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Objective: FAT1 is a large transmembrane protocadherin highly expressed at the slit-diaphragm in podocyte foot-processes. FAT1 deficient mice die perinatally because of massive proteinuria. Ultrastructural analysis demonstrated that the primary defect was located in podocytes that have lost their ability to form actin-based interdigitating foot processes bridged by a slit-diaphragm. In this study, we have undertaken the first investigation of the molecular function of FAT1. Results: Several antisera against FAT1 were raised. In cultured cells, FAT1 was localized at the leading edge of lamellipodial protrusions and in filopodia, both of which are structures based on directed actin polymerization. Indeed, we were able to establish a direct interaction of FAT1 with regulatory proteins of directed actin polymerization: the Ena/VASP proteins. This interaction occurred in an EVH1-domain dependent fashion via a novel tandem repeat of EVH1-binding motifs in the cytoplasmic tail of FAT1. In accordance with our in vitro binding assays, FAT1 and Ena/VASP proteins co-localized in cultured cells. The functional significance of the FAT1-Ena/VASP interaction was investigated using a combination of RNA interference and a lentiviral transduction system. Our experiments indicated that FAT1 deficient cells exhibit a similar phenotype to cells with altered Ena/ VASP function. In addition, FAT1 deficient cells were evaluated in a wound assay. FAT1 deficient cells had lost their ability to polarize towards the denuded area and to migrate into the wound. Using a second experimental approach, we were able to confirm that FAT1 acts as an essential receptor for the orientation of cells in the horizontal plane of epithelia cells (planar cell polarity). Conclusions: These findings have important implications for our understanding of the molecular mechanisms at the slit-diaphragm as well as for the young field of planar cell polarity. [ABSTRACT FROM AUTHOR]

Background: Interstitial fibrosis and tubular atrophy (IFTA), and density and shape of peritubular capillaries (PTCs), are independently prognostic of disease progression. This study aimed to identify novel digital biomarkers of disease progression and assess the clinical relevance of the interplay between a variety of PTC characteristics and their microenvironment in glomerular diseases., Methods: A total of 344 NEPTUNE/CureGN participants were included: 112 minimal change disease, 134 focal segmental glomerulosclerosis, 61 membranous nephropathy, and 37 IgA nephropathy. A PAS-stained whole slide image per patient was manually segmented for cortex, pre- and mature IFTA. Interstitial fractional space (IFS) was computationally quantified. A deep-learning model was applied to segment PTCs. Spatial and shape PTC pathomic features (230) were extracted from the cortex, IFTA, and non-IFTA sub-regions. Participants were divided into training and testing datasets (1:1). Univariate models incorporating IFTA subregions, and IFS-PTC density were constructed. LASSO regression models were used to identify the top PTC features associated with disease progression stratified by IFTA and non-IFTA sub-regions. Machine learning models were built using the top PTC features in IFTA and non-IFTA sub-regions to prognosticate disease progression., Results: PTC density in pre+mature IFTA and IFS, shape features in pre+mature IFTA, and spatial architecture features in the non-IFTA regions associated with disease progression. The machine learning generated risk scores showed a significant association with disease progression on the independent testing set., Conclusion: We uncovered previously underrecognized digital biomarkers of disease progression and the clinical relevance of the complex interplay between the status of the PTCs and the interstitial microenvironment., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Society of Nephrology.)

Purpose: Our purpose is to develop a computer vision approach to quantify intra-arterial thickness on digital pathology images of kidney biopsies as a computational biomarker of arteriosclerosis., Approach: The severity of the arteriosclerosis was scored (0 to 3) in 753 arteries from 33 trichrome-stained whole slide images (WSIs) of kidney biopsies, and the outer contours of the media, intima, and lumen were manually delineated by a renal pathologist. We then developed a multi-class deep learning (DL) framework for segmenting the different intra-arterial compartments (training dataset: 648 arteries from 24 WSIs; testing dataset: 105 arteries from 9 WSIs). Subsequently, we employed radial sampling and made measurements of media and intima thickness as a function of spatially encoded polar coordinates throughout the artery. Pathomic features were extracted from the measurements to collectively describe the arterial wall characteristics. The technique was first validated through numerical analysis of simulated arteries, with systematic deformations applied to study their effect on arterial thickness measurements. We then compared these computationally derived measurements with the pathologists' grading of arteriosclerosis., Results: Numerical validation shows that our measurement technique adeptly captured the decreasing smoothness in the intima and media thickness as the deformation increases in the simulated arteries. Intra-arterial DL segmentations of media, intima, and lumen achieved Dice scores of 0.84, 0.78, and 0.86, respectively. Several significant associations were identified between arteriosclerosis grade and pathomic features using our technique (e.g., intima-media ratio average [ τ = 0.52 , p < 0.0001 ]) through Kendall's tau analysis., Conclusions: We developed a computer vision approach to computationally characterize intra-arterial morphology on digital pathology images and demonstrate its feasibility as a potential computational biomarker of arteriosclerosis., (© 2024 The Authors.)

