Publications by authors named "Sanja Sever"

38 Publications

Role of actin cytoskeleton in podocytes.

Authors:
Sanja Sever

Pediatr Nephrol 2020 Nov 13. Epub 2020 Nov 13.

Harvard Medical School and Massachusetts General Hospital, Boston, MA, USA.

The selectivity of the glomerular filter is established by physical, chemical, and signaling interplay among its three core constituents: glomerular endothelial cells, the glomerular basement membrane, and podocytes. Functional impairment or injury of any of these three components can lead to proteinuria. Podocytes are injured in many forms of human and experimental glomerular disease, including minimal change disease, focal segmental glomerulosclerosis, and diabetes mellitus. One of the earliest signs of podocyte injury is loss of their distinct structure, which is driven by dysregulated dynamics of the actin cytoskeleton. The status of the actin cytoskeleton in podocytes depends on a set of actin binding proteins, nucleators and inhibitors of actin polymerization, and regulatory GTPases. Mutations that alter protein function in each category have been implicated in glomerular diseases in humans and animal models. In addition, a growing body of studies suggest that pharmacological modifications of the actin cytoskeleton have the potential to become novel therapeutics for podocyte-dependent chronic kidney diseases. This review presents an overview of the essential proteins that establish actin cytoskeleton in podocytes and studies demonstrating the feasibility of drugging actin cytoskeleton in kidney diseases.
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http://dx.doi.org/10.1007/s00467-020-04812-zDOI Listing
November 2020

Soluble Urokinase Receptor and Acute Kidney Injury.

N Engl J Med 2020 01;382(5):416-426

From the Division of Cardiology, Department of Medicine, University of Michigan, Ann Arbor (S.S.H.); the Divisions of Renal Medicine (D.E.L., S. Sharma, S.S.W.) and Pulmonary and Critical Care Medicine (R.M.B.), Brigham and Women's Hospital, the Section of Nephrology, Department of Medicine, Boston University School of Medicine (S.S.W.), and the Divisions of Nephrology (S. Sever) and Cardiology (A.C., N.E.I., J.L.J.), Massachusetts General Hospital - all in Boston; Emory Clinical Cardiovascular Research Institute, Emory University School of Medicine, Atlanta (A.S.T., M.R., A.A.Q.); the Department of Medicine, Rush University Medical Center, Chicago (X.W., R.R.D., M.M.A., C.W., J.R.); the Section of Nephrology, Department of Medicine, Baylor College of Medicine, Houston (D.S.-H., J.S.-C.P., M.W.H.); and the Veterans Affairs Pittsburgh Healthcare System and the University of Pittsburgh School of Medicine, Pittsburgh (S.D.W.).

Background: Acute kidney injury is common, with a major effect on morbidity and health care utilization. Soluble urokinase plasminogen activator receptor (suPAR) is a signaling glycoprotein thought to be involved in the pathogenesis of kidney disease. We investigated whether a high level of suPAR predisposed patients to acute kidney injury in multiple clinical contexts, and we used experimental models to identify mechanisms by which suPAR acts and to assess it as a therapeutic target.

Methods: We measured plasma levels of suPAR preprocedurally in patients who underwent coronary angiography and patients who underwent cardiac surgery and at the time of admission to the intensive care unit in critically ill patients. We assessed the risk of acute kidney injury at 7 days as the primary outcome and acute kidney injury or death at 90 days as a secondary outcome, according to quartile of suPAR level. In experimental studies, we used a monoclonal antibody to urokinase plasminogen activator receptor (uPAR) as a therapeutic strategy to attenuate acute kidney injury in transgenic mice receiving contrast material. We also assessed cellular bioenergetics and generation of reactive oxygen species in human kidney proximal tubular (HK-2) cells that were exposed to recombinant suPAR.

Results: The suPAR level was assessed in 3827 patients who were undergoing coronary angiography, 250 who were undergoing cardiac surgery, and 692 who were critically ill. Acute kidney injury developed in 318 patients (8%) who had undergone coronary angiography. The highest suPAR quartile (vs. the lowest) had an adjusted odds ratio of 2.66 (95% confidence interval [CI], 1.77 to 3.99) for acute kidney injury and 2.29 (95% CI, 1.71 to 3.06) for acute kidney injury or death at 90 days. Findings were similar in the surgical and critically ill cohorts. The suPAR-overexpressing mice that were given contrast material had greater functional and histologic evidence of acute kidney injury than wild-type mice. The suPAR-treated HK-2 cells showed heightened energetic demand and mitochondrial superoxide generation. Pretreatment with a uPAR monoclonal antibody attenuated kidney injury in suPAR-overexpressing mice and normalized bioenergetic changes in HK-2 cells.

Conclusions: High suPAR levels were associated with acute kidney injury in various clinical and experimental contexts. (Funded by the National Institutes of Health and others.).
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http://dx.doi.org/10.1056/NEJMoa1911481DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7065830PMC
January 2020

uPAR isoform 2 forms a dimer and induces severe kidney disease in mice.

J Clin Invest 2019 04 2;129(5):1946-1959. Epub 2019 Apr 2.

Department of Medicine, Rush University Medical Center, Chicago, Illinois, USA.

