The 13th International Podocyte Conference

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perspective minimised the carbon footprint of the conference. Different time zones presented challenges to virtual participants aiming to view all sessions live. The on-demand platform allowed participants to view sessions when convenient and also hosted longer pre-recorded talks that speakers prepared in advance. One of the biggest challenges was to continually engage a large virtual audience and keep the interaction with in-person speakers vibrant and engaging. Pivotal to this was the chat room activity and the Q+A platform Slido that facilitated a lively online discussion as well as a speedy transfer of questions into the live session. The feedback was extremely positive but 'nothing replaces F2F'. F2F poster presentations for example would certainly have resulted in more personal and in-depth discussions. Personal connections and networking were more difficult online. Addressing the expense-barrier to science, registration fees were waived for low-and middle-income countries and students from all countries to improve collaborations and to minimise knowledge gaps.
Central to the strategy of hybrid content delivery was the social media strategy of #Podocyte21. Primarily, this was achieved through the Twitter social media platform and the official conference account @podocyte21. Given the earlier planned date of 2020, the account had an early start in stimulating interest and enthusiasm for the meeting within the international podocyte community. Prior to the meeting, 'Podofact Friday' ran weekly with key podocyte biology educational facts to whet the appetite for the scientific meeting to come. During the main meeting, a total of 245 tweets with original content were authored by 140 different individuals, all of which contained picture/slide content and 10 of which included conference video contenthighlighting the free availability of high-quality educational resources for those not registered at the conference.
Of the total authors, 33/140 were registered conference attendees or speakers highlighting that the majority of tweets and interactions originated from outside the attendee pool. 385 Retweets of original material occurred during the main meeting (bringing total tweets to 630 during the main event). A word cloud summary of the meeting (Figure 1.) is available alongside a knowledge transfer map demonstrating the flow of educational content via twitter between participants and the meeting (Figure 2).

Figure 2. Social media knowledge transfer map with individual influencers represented as nodes and connections representing author interactions.
Tweeting about the meeting continued late into November 2021 prior to handover of the account to the organisers of the 14 th International Podocyte Conference in Philadelphia, USA in 2023. The most popular and widely re-tweeted content was the excellent pictographic artistic summaries of the speakers and key presentation points by post-doctoral researcher and science illustrator, Alex Cagan. These detailed and concise summaries were universally praised for their scientific content and artistic flair. Parallel to the scientific submissions, attendees and their contacts were encouraged to submit their 'PodoArt', unique artistic works involving podocyte biology, submitted by researchers and the public. These were available as an online art-show with prizes for winners and runners up.
As F2F meetings re-emerge, hybrid meeting content will continue to provide for those not able to attend in person. In the long term, hybrid models will likely remain a significant component for many events as we learn and benefit from innovations being developed during the ongoing COVID-19 pandemic. The 13 th International Podocyte Conference -Manchester and Onlinewas a successful meeting, bridging not only basic and translational science but the traditional format of conference with the new virtual way of presentation and networking. The podocyte community adapted rapidly to this novel meeting format and as such reflected the adaptive and versatile nature of the glomerular podocyte.