Background: During the COVID-19 pandemic, SARS-CoV-2 monoclonal antibodies for preexposure prophylaxis (SMA-PrEP) offered patients who were immunocompromised another option for protection. However, SMA-PrEP posed administrative, operational, and ethical challenges for health care facilities, resulting in few patients receiving them. Although the first SMA-PrEP medication, tixagevimab and cilgavimab, had its authorization revoked due to compromised in vitro efficacy, new SMA-PrEP medications are currently completing clinical trials. This article provides an operational framework for administrative organization, patient identification and prioritization, equitable medication allocation, medication ordering and administration, and patient tracking., Methods: A retrospective cohort study evaluating our hospital's SMA-PrEP administration strategy was performed. Multivariable logistic regression was used to examine factors associated with receipt of SMA-PrEP., Results: Despite the barriers in administering this medication and the scarcity of resources, our hospital was able to administer at least 1 dose of SMA-PrEP to 1359 of 5902 (23.0%) eligible patients. Even with the steps taken to promote equitable allocation, multivariable logistic regression demonstrated that there were still differences by race, ethnicity, and socioeconomic status. As compared with patients who identified as Black, patients who identified as White (odds ratio [OR], 1.85; 95% CI, 1.46-2.33), Asian (OR, 1.59; 95% CI, 1.03-2.46), and Hispanic (OR, 1.53; 95% CI, 1.02-2.44) were more likely to receive SMA-PrEP. When compared with patients with low socioeconomic status, patients with high socioeconomic status (OR, 1.37; 95% CI, 1.05-1.78) were more likely to be allocated SMA-PrEP., Conclusions: Despite efforts to mitigate health care disparities, differences by race/ethnicity and socioeconomic status still arose in patients receiving SMA-PrEP., Competing Interests: Potential conflicts of interest. D. A. J. has received personal fees for advisory board participation and/or consulting from Biogen, BMS, Cycle Pharma, Horizon, Merck/EMD Serono, Novartis, Roche/Genentech, TG Therapeutics, and Sanofi-Genzyme and grant support to the University of Pennsylvania from Biogen Idec, Roche/Genentech, Merck/EMD Serono, and Novartis. All other authors report no potential conflicts., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Competing Interests: Debbie Gipson received research funding through the University of Michigan from Reata, Travere, Novartis, Boehringer Ingelheim, and Goldfinch Bio and past consulting through the University of Michigan from Novartis, Vertex, and Genentech/Roche. Chia-shi Wang, Eloise Salmon, Rasheed Gbadegesin, Abhijit Naik, and Simone Sanna-Cherchi have no disclosures. Alessia Fornoni is one of the inventors on pending (PCT/US2019/032215; US 17/057,247; PCT/US2019/041730; PCT/US2013/036484; US 17/259,883; US17/259,883; JP501309/2021 and EU19834217.2; CN-201980060078.3; CA2,930,119; CA3,012,773; and CA2,852,904) or issued patents (US10,183,038 and US10,052,345) aimed at preventing and treating renal disease. She stands to gain royalties from their future commercialization. AF is vice president of L&F Health LLC and is a consultant for ZyVersa Therapeutics, Inc. ZyVersa Therapeutics, Inc., has licensed worldwide rights to develop and commercialize hydroxypropyl-beta-cyclodextrin for the treatment of kidney disease from L&F Research, which was partially funded by L&F Health LLC. She also holds equities in Renal 3 River Corporation. Matthias Kretzler has received research support on behalf of the University of Michigan from Boehringer Ingelheim, Novo Nordisk, Certa, Poxel, Astellas, and Janssen, has received research funding from NIH, JDRF, Chan Zuckerburg Initiative, amfAR, AstraZeneca, Boehringer Ingelheim, Elpidera, Gilead, Goldfinch Bio, Eli Lilly, Angion Biomedica, Certa, Novo Nordisk, Janssen, Chinook, RenalytixAI, Regeneron Pharmaceuticals, Travere Therapeutics, and Ionis Pharmaceuticals, is on the editorial boards for J Am Soc Nephrology, Kidney Int, and Kidney Dis, and is on an advisory board for NephCure Kidney International. Sandra Merscher is an inventor on pending and issued patents aimed to diagnose or treat proteinuric renal diseases. She stands to gain royalties from their future commercialization. She holds equity interest in L&F Research and is a shareholder of ZyVersa Pharmaceuticals, Inc., who has licensed worldwide rights to develop and commercialize hydroxypropyl-beta-cyclodextrin for the treatment of kidney diseases from L&F Research. Paul Hoover, Kelley Kidwell, Moin Saleem, Leonard Riella, Lawrence Holzman, Annette Jackson, and Opeyemi Olabisi have no disclosures. Paolo Cravedi is a consultant for Chinook therapeutics, Repertoire Immune Medicines, and Calliditas Therapeutics. Benjamin Freedman is an inventor on a patent and patent applications related to human kidney organoid differentiation and modeling of FSGS in this system (e.g., “three-dimensional differentiation of epiblast spheroids into kidney tubular organoids modeling human micro-physiology, toxicology, and morphogenesis” [Japan, USA, and Australia], licensed to STEMCELL Technologies). He has ownership interest in Plurexa LLC. Hailey Desmond received research funding through the University of Michigan from Boehringer Ingelheim, Travere, Roche, Novartis, and Reata. Howard Trachtman is a consultant to Travere Therapeutics Inc., Walden, Boehringer Ingelheim (pending), Natera, Otsuka, and Aclipse. He is the board of the Kidney Health Initiative and the editorial board of Pediatric Nephrology and Kidney360.