Soluble urokinase plasminogen activator receptor (suPAR) is an immune-derived circulating signaling molecule that has been implicated in chronic kidney disease, such as focal segmental glomerulosclerosis (FSGS). Typically, native uPAR (isoform 1) translates to a 3-domain protein capable of binding and activating integrins, yet the function of additional isoforms generated by alternative splicing is unknown. Here, we characterized mouse uPAR isoform 2 (msuPAR2), encoding domain I and nearly one-half of domain II, as a dimer in solution, as revealed by 3D electron microscopy structural analysis. In vivo, msuPAR2 transgenic mice exhibited signs of severe renal disease characteristic of FSGS with proteinuria, loss of kidney function, and glomerulosclerosis. Sequencing of the glomerular RNAs from msuPAR2-Tg mice revealed a differentially expressed gene signature that includes upregulation of the suPAR receptor Itgb3, encoding β3 integrin. Crossing msuPAR2-transgenic mice with 3 different integrin β3 deficiency models rescued msuPAR2-mediated kidney function. Further analyses indicated a central role for β3 integrin and c-Src in msuPAR2 signaling and in human FSGS kidney biopsies. Administration of Src inhibitors reduced proteinuria in msuPAR2-transgenic mice. In conclusion, msuPAR2 may play an important role in certain forms of scarring kidney disease.
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http://dx.doi.org/10.1172/JCI124793DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6486353PMC
April 2019

Actin dynamics at focal adhesions: a common endpoint and putative therapeutic target for proteinuric kidney diseases.

Kidney Int 2018 06 17;93(6):1298-1307. Epub 2018 Apr 17.

Department of Nephrology, Hannover Medical School, Hannover, Germany. Electronic address:

Proteinuria encompasses diverse causes including both genetic diseases and acquired forms such as diabetic and hypertensive nephropathy. The basis of proteinuria is a disturbance in size selectivity of the glomerular filtration barrier, which largely depends on the podocyte: a terminally differentiated epithelial cell type covering the outer surface of the glomerulus. Compromised podocyte structure is one of the earliest signs of glomerular injury. The phenotype of diverse animal models and podocyte cell culture firmly established the essential role of the actin cytoskeleton in maintaining functional podocyte structure. Podocyte foot processes, actin-based membrane extensions, contain 2 molecularly distinct "hubs" that control actin dynamics: a slit diaphragm and focal adhesions. Although loss of foot processes encompasses disassembly of slit diaphragm multiprotein complexes, as long as cells are attached to the glomerular basement membrane, focal adhesions will be the sites in which stress due to filtration flow is counteracted by forces generated by the actin network in foot processes. Numerous studies within last 20 years have identified actin binding and regulatory proteins as well as integrins as essential components of signaling and actin dynamics at focal adhesions in podocytes, suggesting that some of them may become novel, druggable targets for proteinuric kidney diseases. Here we review evidence supporting the idea that current treatments for chronic kidney diseases beneficially and directly target the podocyte actin cytoskeleton associated with focal adhesions and suggest that therapeutic reagents that target the focal adhesion-regulated actin cytoskeleton in foot processes have potential to modernize treatments for chronic kidney diseases.
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http://dx.doi.org/10.1016/j.kint.2017.12.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5967993PMC
June 2018

Rituximab and Therapeutic Plasma Exchange in Recurrent Focal Segmental Glomerulosclerosis Postkidney Transplantation.

Transplantation 2018 03;102(3):e115-e120

Division of Nephrology, Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD.

Background: Focal segmental glomerulosclerosis (FSGS) is a common cause of end-stage renal disease with a high rate of recurrence after kidney transplantation. Several factors, such as white race, rapid progression, and previous allograft failure due to recurrence, were found to be risks of recurrence. Data are limited on the benefits of rituximab and/or therapeutic plasma exchange (TPE) in preventing recurrence. In this study, we sought to assess the efficacy of rituximab and TPE for the prevention and treatment of recurrent FSGS after kidney transplantation.

Methods: We enrolled 66 patients with FSGS in this prospective observational study and followed their outcomes. Patients with high risk for recurrence received preventative therapy with TPE and/or rituximab.

Results: Twenty-three (62%) of the 37 patients who received preventative therapy developed recurrence compared with 14 (51%) recurrences of the 27 patients who did not receive any therapy (P = 0.21). There was a trend for less relapse when rituximab was used as a therapy for recurrent FSGS (6/22 vs 9/18, P = 0.066). We used a clinical score of 5 values to assess the prediction of FSGS recurrence. A score of 3 or more had a predictive receiver operating characteristic curve of 0.72. Treatment with TPE and/or rituximab resulted in better allograft survival than historical studies. Allograft failure because of recurrent FSGS occurred in only 6 (9%) patients.

Conclusions: Preventative therapies do not decrease the recurrence rate of recurrent FSGS. However, prompt treatment of recurrence with these therapies may result in improved outcomes.
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http://dx.doi.org/10.1097/TP.0000000000002008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5820165PMC
March 2018

Cardiovascular Disease Biomarkers and suPAR in Predicting Decline in Renal Function: A Prospective Cohort Study.

Kidney Int Rep 2017 May 9;2(3):425-432. Epub 2017 Feb 9.

Division of Cardiology, Emory University School of Medicine, Atlanta, Georgia, USA.

Introduction: Soluble urokinase-type plasminogen activator receptor (suPAR) strongly predicts outcomes and incident chronic kidney disease (CKD) in patients with cardiovascular disease (CVD). Whether the association between suPAR and CKD is a reflection of its overall association with chronic inflammation and poor CVD outcomes is unclear. We examined whether CVD biomarkers, including high-sensitivity C-reactive protein (hs-CRP), fibrin-degradation products (FDPs), heat-shock protein 70 (HSP-70), and high-sensitivity troponin I (hs-TnI) were associated with a decline in kidney function in the Emory Cardiovascular Biobank cohort, in which suPAR levels were shown to be predictive of both incident CKD and CVD outcomes.

