograft nephropathy, also contributes to capillary dropout. Chronic tissue hypoxia, which ensues from the rarefaction of the peritubular capillary network, can generate both an angiogenic and a fibrogenic response. However, in CKD, the balance is strongly tipped toward fibrogenesis. Understanding the underlying mechanisms for failed angiogenesis in CKD and harnessing endothelial-specific survival and pro-angiogenic mechanisms for therapy should be our goal if we are to reduce the disease burden from CKD.

Phosphorylation of Rac1 T108 by extracellular signal-regulated kinase in response to epidermal growth factor: a novel mechanism to regulate Rac1 function.

Accumulating evidence has implicated Rho GTPases, including Rac1, in many aspects of cancer development. Recent findings suggest that phosphorylation might further contribute to the tight regulation of Rho GTPases. Interestingly, sequence analysis of Rac1 shows that Rac1 T108 within the (106)PNTP(109) motif is likely an extracellular signal-regulated kinase (ERK) phosphorylation site and that Rac1 also has an ERK docking site, (183)KKRKRKCLLL(192) (D site), at the C terminus. Indeed, we show here that both transfected and endogenous Rac1 interacts with ERK and that this interaction is mediated by its D site. Green fluorescent protein (GFP)-Rac1 is threonine (T) phosphorylated in response to epidermal growth factor (EGF), and EGF-induced Rac1 threonine phosphorylation is dependent on the activation of ERK. Moreover, mutant Rac1 with the mutation of T108 to alanine (A) is not threonine phosphorylated in response to EGF. In vitro ERK kinase assay further shows that pure active ERK phosphorylates purified Rac1 but not mutant Rac1 T108A. We also show that Rac1 T108 phosphorylation decreases Rac1 activity, partially due to inhibiting its interaction with phospholipase C-γ1 (PLC-γ1). T108 phosphorylation targets Rac1 to the nucleus, which isolates Rac1 from other guanine nucleotide exchange factors (GEFs) and hinders Rac1's role in cell migration. We conclude that Rac1 T108 is phosphorylated by ERK in response to EGF, which plays an important role in regulating Rac1.

Multi-directional function of the protein phosphatase 1 regulatory subunit TIMAP.

TIMAP is an endothelial-cell predominant member of the MYPT family of PP1c regulatory subunits. This study explored the TIMAP-PP1c interaction and substrate specificity in vitro. TIMAP associated with all three PP1c isoforms, but endogenous endothelial cell TIMAP preferentially co-immunoprecipitated with PP1cß. Structural modeling of the TIMAP/PP1c complex predicts that the PP1c C-terminus is buried in the TIMAP ankyrin cluster, and that the PP1c active site remains accessible. Consistent with this model, C-terminal PP1c phosphorylation by cdk2-cyclinA was masked by TIMAP, and PP1c bound TIMAP when the active site was occupied by the inhibitor microcystin. TIMAP inhibited PP1c activity toward phosphorylase a in a concentration-dependent manner, with half-maximal inhibition in the 0.4-1.2 nM range, an effect modulated by the length, and by Ser333/Ser337 phosphomimic mutations of the TIMAP C-terminus. TIMAP-bound PP1cß effectively dephosphorylated MLC2 and TIMAP itself. By contrast, TIMAP inhibited the PP1cß activity toward the putative substrate LAMR1, and instead masked LAMR1 PKA- and PKC-phosphorylation sites. This is direct evidence that MLC2 is a TIMAP/PP1c substrate. The data also indicate that TIMAP can modify protein phosphorylation independent of its function as a PP1c regulatory subunit, namely by masking phosphorylation sites of binding partners like PP1c and LAMR1.

Glomerular endothelium: a porous sieve and formidable barrier.