Background: FSGS is a heterogeneous diagnosis with a guarded prognosis. Polymorphisms in the apolipoprotein L1 ( APOL1 ) gene are associated with developing FSGS and faster progression to kidney failure in affected patients. Better understanding the natural history of patients with FSGS and APOL1 risk alleles is essential to improve patient care and support the design and interpretation of interventional studies. The objective of this study was to evaluate the quantitative association between APOL1 and kidney disease progression and the interaction with other clinical and laboratory factors., Methods: CureGN cohort study participants with biopsy diagnosis of FSGS, regardless of self-identified race, were included. The exposure of interest was two APOL1 risk alleles (high risk) versus zero to one risk alleles (low risk). The primary outcome was eGFR slope categorized as rapid progressor (eGFR slope ≤-5 ml/min per year), intermediate progressor (slope between 0 and -5), or nonprogressor (slope ≥0). Multivariable ordinal logistic and linear regressions were used for adjusted analyses. Missing data were addressed using multiple imputation., Results: Of 650 participants, 476 (73%) had genetic testing, among whom 87 (18%) were high risk. High-risk participants were more likely to have lower median eGFR (62 [interquartile range, 36-81] versus low-risk participants 76 ml/min per 1.73 m 2 [interquartile range, 44-106]; P <0.01). In adjusted analysis, the odds of more rapid progression of eGFR was 2.75 times higher (95% confidence interval, 1.67 to 4.53; P <0.001) in the high-risk versus low-risk groups., Conclusions: In patients with FSGS, high-risk APOL1 genotype is the predominant factor associated with more rapid loss of kidney function., (Copyright © 2023 by the American Society of Nephrology.)

Introduction: Treatment-resistant nephrotic syndrome is a rare form of glomerular disease that occurs in children and adults. No Food and Drug Administration-approved treatments consistently achieve remission of proteinuria and preservation of kidney function. CD80 (B7-1) can be expressed on injured podocytes, and administration of abatacept (modified CTLA4-Ig based on a natural ligand to CD80) has been associated with sustained normalization of urinary protein excretion and maintenance of glomerular filtration rate in experimental and clinical settings., Methods: In this report, we describe the rationale for and design of a randomized, placebo-controlled, clinical trial of abatacept in patients with treatment-resistant nephrotic syndrome caused by focal segmental glomerulosclerosis or minimal change disease. The design is a hybrid of a parallel-group and crossover design (switchover) with the primary objectives assessed in the first period of the study and the secondary objectives assessed using data from both periods. All participants will receive the active agent in 1 of the periods. The duration of treatment will be 4 months per period., Results: The primary outcome will be improvement in nephrotic-range proteinuria to subnephrotic range, that is, reduction from baseline to 4 months in urine protein:creatinine ratio ≥ 50% and to a level < 3. The projected sample size is 90 patients, which has 80% power to detect a treatment difference of 28%., Conclusion: This study advances efforts to validate CD80 as a therapeutic target for treatment-resistant nephrotic syndrome, and implements a precision medicine-based approach to this serious kidney condition in which the selection of a therapeutic agent is guided by the underlying disease mechanism operating in individual patients.