Methods: We measured suPAR, hs-CRP, HSP-70, FDP, and hs-TnI plasma levels in 3282 adults (mean age 63 years, 64% male, 75% estimated glomerular filtration rate [eGFR] >60 ml/min per 1.73 m). Glomerular filtration rate was estimated using Chronic Kidney Disease-Epidemiology Collaboration (eGFR) at enrollment (n = 3282) and follow-up (n = 2672; median 3.5 years). Urine protein by dipstick at baseline was available for 1335 subjects.

Results: There was a weak correlation among biomarkers (r range: 0.17-0.28). hs-CRP, FDPs, hs-TnI, and suPAR were independently associated with baseline eGFR and proteinuria. The median yearly decline in eGFR was -0.6 ml/min per 1.73 m. hs-CRP (β: -0.04;  = 0.46), FDPs (β: -0.13;  = 0.08), HSP-70 (β: 0.05;  = 0.84), or hs-TnI (β: -0.01;  = 0.76) were associated with eGFR decline. suPAR remained predictive of eGFR decline even after adjusting for all biomarkers.

Discussion: hs-CRP, FDP, HSP-70, and hs-TnI were not associated with eGFR decline. The specific association of suPAR with eGFR decline supported its involvement in pathways specific to the pathogenesis of kidney disease.
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http://dx.doi.org/10.1016/j.ekir.2017.02.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5678674PMC
May 2017

Association of Serum Soluble Urokinase Receptor Levels With Progression of Kidney Disease in Children.

JAMA Pediatr 2017 11 6;171(11):e172914. Epub 2017 Nov 6.

Department of Medicine, Rush University Medical Center, Chicago, Illinois.

Importance: Conventional methods to diagnose and monitor chronic kidney disease (CKD) in children, such as creatinine level and cystatin C-derived estimated glomerular filtration rate (eGFR) and assessment of proteinuria in spot or timed urine samples, are of limited value in identifying patients at risk of progressive kidney function loss. Serum soluble urokinase receptor (suPAR) levels strongly predict incident CKD stage 3 in adults.

Objective: To determine whether elevated suPAR levels are associated with renal disease progression in children with CKD.

Design, Setting, And Participants: Post hoc analysis of 2 prospectively followed up pediatric CKD cohorts, ie, the ESCAPE Trial (1999-2007) and the 4C Study (2010-2016), with serum suPAR level measured at enrollment and longitudinal eGFR measured prospectively. In the 2 trials, a total of 898 children were observed at 30 (ESCAPE Trial; n = 256) and 55 (4C Study; n = 642) tertiary care hospitals in 13 European countries. Renal diagnoses included congenital anomalies of the kidneys and urinary tract (n = 637 [70.9%]), tubulointerstitial nephropathies (n = 92 [10.2%]), glomerulopathies (n = 69 [7.7%]), postischemic CKD (n = 42 [4.7%]), and other CKD (n = 58 [6.5%]). Total follow-up duration was up to 7.9 years, and median follow-up was 3.1 years. Analyses were conducted from October 2016 to December 2016.

Exposures: Serum suPAR level was measured at enrollment, and eGFR was measured every 2 months in the ESCAPE Trial and every 6 months in the 4C Study. The primary end point of CKD progression was a composite of 50% eGFR loss, eGFR less than 10 mL/min/1.73 m2, or initiation of renal replacement therapy.

Main Outcomes And Measures: The primary end point in this study was renal survival, defined as a composite of 50% loss of GFR that persisted for at least 1 month, the start of renal replacement therapy, or an eGFR less than 10 mL/min/1.73 m2.

Results: Of the 898 included children, 560 (62.4%) were male, and the mean (SD) patient age at enrollment was 11.9 (3.5) years. The mean (SD) eGFR was 34 (16) mL/min/1.73 m2. The 5-year end point-free renal survival was 64.5% (95% CI, 57.4-71.7) in children with suPAR levels in the lowest quartile compared with 35.9% (95% CI, 28.7-43.0) in those in the highest quartile (P < .001). By multivariable analysis, the risk of attaining the end point was higher in children with glomerulopathies and increased with age, blood pressure, proteinuria, and lower eGFR at baseline. In patients with baseline eGFR greater than 40 mL/min/1.73 m2, higher log-transformed suPAR levels were associated with a higher risk of CKD progression after adjustment for traditional risk factors (hazard ratio, 5.12; 95% CI, 1.56-16.7; P = .007).

Conclusions And Relevance: Patients with high suPAR levels were more likely to have progression of their kidney disease. Further studies should determine whether suPAR levels can identify children at risk for future CKD.
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http://dx.doi.org/10.1001/jamapediatrics.2017.2914DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6121753PMC
November 2017

A tripartite complex of suPAR, APOL1 risk variants and αβ integrin on podocytes mediates chronic kidney disease.

Nat Med 2017 Aug 26;23(8):945-953. Epub 2017 Jun 26.

Department of Internal Medicine, Rush University Medical Center, Chicago, Illinois, USA.

Soluble urokinase plasminogen activator receptor (suPAR) independently predicts chronic kidney disease (CKD) incidence and progression. Apolipoprotein L1 (APOL1) gene variants G1 and G2, but not the reference allele (G0), are associated with an increased risk of CKD in individuals of recent African ancestry. Here we show in two large, unrelated cohorts that decline in kidney function associated with APOL1 risk variants was dependent on plasma suPAR levels: APOL1-related risk was attenuated in patients with lower suPAR, and strengthened in those with higher suPAR levels. Mechanistically, surface plasmon resonance studies identified high-affinity interactions between suPAR, APOL1 and αβ integrin, whereby APOL1 protein variants G1 and G2 exhibited higher affinity for suPAR-activated avb3 integrin than APOL1 G0. APOL1 G1 or G2 augments αβ integrin activation and causes proteinuria in mice in a suPAR-dependent manner. The synergy of circulating factor suPAR and APOL1 G1 or G2 on αβ integrin activation is a mechanism for CKD.
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http://dx.doi.org/10.1038/nm.4362DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6019326PMC
August 2017

Soluble Urokinase Plasminogen Activator Receptor and Outcomes in Patients with Diabetes on Hemodialysis.