Funding
The International Organising Committee of the Podocyte Conference and the authors of this article acknowledge the support of NephCure Kidney International for assistance with conference planning, advertising, fundraising and delivery. The meeting itself was supported with funding from: Travere Therapeutics, Astrazeneca, Goldfinch Bio, Nipoka, Chinook, Sanofi-Aventis, Novartis, Caliditas Therapeutics, University of Cologne, Wellcome Trust, Kidney Research UK, Kidneys for Life. making it a potential target for new therapeutics. However, the role of VEGFR3 signalling in kidney development, how it influences kidney disease, and the vascular beds involved remains uncertain.
We performed a detailed expression profile of VEGFR3 in the developing mouse kidney. We generated a new transgenic mouse model to investigate the role of Vegfr3 in the kidney vasculature (Vegfr3Flx). Conditional and cell-specific excision of the floxed allele was performed using the Rosa-rtTA-TetOCre, Cdh5-Cre/ERT2, and Prox1-Cre/ERT2 driver strains to evaluate global, pan-endothelial, and lymphatic endothelial cell deletion of Vegfr3 respectively. Mice underwent a detailed phenotypic evaluation and kidney sections were processed for histology.
VEGFR3 undergoes dynamic expression through development in several fenestrated blood microvascular beds of the kidney including glomerular endothelial cells (GECs). Constitutive deletion of Vegfr3 during mid-embryonic development resulted in reduced viability, lymphatic vascular defects, and a reduction in kidney size. Additionally, deletion of Vegfr3 at embryonic day 11.5 resulted in marked disruption of glomerular development with cavernous capillary malformations. Immunofluorescence and electron microscopy revealed glomerular structures surrounded by simplified podocytes, abnormal attachment of endothelial cells with reduced fenestrations, and poor formation of the glomerular basement membrane.
VEGFR3 is expressed in GECs and is integral to normal glomerular development. The mechanisms of VEGFR3 signalling in GEC crosstalk with podocytes will be essential to define prior to the development of therapeutics targeting this pathway.
downregulation of Myo1e and Ptk2, suggesting an adaptive cellular response to mechanical load.
Whilst in wild-type mice albumin-creatinine ratios were preserved, a rapid decline in kidney function was observed in Alport mice post-surgery. Moreover, EM demonstrated disorganisation of the glomerular filtration barrier. MS analysis revealed opposing regulation of proteins in the Alport model, indicating a protective role for specific mechanoregulators in wild-type mice.
Conclusions: This study provides deep molecular resolution of glomerular response to mechanical load. We identify new force-responsive proteins, which could be targeted in glomerular disease. Results: Whole mount immunofluorescence demonstrated that kidney organoids differentiate into glomerular structures containing podocytes (WT1+, NPHS1+), proximal tubule (LTL+), distal tubule (ECAD+), and endothelial cells (CD31+). Notably, immunostaining of the key BM markers showed that organoids form BM during differentiation, and more importantly, recapitulate a sequence of assembly events with initial deposition of laminin followed by incorporation of type IV collagen, nidogen and perlecan. Single-cell RNA sequencing analysis from day 25 kidney organoids (Combes et al., 2019) confirmed NPHS2+/PODXL+ podocytes were the main source of COL4A3, COL4A4.
Conclusions: These findings fit with the current understanding of kidney development in vivo and demonstrate that kidney organoids can efficiently recapitulate the spatiotemporal emergence of GBM components during glomerulogenesis. Results: We demonstrate that podocytes isolated from our Myo1e-KO mice exhibit lower levels of fibronectin in the ECM than myo1e heterozygous or wild type (WT) podocytes. Myo1e-KO podocytes also exhibited altered localization of the focal adhesion protein vinculin.
Additionally, fibronectin mRNA expression appeared to be less upregulated in response to flow in Myo1e-KO podocytes than in the WT podocytes.
Conclusions: The observed vinculin mislocalization and the decreased levels of extracellular fibronectin may affect podocyte adhesion to the GBM. Though the results of our flow experiments are preliminary, they may indicate that myo1e-deficient podocytes are less adaptable to mechanical stress. Ultimately, our findings suggest that the pathology observed in both mice and patients with myo1e deficiencies may be the result of an altered GBM composition.

Circulating factors and disease biomarkers -Poster
1 Soluble RARRES1 induces apoptosis of podocytes to promote progression of kidney disease Dr Anqun Chen 1 , Dr Tianjun Guan 1 , Dr Kyung Lee 2 , Dr John Cijiang He 2,3 Background: Podocyte loss is a major event leading to the progression of focal segmental glomerulosclerosis (FSGS) and diabetic nephropathy (DN). Here, we found that retinoic acid receptor responder protein 1 (RARRES1) contributes to the podocyte loss in FSGS and DN.
Methods: We determined the role of RARRES1 in human and mouse with FSGS and DN. We investigated the mechanisms of RARRES1 in cultured human podocytes.
Results: We found that expression of RARRES1 increased in the glomeruli of patients with FSGS and DN and correlated with the eGFR. Single-cell RNA-sequencing of the kidney showed that RARRES1 expressed highly in podocytes. Immunostaining confirmed that podocyte expression of RARRES1 increased in patients with DN and FSGS as compared to minimal change disease. Expression of RARRES1 in cultured human podocytes was upregulated by TNF-α. RNA-sequencing of podocytes with RARRES1 overexpression revealed genes enriched in apoptosis and overexpression of RARRES1 in podocytes induced apoptosis. Mechanistically, RARRES1 induced podocyte apoptosis through the cleavage into a soluble form which underwent endocytosis and then interacted with atypical protein kinase RIOK1 to activate p53. We mapped the cleavage site at 71 st amino acid of RARRES1 and mutation of this site abolished its apoptotic effect. In vivo, induction of RARRES1 overexpression in podocytes caused albuminuria and glomerular injury in normal mice and aggravated kidney injury in mice with DN and FSGS whereas induction of RARRES1 with 71 st amino acid mutation did not.
Conclusion: Our study reveals a novel role and mechanism of RARRES1 in podocyte injury in glomerular disease.  (DN), which in later stages often requires renal replacement therapy. Therefore, there is a great need for early DN biomarkers, which is still not met. Because the anti-aging and anti-inflammatory α-Klotho (KL) protein level was decreased in serum and urine of individuals with acute kidney injury, we analyzed its usefulness as the early biomarker of DN. Therefore, we measured Klotho level in serum, urine (ELISA), and renal tissue (Western blot, immunohistochemistry) in male Wistar rats with streptozotocin-induced (STZ) diabetes and age-matched healthy controls. We measured mRNA (Real-time PCR) and total KL protein expression (Western blot, immunofluorescence) in human immortalized podocytes, cultured in diabetic (hyperglycemic, HG) or normoglycemic (NG) conditions, and membrane KL (mbKL) level in these cells after soluble KL (sKL) shedding to the cell medium (biotinylation, Western blot).