Nephrology has conducted few high-quality clinical trials, and the trials that have been conducted have not resulted in the approval of new treatments for primary or inflammatory glomerular diseases. There are overarching process issues that affect the conduct of all clinical trials, but there are also some specialty-specific issues. Within nephrology, primary glomerular diseases are rare, making adequate recruitment for meaningful trials difficult. Nephrologists need better ways, beyond histopathology, to phenotype patients with glomerular diseases and stratify the risk for progression to ESRD. Rigorous trial design is needed for the testing of new therapies, where most patients with glomerular diseases are offered the opportunity to enroll in a clinical trial if standard therapies have failed or are lacking. Training programs to develop a core group of kidney specialists with expertise in the design and implementation of clinical trials are also needed. Registries of patients with glomerular disease and observational studies can aid in the ability to determine realistic estimates of disease prevalence and inform trial design through a better understanding of the natural history of disease. Some proposed changes to the Common Rule, the federal regulations governing the ethical conduct of research involving humans, and the emerging use of electronic health records may facilitate the efficiency of initiating multicenter clinical trials. Collaborations among academia, government scientific and regulatory agencies, industry, foundations, and patient advocacy groups can accelerate therapeutic development for these complex diseases., (Copyright © 2016 by the American Society of Nephrology.)

The multicenter Nephrotic Syndrome Study Network (NEPTUNE) digital pathology scoring system employs a novel and comprehensive methodology to document pathologic features from whole-slide images, immunofluorescence and ultrastructural digital images. To estimate inter- and intra-reader concordance of this descriptor-based approach, data from 12 pathologists (eight NEPTUNE and four non-NEPTUNE) with experience from training to 30 years were collected. A descriptor reference manual was generated and a webinar-based protocol for consensus/cross-training implemented. Intra-reader concordance for 51 glomerular descriptors was evaluated on jpeg images by seven NEPTUNE pathologists scoring 131 glomeruli three times (Tests I, II, and III), each test following a consensus webinar review. Inter-reader concordance of glomerular descriptors was evaluated in 315 glomeruli by all pathologists; interstitial fibrosis and tubular atrophy (244 cases, whole-slide images) and four ultrastructural podocyte descriptors (178 cases, jpeg images) were evaluated once by six and five pathologists, respectively. Cohen's kappa for inter-reader concordance for 48/51 glomerular descriptors with sufficient observations was moderate (0.40

Heparan sulfate proteoglycans have been shown to modulate podocyte adhesion to--and pedicel organization on--the glomerular basement membrane. Recent studies showed that foot process effacement developed in a mutant mouse model whose podocytes were unable to assemble heparan sulfate glycosaminoglycan chains. This study, a further refinement, explored the role of heparan N-sulfation on podocyte behavior. A novel mutant mouse (Ndst1(-/-)) was developed, having podocyte-specific deletion of Ndst1, the enzyme responsible for N-sulfation of heparan sulfate chains. Podocytes having this mutation had foot process effacement and abnormal adhesion to Bowman's capsule. Although glomerular hypertrophy did develop in the kidneys of mutant animals, mesangial expansion was not seen. The lack of heparan N-sulfation did not affect the expression of agrin or perlecan proteoglycan core proteins. Loss of N-sulfation did not result in significant proteinuria, but the increase in the albumin/creatinine ratio was coincident with the development of the enlarged lysosomes in the proximal tubules. Thus, although the renal phenotype of the Ndst1(-/-) mouse is mild, the data show that heparan chain N-sulfation plays a key role in podocyte organization.