Clin J Am Soc Nephrol 2017 Aug 11;12(8):1265-1273. Epub 2017 May 11.

Due to the number of contributing authors, the affiliations are provided in the Supplemental Material.

Background And Objectives: Soluble urokinase plasminogen activator receptor is a novel biomarker strongly predictive of cardiovascular outcomes implicated in the pathogenesis of kidney disease. Soluble urokinase plasminogen activator receptor levels, however, correlate with declining kidney function. It is unclear whether soluble urokinase plasminogen activator receptor levels remain associated with outcomes in patients with ESRD.

Design, Setting, Participants, & Measurements: We measured plasma soluble urokinase plasminogen activator receptor levels in 1175 patients (mean age =66±8 years old, 54% men) with type 2 diabetes mellitus on hemodialysis participating in the German Diabetes and Dialysis Study followed for a median of 4 years for outcomes including all-cause death, cardiovascular events, and infection-related mortality. Survival analysis was performed using stepwise Cox proportional hazards models adjusted for potential confounders. Also, adjustments were made for inflammatory markers (C-reactive protein and leukocyte count) and the oxidative stress marker asymmetric dimethyl arginine to investigate potential mediators of the relationship between soluble urokinase plasminogen activator receptor and outcomes.

Results: Median soluble urokinase plasminogen activator receptor levels were 10,521 pg/ml (interquartile range, 9105-12,543 pg/ml). When stratified by tertiles, patients with soluble urokinase plasminogen activator receptor >11,633 pg/ml (third tertile) had adjusted 1.6-fold higher mortality (hazard ratio, 1.60; 95% confidence interval, 1.27 to 2.03) compared with those with low soluble urokinase plasminogen activator receptor <9599 pg/ml (first tertile). Risks of sudden death and stroke were higher (adjusted hazard ratio, 1.98; 95% confidence interval, 1.27 to 3.09 and adjusted hazard ratio, 1.74; 95% confidence interval, 1.05 to 2.90, respectively), together accounting for higher incidence of cardiovascular events (adjusted hazard ratio, 1.48; 95% confidence interval, 1.15 to 1.89). Associations with outcomes persisted after adjusting for C-reactive protein, leukocyte count, and asymmetric dimethyl arginine. Addition of soluble urokinase plasminogen activator receptor to a risk factor model modestly improved risk discrimination for all-cause death ( statistic, 0.02; 95% confidence interval, 0.00 to 0.03) and cardiovascular events ( statistic, 0.02; 95% confidence interval, 0.00 to 0.05).

Conclusions: The association of soluble urokinase plasminogen activator receptor levels with outcomes persists in patients on hemodialysis. Additional study is warranted to characterize the underlying pathways of that association, which may yield opportunities to develop new therapeutic strategies.
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http://dx.doi.org/10.2215/CJN.10881016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5544516PMC
August 2017

Bone marrow-derived immature myeloid cells are a main source of circulating suPAR contributing to proteinuric kidney disease.

Nat Med 2017 01 12;23(1):100-106. Epub 2016 Dec 12.

Department of Internal Medicine, Rush University Medical Center, Chicago, Illinois, USA.

Excess levels of protein in urine (proteinuria) is a hallmark of kidney disease that typically occurs in conjunction with diabetes, hypertension, gene mutations, toxins or infections but may also be of unknown cause (idiopathic). Systemic soluble urokinase plasminogen activator receptor (suPAR) is a circulating factor implicated in the onset and progression of chronic kidney disease (CKD), such as focal segmental glomerulosclerosis (FSGS). The cellular source(s) of elevated suPAR associated with future and progressing kidney disease is unclear, but is likely extra-renal, as the pathological uPAR is circulating and FSGS can recur even after a damaged kidney is replaced with a healthy donor organ. Here we report that bone marrow (BM) Gr-1 immature myeloid cells are responsible for the elevated, pathological levels of suPAR, as evidenced by BM chimera and BM ablation and cell transfer studies. A marked increase of Gr-1 myeloid cells was commonly found in the BM of proteinuric animals having high suPAR, and these cells efficiently transmit proteinuria when transferred to healthy mice. In accordance with the results seen in suPAR-associated proteinuric animal models, in which kidney damage is caused not by local podocyte-selective injury but more likely by systemic insults, a humanized xenograft model of FSGS resulted in an expansion of Gr-1 cells in the BM, leading to high plasma suPAR and proteinuric kidney disease. Together, these results identify suPAR as a functional connection between the BM and the kidney, and they implicate BM immature myeloid cells as a key contributor to glomerular dysfunction.
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http://dx.doi.org/10.1038/nm.4242DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5405698PMC
January 2017

Anks1a regulates COPII-mediated anterograde transport of receptor tyrosine kinases critical for tumorigenesis.

Nat Commun 2016 09 13;7:12799. Epub 2016 Sep 13.

Department of Biological Science, Sookmyung Women's University, Chungpa-ro 47gil 100, Yongsan-gu, Seoul 140-742, Korea.