The glomerular capillary endothelium is highly specialized to support the selective filtration of massive volumes of plasma. Filtration is driven by Starling forces acting across the glomerular capillary wall, and depends on its large surface area and extremely high water permeability. Glomerular endothelial cells are extremely flat and perforated by dense arrays of trans-cellular pores, the fenestrae. This phenotype is critical for the high glomerular water permeability and depends on podocyte-derived VEGF, as well as TGF-beta. Endothelial cell-derived PDGFB, in turn, is necessary for the establishment of mesangial cells, which sculpt the glomerular loop structure that underlies the large filtration surface area. In pre-eclampsia, inhibition of the VEGF- and TGF-beta signaling pathways leads to endothelial swelling and loss of fenestrae, reducing the glomerular filtration rate. Similarly, in the thrombotic microangiopathies, glomerular endothelial cell injury coupled with inappropriate VWF activation leads to intracapillary platelet aggregation and loss of the flat, fenestrated phenotype, thus reducing the glomerular filtration rate. Normally, a remarkably small fraction of albumin and other large plasma proteins passes across the glomerular capillary wall despite the massive filtration of water and small solutes. An elaborate glycocalyx, which covers glomerular endothelial cells and their fenestrae forms an impressive barrier that, together with other components of the glomerular capillary wall, prevents loss of plasma proteins into the urine. Indeed, microalbuminuria is a marker for endothelial glycocalyx disruption, and most forms of glomerular endothelial cell injury including pre-eclampsia and thrombotic microangiopaties can cause proteinuria.

Repressors NFI and NFY participate in organ-specific regulation of von Willebrand factor promoter activity in transgenic mice.

OBJECTIVE: To determine the role of repressors in cell type and organ-specific activation of von Willebrand factor (VWF) promoter sequences -487 to 247 in vivo. METHODS AND RESULTS: Activation patterns of wild-type and mutant VWF promoters (sequences -487 to 247) containing mutations in repressors nuclear factor-I (NFI)- and nuclear factor Y (NFY)-binding sites were analyzed in transgenic mice. Mutation of the NFI-binding site activated the promoter in heart and lung endothelial cells, whereas mutation of the NFY-binding site activated the promoter in kidney vasculature. Immunofluorescence analyses showed that NFIB was predominant in heart and lung endothelial cells, whereas NFIX was predominantly detected in kidney endothelial cell nuclei. By using chromatin immunoprecipitation, we demonstrated that the distal lung-specific enhancer (containing a YY1 site) of the VWF gene is brought in proximity to the NFI binding site. CONCLUSIONS: The NFI and NFY repressors contribute differentially to organ-specific regulation of the VWF promoter, and the organ-specific action of NFI may reflect its organ-specific isoform distribution. In addition, the lung-specific enhancer region of the endogenous VWF gene may inhibit NFI repressor function through chromatin looping, which can approximate the 2 regions.

CLIC5A, a component of the ezrin-podocalyxin complex in glomeruli, is a determinant of podocyte integrity.

The chloride intracellular channel 5A (CLIC5A) protein, one of two isoforms produced by the CLIC5 gene, was isolated originally as part of a cytoskeletal protein complex containing ezrin from placental microvilli. Whether CLIC5A functions as a bona fide ion channel is controversial. We reported previously that a CLIC5 transcript is enriched approximately 800-fold in human renal glomeruli relative to most other tissues. Therefore, this study sought to explore CLIC5 expression and function in glomeruli. RT-PCR and Western blots show that CLIC5A is the predominant CLIC5 isoform expressed in glomeruli. Confocal immunofluorescence and immunogold electron microscopy reveal high levels of CLIC5A protein in glomerular endothelial cells and podocytes. In podocytes, CLIC5A localizes to the apical plasma membrane of foot processes, similar to the known distribution of podocalyxin and ezrin. Ezrin and podocalyxin colocalize with CLIC5A in glomeruli, and podocalyxin coimmunoprecipitates with CLIC5A from glomerular lysates. In glomeruli of jitterbug (jbg/jbg) mice, which lack the CLIC5A protein, ezrin and phospho-ERM levels in podocytes are markedly lower than in wild-type mice. Transmission electron microscopy reveals patchy broadening and effacement of podocyte foot processes as well as vacuolization of glomerular endothelial cells. These ultrastructural changes are associated with microalbuminuria at baseline and increased susceptibility to adriamycin-induced glomerular injury compared with wild-type mice. Together, the data suggest that CLIC5A is required for the development and/or maintenance of the proper glomerular endothelial cell and podocyte architecture. We postulate that the interaction between podocalyxin and subjacent filamentous actin, which requires ezrin, is compromised in podocytes of CLIC5A-deficient mice, leading to dysfunction under unfavorable genetic or environmental conditions.