The transmembrane protein nephrin is an essential component of slit diaphragms, the specialized cell junctions that link podocyte foot processes. Podocytes are epithelial cells that surround the glomerular capillaries in the kidney and are necessary for the organ-filtering function. Nephrin signaling complex transduces extracellular cues to the podocyte cytoskeleton and regulates podocyte shape and function. Vascular endothelial growth factor A (VEGF-A) is a required growth factor produced and secreted by podocytes. Accumulating evidence suggests a cross-talk between VEGF-A and nephrin signaling pathways. We previously showed that in vivo nephrin associates with VEGF receptor-2 (VEGFR2), the signaling receptor for VEGF-A. In the present work, we characterized the interaction between nephrin and VEGFR2 in cultured cells and in vitro. We demonstrate that nephrin-VEGFR2 interaction is direct using mass spectrometry, immunoprecipitation, GST-binding assays, and blot overlay experiments. This interaction occurs through VEGFR2 and nephrin cytoplasmic domains. Nephrin-VEGFR2 interaction is modulated by tyrosine phosphorylation of both cytoplasmic domains. Furthermore, the nephrin-VEGFR2 complex involves Nck and actin. VEGF-A signaling via this complex results in decreased cell size. We provide evidence that this multiprotein interaction occurs in cultured podocytes. We propose that the nephrin-VEGFR2 complex acts as a key mediator to transduce local VEGF-A signals to the podocyte actin cytoskeleton, regulating the foot process structure and glomerular filter integrity.

Podocytes adhere to the glomerular basement membrane by cell surface receptors. Since in other cells these adhesions are enhanced by cell surface proteoglycans, we examined the contribution of these molecules and their glycosaminoglycan side chains to podocyte adhesion by developing immortalized podocyte cell lines with (control) or without (mutant) heparan sulfate glycosaminoglycan chains. In adhesion assays control podocytes attached, spread, and migrated more efficiently compared with mutants, indicating a requirement for heparan sulfate chains in these processes. The proteoglycan syndecan-4 is known to have direct effects on cell attachment, spreading, and cytoskeletal organization. We found it localized to focal adhesions in control podocytes coincident with stress fiber formation. In mutant cells, syndecan-4 was associated with smaller focal contacts and cortical actin organization. Analysis by flow cytometry showed that mutant cells had twice the amount of surface syndecan-4 of control cells. Protein kinase Cα, a signaling molecule bound to and activated by syndecan-4, showed a fourfold increase in membrane localization-activation than that seen in control cells. In vivo, the loss of heparan sulfate glycosaminoglycans in PEXTKO mice led to a loss of glomerular syndecan-4. Overall, our study provides further evidence for a dynamic role of cell surface heparan sulfate glycosaminoglycans in podocyte activity.

Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase that plays a critical role in cell motility. Movement and retraction of podocyte foot processes, which accompany podocyte injury, suggest focal adhesion disassembly. To understand better the mechanisms by which podocyte foot process effacement leads to proteinuria and kidney failure, we studied the function of FAK in podocytes. In murine models, glomerular injury led to activation of podocyte FAK, followed by proteinuria and foot process effacement. Both podocyte-specific deletion of FAK and pharmacologic inactivation of FAK abrogated the proteinuria and foot process effacement induced by glomerular injury. In vitro, podocytes isolated from conditional FAK knockout mice demonstrated reduced spreading and migration; pharmacologic inactivation of FAK had similar effects on wild-type podocytes. In conclusion, FAK activation regulates podocyte foot process effacement, suggesting that pharmacologic inhibition of this signaling cascade may have therapeutic potential in the setting of glomerular injury.

Background: Cell culture of podocytes has become an indispensable tool in the study of podocyte biology. To date, however, podocyte cell lines with stable expression of the crucial slit diaphragm protein nephrin and localization of nephrin to cell-cell contacts are not available., Methods: Conditionally immortalized cells were grown from isolated glomeruli of mice, harboring the temperature-sensitive SV40 large T antigen. About 60 clonal cell lines were generated by limiting dilution., Results: Among 30 Wilm's tumor (WT)-1- and podocalyxin-positive cell clones, two cell clones stably expressed nephrin as assessed by reverse transcription-polymerase chain reaction (RT-PCR), Northern and Western blotting, and immunofluorescence. In addition, expression of the following podocyte proteins was demonstrated: NEHP1, FAT, P-cadherin, podocin, CD2AP, ZO-1 (alpha(-) isoform), Lmx1b, podoplanin, synaptopodin, cortactin, and vimentin. The nephrin-positive podocyte cell lines formed a monolayer with abundant cell-cell contacts. Transmission electron microscopy revealed formation of primitive foot process-like interdigitations and slit diaphragm-like junctions. Nephrin colocalized with F-actin at cell-cell contacts as demonstrated by immunofluorescence. Intriguingly, nephrin and actin-associated proteins (synaptopodin, CD2AP, and cortactin) were recruited to and accumulated at the entire cell margin only in confluent cells, but not in dispersed cells., Conclusion: We present novel murine podocyte cell lines with stable expression of nephrin and abundant formation of cell-cell contacts, possessing several features of in situ podocyte cell-cell contacts. Furthermore, our data suggest that the accumulation of certain proteins in podocyte foot processes is linked to formation of cell-cell contacts.