ErbB2 signalling, which is amplified by EphA2 binding, is an important therapeutic target for breast cancer. Despite the importance of the EphA2/ErbB2 complex in promoting breast tumorigenesis, the mechanism by which these receptor tyrosine kinases (RTKs) are exported from the endoplasmic reticulum (ER) remains poorly understood. Here we report that the PTB adaptor Anks1a is specifically localized to the ER on its own serine phosphorylation. Once there, Anks1a acts as an important regulator of COPII-mediated EphA2 ER export. The Anks1a ankyrin repeat domain binds EphA2 and causes it to accumulate at sites of ER exit. Simultaneously, the Anks1a PTB domain binds Sec23. This induces internalization of EphA2 via COPII vesicles, while Anks1a remains behind on the ER membrane. EphA2 also binds ErbB2 in the ER and seems to load ErbB2 into growing COPII carriers. Together, our study reveals a novel mechanism that regulates the loading of RTKs into COPII vesicles.
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http://dx.doi.org/10.1038/ncomms12799DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5027278PMC
September 2016

Dynamin Autonomously Regulates Podocyte Focal Adhesion Maturation.

J Am Soc Nephrol 2017 02 18;28(2):446-451. Epub 2016 Jul 18.

Department of Medicine, Harvard Medical School, Division of Nephrology, Massachusetts General Hospital, Charlestown, Massachusetts; and

Rho family GTPases, the prototypical members of which are Cdc42, Rac1, and RhoA, are molecular switches best known for regulating the actin cytoskeleton. In addition to the canonical small GTPases, the large GTPase dynamin has been implicated in regulating the actin cytoskeleton via direct dynamin-actin interactions. The physiologic role of dynamin in regulating the actin cytoskeleton has been linked to the maintenance of the kidney filtration barrier. Additionally, the small molecule Bis-T-23, which promotes actin-dependent dynamin oligomerization and thus, increases actin polymerization, improved renal health in diverse models of CKD, implicating dynamin as a potential therapeutic target for the treatment of CKD. Here, we show that treating cultured mouse podocytes with Bis-T-23 promoted stress fiber formation and focal adhesion maturation in a dynamin-dependent manner. Furthermore, Bis-T-23 induced the formation of focal adhesions and stress fibers in cells in which the RhoA signaling pathway was downregulated by multiple experimental approaches. Our study suggests that dynamin regulates focal adhesion maturation by a mechanism parallel to and synergistic with the RhoA signaling pathway. Identification of dynamin as one of the essential and autonomous regulators of focal adhesion maturation suggests a molecular mechanism that underlies the beneficial effect of Bis-T-23 on podocyte physiology.
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http://dx.doi.org/10.1681/ASN.2016010008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5280009PMC
February 2017

Soluble Urokinase Receptor and Chronic Kidney Disease.

N Engl J Med 2015 Nov 5;373(20):1916-25. Epub 2015 Nov 5.

From the Division of Cardiology, Emory Clinical Cardiovascular Research Institute, Emory University School of Medicine (S.S.H., Y.-A.K., M.A., L.S.S., S.L., A.A.Q.), and the Department of Biostatistics and Bioinformatics, Emory University (Y.-A.K.) - both in Atlanta; the Department of Medicine, Harvard Medical School, Boston, and Division of Nephrology, Massachusetts General Hospital, Charlestown - both in Massachusetts (S.S.); the Department of Pediatrics, NYU Langone Medical Center, New York (H.T.); and the Department of Medicine, Rush University Medical Center (S.W., M.M.A., C.W., A.L.F., J.R.), and the Women's Interagency HIV Study/CORE Center of Cook County (A.L.H., A.L.F.) - both in Chicago.

Background: Relatively high plasma levels of soluble urokinase-type plasminogen activator receptor (suPAR) have been associated with focal segmental glomerulosclerosis and poor clinical outcomes in patients with various conditions. It is unknown whether elevated suPAR levels in patients with normal kidney function are associated with future decline in the estimated glomerular filtration rate (eGFR) and with incident chronic kidney disease.

Methods: We measured plasma suPAR levels in 3683 persons enrolled in the Emory Cardiovascular Biobank (mean age, 63 years; 65% men; median suPAR level, 3040 pg per milliliter) and determined renal function at enrollment and at subsequent visits in 2292 persons. The relationship between suPAR levels and the eGFR at baseline, the change in the eGFR over time, and the development of chronic kidney disease (eGFR <60 ml per minute per 1.73 m(2) of body-surface area) were analyzed with the use of linear mixed models and Cox regression after adjustment for demographic and clinical variables.

Results: A higher suPAR level at baseline was associated with a greater decline in the eGFR during follow-up; the annual change in the eGFR was -0.9 ml per minute per 1.73 m(2) among participants in the lowest quartile of suPAR levels as compared with -4.2 ml per minute per 1.73 m(2) among participants in the highest quartile (P<0.001). The 921 participants with a normal eGFR (≥ 90 ml per minute per 1.73 m(2)) at baseline had the largest suPAR-related decline in the eGFR. In 1335 participants with a baseline eGFR of at least 60 ml per minute per 1.73 m(2), the risk of progression to chronic kidney disease in the highest quartile of suPAR levels was 3.13 times as high (95% confidence interval, 2.11 to 4.65) as that in the lowest quartile.

Conclusions: An elevated level of suPAR was independently associated with incident chronic kidney disease and an accelerated decline in the eGFR in the groups studied. (Funded by the Abraham J. and Phyllis Katz Foundation and others.).
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http://dx.doi.org/10.1056/NEJMoa1506362DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4701036PMC
November 2015

CD2AP, dendrin, and cathepsin L in the kidney.

Am J Pathol 2015 Nov;185(11):3129-30

Department of Medicine, Rush University Medical Center, Chicago, Illinois. Electronic address:

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http://dx.doi.org/10.1016/j.ajpath.2015.06.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4630172PMC
November 2015

Pharmacological targeting of actin-dependent dynamin oligomerization ameliorates chronic kidney disease in diverse animal models.