A human glomerular SAGE transcriptome database.

BACKGROUND: To facilitate in the identification of gene products important in regulating renal glomerular structure and function, we have produced an annotated transcriptome database for normal human glomeruli using the SAGE approach. DESCRIPTION: The database contains 22,907 unique SAGE tag sequences, with a total tag count of 48,905. For each SAGE tag, the ratio of its frequency in glomeruli relative to that in 115 non-glomerular tissues or cells, a measure of transcript enrichment in glomeruli, was calculated. A total of 133 SAGE tags representing well-characterized transcripts were enriched 10-fold or more in glomeruli compared to other tissues. Comparison of data from this study with a previous human glomerular Sau3A-anchored SAGE library reveals that 47 of the highly enriched transcripts are common to both libraries. Among these are the SAGE tags representing many podocyte-predominant transcripts like WT-1, podocin and synaptopodin. Enrichment of podocyte transcript tags SAGE library indicates that other SAGE tags observed at much higher frequencies in this glomerular compared to non-glomerular SAGE libraries are likely to be glomerulus-predominant. A higher level of mRNA expression for 19 transcripts represented by glomerulus-enriched SAGE tags was verified by RT-PCR comparing glomeruli to lung, liver and spleen. CONCLUSION: The database can be retrieved from, or interrogated online at http://cgap.nci.nih.gov/SAGE. The annotated database is also provided as an additional file with gene identification for 9,022, and matches to the human genome or transcript homologs in other species for 1,433 tags. It should be a useful tool for in silico mining of glomerular gene expression.

Resolved: capillary endothelium is a major contributor to the glomerular filtration barrier.

There is growing debate as to the primal role of capillary endothelial cells in the barrier function of the glomerular filter. Two experts argue the points for and against. Clearly unresolved is agreement whether glomerular capillary endothelium has fenestrae subtended by diaphragms.

Phosphorylation of TIMAP by glycogen synthase kinase-3beta activates its associated protein phosphatase 1.

TIMAP (TGF-beta1 inhibited, membrane-associated protein) is a prenylated, endothelial cell-predominant protein phosphatase 1 (PP1c) regulatory subunit that localizes to the plasma membrane of filopodia. Here, we determined whether phosphorylation regulates TIMAP-associated PP1c function. Phosphorylation of TIMAP was observed in cells metabolically labeled with [32P]orthophosphate and was reduced by inhibitors of protein kinase A (PKA) and glycogen synthase kinase-3 (GSK-3). In cell-free assays, immunopurified TIMAP was phosphorylated by PKA and, after PKA priming, by GSK-3beta. Site-specific Ser to Ala substitution identified amino acid residues Ser333/Ser337 as the likely PKA/GSK-3beta phosphorylation site. Substitution of Ala for Val and Phe in the KVSF motif of TIMAP (TIMAPV64A/F66A) abolished PP1c binding and TIMAP-associated PP1c activity. TIMAPV64A/F66A was hyper-phosphorylated in cells, indicating that TIMAP-associated PP1c auto-dephosphorylates TIMAP. Constitutively active GSK-3beta stimulated phosphorylation of TIMAPV64A/F66A, but not wild-type TIMAP, suggesting that the PKA/GSK-3beta site may be subject to dephosphorylation by TIMAP-associated PP1c. Substitution of Asp or Glu for Ser at amino acid residues 333 and 337 to mimic phosphorylation reduced the PP1c association with TIMAP. Conversely, GSK-3 inhibitors augmented PP1c association with TIMAP-PP1c in cells. The 333/337 phosphomimic mutations also increased TIMAP-associated PP1c activity in vitro and against the non-integrin laminin receptor 1 in cells. Finally, TIMAP mutants with reduced PP1c activity strongly stimulated endothelial cell filopodia formation, an effect mimicked by the GSK-3 inhibitor LiCl. We conclude that TIMAP is a target for PKA-primed GSK-3beta-mediated phosphorylation. This phosphorylation controls TIMAP association and activity of PP1c, in turn regulating extension of filopodia in endothelial cells.