NPHS2 was recently identified as a gene whose mutations cause autosomal recessive steroid-resistant nephrotic syndrome. Its product, podocin, is a new member of the stomatin family, which consists of hairpin-like integral membrane proteins with intracellular NH(2)- and COOH-termini. Podocin is expressed in glomerular podocytes, but its subcellular distribution and interaction with other proteins are unknown. Here we show, by immunoelectron microscopy, that podocin localizes to the podocyte foot process membrane, at the insertion site of the slit diaphragm. Podocin accumulates in an oligomeric form in lipid rafts of the slit diaphragm. Moreover, GST pull-down experiments reveal that podocin associates via its COOH-terminal domain with CD2AP, a cytoplasmic binding partner of nephrin, and with nephrin itself. That podocin interacts with CD2AP and nephrin in vivo is shown by coimmunoprecipitation of these proteins from glomerular extracts. Furthermore, in vitro studies reveal direct interaction of podocin and CD2AP. Hence, as with the erythrocyte lipid raft protein stomatin, podocin is present in high-order oligomers and may serve a scaffolding function. We postulate that podocin serves in the structural organization of the slit diaphragm and the regulation of its filtration function.

Background: Podocytes are highly differentiated glomerular epithelial cells with limited potential to divide. They are responsible for maintaining and supporting the glomerular basement membrane so as to facilitate efficient filtration. The hypothesis tested was whether the development of glomerulosclerosis in the puromycin aminonucleoside (PAN)-treated rat could be attributed to podocyte depletion., Methods: PAN was injected in Sprague-Dawley rats once, twice, or three times at 30-day intervals. Podocytes were counted in glomeruli using immunoperoxidase histochemistry and antibodies to both GLEPP1 (PTPRO) and WT-1. Podocytes were assayed in urine using reverse transcription-quantitative polymerase chain reaction (RT-QPCR). Glomerular areas were measured by computerized morphometry., Results: In a preliminary experiment, a single injection of PAN caused a reduction in the glomerular podocyte count by 25%. Additional independent confirmation that podocytes were lost from glomeruli after PAN injection was obtained identifying detached podocytes in Bowman's space, measurement of nephrin and GLEPP1 mRNAs in urine, ultrastructural analysis of glomeruli, and identification of TUNEL-positive apoptotic podocytes in glomeruli. In a second experiment, sequential podocyte depletion by 15, 31, and 53% was achieved by the administration of one, two, or three injections of PAN at 30-day intervals. The region of the glomerulus devoid of podocytes developed glomerulosclerosis, and this area progressively increased as podocytes were progressively depleted. The correlation coefficient (r(2)) value for the relationship between percent podocyte depletion and glomerulosclerotic area was 0.99. The Y intercept of this plot showed that glomerulosclerosis was initiated when only 10 to 20% of podocytes were lost., Conclusion: This report supports the growing body of data linking glomerulosclerosis directly to a reduction in relative podocyte number [increased glomerular area per podocyte (GAPP)]. It raises important questions related to the mechanisms of podocyte loss, strategies for prevention of podocyte depletion, and the prevention of progression of glomerular diseases.

It has been proposed that JNK-interacting proteins (JIP) facilitate mixed lineage kinase-dependent signal transduction to JNK by aggregating the three components of a JNK module. A new model for the assembly and regulation of these modules is proposed based on several observations. First, artificially induced dimerization of dual leucine zipper-bearing kinase (DLK) confirmed that DLK dimerization is sufficient to induce DLK activation. Secondly, under basal conditions, DLK associated with JIP is held in a monomeric, unphosphorylated and catalytically inactive state. Thirdly, JNK recruitment to JIP coincided with significantly decreased affinity of JIP and DLK. JNK promoted the dimerization, phosphorylation and activation of JIP-associated DLK. Similarly, treatment of cells with okadaic acid inhibited DLK association with JIP and resulted in DLK dimerization in the presence of JIP. In summary, JIP maintains DLK in a monomeric, unphosphorylated, inactive state. Upon stimulation, JNK-JIP binding affinity increases while JIP-DLK interaction affinity is attenuated. Dissociation of DLK from JIP results in subsequent DLK dimerization, autophosphorylation and module activation. Evidence is provided that this model holds for other MLK-dependent JNK modules.