Nat Med 2015 Jun 11;21(6):601-9. Epub 2015 May 11.

Department of Medicine, Harvard Medical School and Division of Nephrology, Massachusetts General Hospital, Charlestown, Massachusetts, USA.

Dysregulation of the actin cytoskeleton in podocytes represents a common pathway in the pathogenesis of proteinuria across a spectrum of chronic kidney diseases (CKD). The GTPase dynamin has been implicated in the maintenance of cellular architecture in podocytes through its direct interaction with actin. Furthermore, the propensity of dynamin to oligomerize into higher-order structures in an actin-dependent manner and to cross-link actin microfilaments into higher-order structures has been correlated with increased actin polymerization and global organization of the actin cytoskeleton in the cell. We found that use of the small molecule Bis-T-23, which promotes actin-dependent dynamin oligomerization and thus increased actin polymerization in injured podocytes, was sufficient to improve renal health in diverse models of both transient kidney disease and CKD. In particular, administration of Bis-T-23 in these renal disease models restored the normal ultrastructure of podocyte foot processes, lowered proteinuria, lowered collagen IV deposits in the mesangial matrix, diminished mesangial matrix expansion and extended lifespan. These results further establish that alterations in the actin cytoskeleton of kidney podocytes is a common hallmark of CKD, while also underscoring the substantial regenerative potential of injured glomeruli and identifying the oligomerization cycle of dynamin as an attractive potential therapeutic target to treat CKD.
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http://dx.doi.org/10.1038/nm.3843DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4458177PMC
June 2015

A Podocyte-Based Automated Screening Assay Identifies Protective Small Molecules.

J Am Soc Nephrol 2015 Nov 9;26(11):2741-52. Epub 2015 Apr 9.

Department of Medicine, Rush University Medical Center, Chicago, Illinois;

Podocyte injury and loss mark an early step in the pathogenesis of various glomerular diseases, making these cells excellent targets for therapeutics. However, cell-based high-throughput screening assays for the rational development of podocyte-directed therapeutics are currently lacking. Here, we describe a novel high-content screening-based phenotypic assay that analyzes thousands of podocytes per assay condition in 96-well plates to quantitatively measure dose-dependent changes in multiple cellular features. Our assay consistently produced a Z' value >0.44, making it suitable for compound screening. On screening with >2100 pharmacologically active agents, we identified 24 small molecules that protected podocytes against injury in vitro (1% hit rate). Among the identified hits, we confirmed an β1-integrin agonist, pyrintegrin, as a podocyte-protective agent. Treatment with pyrintegrin prevented damage-induced decreases in F-actin stress fibers, focal adhesions, and active β1-integrin levels in cultured cells. In vivo, administration of pyrintegrin protected mice from LPS-induced podocyte foot process effacement and proteinuria. Analysis of the murine glomeruli showed that LPS administration reduced the levels of active β1 integrin in the podocytes, which was prevented by cotreatment with pyrintegrin. In rats, pyrintegrin reduced peak proteinuria caused by puromycin aminonucleoside-induced nephropathy. Our findings identify pyrintegrin as a potential therapeutic candidate and show the use of podocyte-based screening assays for identifying novel therapeutics for proteinuric kidney diseases.
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http://dx.doi.org/10.1681/ASN.2014090859DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4625676PMC
November 2015

The grand challenge of nephrology.

Front Med (Lausanne) 2014 18;1:28. Epub 2014 Sep 18.

Department of Medicine, Rush University , Chicago, IL , USA.

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http://dx.doi.org/10.3389/fmed.2014.00028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4291843PMC
January 2015

Regulation of dynamin oligomerization in cells: the role of dynamin-actin interactions and its GTPase activity.

Traffic 2014 Aug 24;15(8):819-38. Epub 2014 Jun 24.

Division of Nephrology, Massachusetts General Hospital, Charlestown, MA, 02129, USA.

Dynamin is a 96-kDa protein that has multiple oligomerization states that influence its GTPase activity. A number of different dynamin effectors, including lipids, actin filaments, and SH3-domain-containing proteins, have been implicated in the regulation of dynamin oligomerization, though their roles in influencing dynamin oligomerization have been studied predominantly in vitro using recombinant proteins. Here, we identify higher order dynamin oligomers such as rings and helices in vitro and in live cells using fluorescence lifetime imaging microscopy (FLIM). FLIM detected GTP- and actin-dependent dynamin oligomerization at distinct cellular sites, including the cell membrane and transition zones where cortical actin transitions into stress fibers. Our study identifies a major role for direct dynamin-actin interactions and dynamin's GTPase activity in the regulation of dynamin oligomerization in cells.
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http://dx.doi.org/10.1111/tra.12178DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4107071PMC
August 2014

Reduction of proteinuria through podocyte alkalinization.

J Biol Chem 2014 Jun 9;289(25):17454-67. Epub 2014 May 9.

From the Department of Medicine, Rush University Medical Center, Chicago, Illinois 60035,

Podocytes are highly differentiated cells and critical elements for the filtration barrier of the kidney. Loss of their foot process (FP) architecture (FP effacement) results in urinary protein loss. Here we show a novel role for the neutral amino acid glutamine in structural and functional regulation of the kidney filtration barrier. Metabolic flux analysis of cultured podocytes using genetic, toxic, and immunologic injury models identified increased glutamine utilization pathways. We show that glutamine uptake is increased in diseased podocytes to couple nutrient support to increased demand during the disease state of FP effacement. This feature can be utilized to transport increased amounts of glutamine into damaged podocytes. The availability of glutamine determines the regulation of podocyte intracellular pH (pHi). Podocyte alkalinization reduces cytosolic cathepsin L protease activity and protects the podocyte cytoskeleton. Podocyte glutamine supplementation reduces proteinuria in LPS-treated mice, whereas acidification increases glomerular injury. In summary, our data provide a metabolic opportunity to combat urinary protein loss through modulation of podocyte amino acid utilization and pHi.
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http://dx.doi.org/10.1074/jbc.M114.568998DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4067184PMC
June 2014

Signal transduction in podocytes--spotlight on receptor tyrosine kinases.

Nat Rev Nephrol 2014 Feb 7;10(2):104-15. Epub 2014 Jan 7.

Division of Nephrology and Hypertension, University of Miami Miller School of Medicine, 1580 North West 10th Avenue (R-762), Batchelor Building 626, Miami, FL 33136, USA.

The mammalian kidney filtration barrier is a complex multicellular, multicomponent structure that maintains homeostasis by regulating electrolytes, acid-base balance, and blood pressure (via maintenance of salt and water balance). To perform these multiple functions, podocytes--an important component of the filtration apparatus--must process a series of intercellular signals. Integrating these signals with diverse cellular responses enables a coordinated response to various conditions. Although mature podocytes are terminally differentiated and cannot proliferate, they are able to respond to growth factors. It is possible that the initial response of podocytes to growth factors is beneficial and protective, and might include the induction of hypertrophic cell growth. However, extended and/or uncontrolled growth factor signalling might be maladaptive and could result in the induction of apoptosis and podocyte loss. Growth factors signal via the activation of receptor tyrosine kinases (RTKs) on their target cells and around a quarter of the 58 RTK family members that are encoded in the human genome have been identified in podocytes. Pharmacological inhibitors of many RTKs exist and are currently used in experimental and clinical cancer therapy. The identification of pathological RTK-mediated signal transduction pathways in podocytes could provide a starting point for the development of novel therapies for glomerular disorders.
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http://dx.doi.org/10.1038/nrneph.2013.274DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4109315PMC
February 2014

Transient receptor potential channel 6 (TRPC6) protects podocytes during complement-mediated glomerular disease.

J Biol Chem 2013 Dec 5;288(51):36598-609. Epub 2013 Nov 5.

From the Division of Nephrology and Hypertension, Department of Medicine, University of Miami Miller School of Medicine, Miami, Florida 33136.

Gain-of-function mutations in the calcium channel TRPC6 lead to autosomal dominant focal segmental glomerulosclerosis and podocyte expression of TRPC6 is increased in some acquired human glomerular diseases, particularly in membranous nephropathy. These observations led to the hypothesis that TRPC6 overactivation is deleterious to podocytes through pathological calcium signaling, both in genetic and acquired diseases. Here, we show that the effects of TRPC6 on podocyte function are context-dependent. Overexpression of TRPC6 alone did not directly affect podocyte morphology and cytoskeletal structure. Unexpectedly, however, overexpression of TRPC6 protected podocytes from complement-mediated injury, whereas genetic or pharmacological TRPC6 inactivation increased podocyte susceptibility to complement. Mechanistically, this effect was mediated by Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) activation. Podocyte-specific TRPC6 transgenic mice showed stronger CaMKII activation, reduced podocyte foot process effacement and reduced levels of proteinuria during nephrotoxic serum nephritis, whereas TRPC6 null mice exhibited reduced CaMKII activation and higher levels of proteinuria compared with wild type littermates. Human membranous nephropathy biopsy samples showed podocyte staining for active CaMKII, which correlated with the degree of TRPC6 expression. Together, these data suggest a dual and context dependent role of TRPC6 in podocytes where acute activation protects from complement-mediated damage, but chronic overactivation leads to focal segmental glomerulosclerosis.
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http://dx.doi.org/10.1074/jbc.M113.488122DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3868772PMC
December 2013

Dynamin rings: not just for fission.

Traffic 2013 Dec 19;14(12):1194-9. Epub 2013 Sep 19.

Nephrology Division, Massachusetts General Hospital, CNY 149 8.113, 149 13th Street, Charlestown, MA, 02129, USA.

The GTPase dynamin has captivated researchers for over two decades, even managing to establish its own research field. Dynamin's allure is partly due to its unusual biochemical properties as well as its essential role in multiple cellular processes, which include the regulation of clathrin-mediated endocytosis and of actin cytoskeleton. On the basis of the classic model, dynamin oligomerization into higher order oligomers such as rings and helices directly executes the final fission reaction in endocytosis, which results in the generation of clathrin-coated vesicles. Dynamin's role in the regulation of actin cytoskeleton is mostly explained by its interactions with a number of actin-binding and -regulating proteins; however, the molecular mechanism of dynamin's action continues to elude us. Recent insights into the mechanism and role of dynamin oligomerization in the regulation of actin polymerization point to a novel role for dynamin oligomerization in the cell.
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http://dx.doi.org/10.1111/tra.12116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3830594PMC
December 2013

Podocyte biology and pathogenesis of kidney disease.

Annu Rev Med 2013 26;64:357-66. Epub 2012 Nov 26.

Department of Medicine, Rush University Medical Center, Chicago, Illinois 60612, USA.

Proteinuric chronic kidney disease (CKD), once a rare affliction believed to be mainly caused by genetic mutations, has become a global pandemic that severely diminishes the quality of life for millions. Despite the changing face of CKD, treatment options and resources remain woefully antiquated and have failed to arrest or reverse the effects of kidney-related diseases. Histological and genetic data strongly implicate one promising target: the podocyte. Podocytes are terminally differentiated cells of the kidney glomerulus that are essential for the integrity of the kidney filter. Their function is primarily based on their intricate structure, which includes foot processes. Loss of these actin-driven membrane extensions is tightly connected to the presence of protein in the urine, podocyte loss, development of CKD, and ultimately renal failure.
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http://dx.doi.org/10.1146/annurev-med-050311-163340DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3736800PMC
July 2013

CD2AP in mouse and human podocytes controls a proteolytic program that regulates cytoskeletal structure and cellular survival.

J Clin Invest 2011 Oct 12;121(10):3965-80. Epub 2011 Sep 12.

Nephrology Division, Department of Medicine, Harvard Medical School and Massachusetts General Hospital, Charlestown, Massachusetts, USA.

Kidney podocytes are highly differentiated epithelial cells that form interdigitating foot processes with bridging slit diaphragms (SDs) that regulate renal ultrafiltration. Podocyte injury results in proteinuric kidney disease, and genetic deletion of SD-associated CD2-associated protein (CD2AP) leads to progressive renal failure in mice and humans. Here, we have shown that CD2AP regulates the TGF-β1-dependent translocation of dendrin from the SD to the nucleus. Nuclear dendrin acted as a transcription factor to promote expression of cytosolic cathepsin L (CatL). CatL proteolyzed the regulatory GTPase dynamin and the actin-associated adapter synaptopodin, leading to a reorganization of the podocyte microfilament system and consequent proteinuria. CD2AP itself was proteolyzed by CatL, promoting sustained expression of the protease during podocyte injury, and in turn increasing the apoptotic susceptibility of podocytes to TGF-β1. Our study identifies CD2AP as the gatekeeper of the podocyte TGF-β response through its regulation of CatL expression and defines a molecular mechanism underlying proteinuric kidney disease.
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http://dx.doi.org/10.1172/JCI58552DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3195478PMC
October 2011

Direct dynamin-actin interactions regulate the actin cytoskeleton.

EMBO J 2010 Nov 8;29(21):3593-606. Epub 2010 Oct 8.

Nephrology Division, Department of Medicine, Harvard Medical School and Massachusetts General Hospital, Charlestown, MA 02129, USA.

The large GTPase dynamin assembles into higher order structures that are thought to promote endocytosis. Dynamin also regulates the actin cytoskeleton through an unknown, GTPase-dependent mechanism. Here, we identify a highly conserved site in dynamin that binds directly to actin filaments and aligns them into bundles. Point mutations in the actin-binding domain cause aberrant membrane ruffling and defective actin stress fibre formation in cells. Short actin filaments promote dynamin assembly into higher order structures, which in turn efficiently release the actin-capping protein (CP) gelsolin from barbed actin ends in vitro, allowing for elongation of actin filaments. Together, our results support a model in which assembled dynamin, generated through interactions with short actin filaments, promotes actin polymerization via displacement of actin-CPs.
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http://dx.doi.org/10.1038/emboj.2010.249DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2982766PMC
November 2010

Nucleoside diphosphate kinase Nm23-H1 regulates chromosomal stability by activating the GTPase dynamin during cytokinesis.

Proc Natl Acad Sci U S A 2010 Aug 16;107(35):15461-6. Epub 2010 Aug 16.

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.

Chromosomal instability and the subsequent genetic mutations are considered to be critical factors in the development of the majority of solid tumors. Here, we describe how the nucleoside diphosphate kinase Nm23-H1, a protein with a known link to cancer progression, regulates a critical step during cytokinesis. Nm23-H1 acts to provide a local source of GTP for the GTPase dynamin. Loss of Nm23-H1 in diploid cells leads to cytokinetic furrow regression, followed by cytokinesis failure and generation of tetraploid cells. Loss of dynamin phenocopies loss of Nm23-H1, and ectopic overexpression of WT dynamin complements the loss of Nm23-H1. In the absence of p53 signaling, the tetraploid cells resulting from loss of Nm23-H1 continue cycling and develop classic hallmarks of tumor cells. We thus provide evidence that the loss of Nm23-H1, an event suspected to promote metastasis, may additionally function at an earlier stage of tumor development to drive the acquisition of chromosomal instability.
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http://dx.doi.org/10.1073/pnas.1010633107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2932595PMC
August 2010

Establishment of protein delivery systems targeting podocytes.

PLoS One 2010 Jul 29;5(7):e11837. Epub 2010 Jul 29.

Department of Medicine, Massachusetts General Hospital, Boston, Massachusetts, United States of America.

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 beta-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 beta-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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0011837PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2912276PMC
July 2010

Synaptotagmin-mediated vesicle fusion regulates cell migration.

Nat Immunol 2010 Jun 16;11(6):495-502. Epub 2010 May 16.

Center for Immunology and Inflammatory Diseases, Massachusetts General Hospital, Boston, Massachusetts, USA.

Chemokines and other chemoattractants direct leukocyte migration and are essential for the development and delivery of immune and inflammatory responses. To probe the molecular mechanisms that underlie chemoattractant-guided migration, we did an RNA-mediated interference screen that identified several members of the synaptotagmin family of calcium-sensing vesicle-fusion proteins as mediators of cell migration: SYT7 and SYTL5 were positive regulators of chemotaxis, whereas SYT2 was a negative regulator of chemotaxis. SYT7-deficient leukocytes showed less migration in vitro and in a gout model in vivo. Chemoattractant-induced calcium-dependent lysosomal fusion was impaired in SYT7-deficient neutrophils. In a chemokine gradient, SYT7-deficient lymphocytes accumulated lysosomes in their uropods and had impaired uropod release. Our data identify a molecular pathway required for chemotaxis that links chemoattractant-induced calcium flux to exocytosis and uropod release.
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http://dx.doi.org/10.1038/ni.1878DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2951881PMC
June 2010