Serum and urine levels of Klotho as early biomarkers of diabetic nephropathy
We found that serum sKL levels increased, whereas Klotho urine excretion, Klotho level in stained renal tissue and Klotho protein expression in glomeruli decreased in STZ vs. control rats. mRNA and total protein expression of KL in human podocytes was not dependent on NG/HG conditions. However, mbKL level in podocytes decreased, while sKL shedding increased in HG.
As previous studies showed no disruption in serum Klotho level in DM patients without nephropathy, and a link between low urinary KL levels and podocyte dysfunction, the revealed increased serum and decreased urinary Klotho levels could be considered as early biomarkers of DN. Background: Idiopathic nephrotic syndrome (INS) comprises a group of rare glomerulopathies attributed to direct or indirect interaction of putative circulating factors with podocytes. One of the pathological manifestations of INS is the rapid recurrence of focal and segmental glomerulosclerosis (rFSGS) after renal transplant in certain individuals. To date, the nature and identity of circulating factors has not been confirmed, while the pathogenic mechanisms in the podocyte are partially understood. We aimed to study the early proximal signalling events in podocytes in response to plasma from post-transplant rFSGS patients, by a combination of phosphoproteomics and lipid raft proteomics (raftomics).

Phosphoproteome and raft proteome changes of human podocytes in response to recurrent idiopathic nephrotic syndrome plasma
Methods: We incubated differentiated human podocytes for 30min with 10% of first posttransplant plasma exchange fluid from either rFSGS or non-INS patients, the latter used as controls. We performed differential phosphoproteomics after phosphopeptide enrichment in a TiO2 column. In parallel, we isolated lipid rafts by a detergent-free method and performed differential proteomics by LC-MS/MS analysis.
Results: The ensemble of results showed specific differences in protein expression and phosphorylation associated with rFSGS plasma incubation, suggesting alterations in the mTOR pathway, in autophagy, in mitochondrial metabolism, and in cytoskeleton organization.
In particular, validation experiments suggested an altered phosphorylation pattern of Hsp27.
Conclusions: The observed changes associated with rFSGS plasma incubation highlight some of the mechanisms involved in FSGS recurrence and could be used as specific early markers of circulating factor activity on podocytes.

Manchester, United Kingdom
Background: Proteinuria is a key primary clinical indicator of kidney disease. In humans, measuring proteinuria is relatively simple, but in model organisms used in laboratory settings this can be more difficult. This is particularly the case for the zebrafish, which has become an important system to investigate kidney development and disease. Their aquatic environment and small urine volume makes quantitative analysis of urine difficult.
Methods: We have used Tol2 transgenics to develop a Nanoluciferase transgenic zebrafish reporter of proteinuria. Using a luminometer, analysis of embryonic media can be assayed in a high-throughput manner. We used morpholino and CRISPR-Cas9 knockdown approaches as well as chemical treatments to demonstrate the usability of our reporter. Proteomics was used to identify the glomerular proteome in 5 day-old zebrafish.
Results: Our proteinuria reporter is able to detect dysfunction in both the glomerulus and proximal tubules. We establish the zebrafish as a model of Alport syndrome, which is caused by mutations in the COL4A3/COL4A4/COL4A5 genes in humans. Orthologs to these genes are expressed in the zebrafish glomerulus and caused proteinuria upon depletion.
Conclusions: Our new tool for quantifying proteinuria in the zebrafish enables this system to be used to assay the effects of genetic alterations or chemical treatments on glomerular and proximal tubule function in a high-throughput manner. The viability of the zebrafish as a vertebrate model with which screening of chemicals can be performed means that this new tool has the potential to establish the zebrafish as a key model for future drug testing. Background: Nephrotic syndrome (NS) is one of the most common monogenic kidney diseases. NPHS2 missense mutations were reported to result in misfolding and mistrafficking of Podocin. Such studies overexpressed mutant proteins in immortalised podocyte and nonpodocyte cell lines, which may not reflect the in vivo consequences of the mutation. We generated NPHS2 mutant iPSC-derived kidney organoids as a model to dissect the pathogenic process of NS.
Methods: A control human fibroblast line was simultaneously reprogrammed and CRISPR/Cas9 gene edited, generating 5 iPSC lines containing mutations of the endogenous NPHS2 locus (G92C, P118L, R138Q, R168H, R291W) as well as a control wild-type line. All lines were differentiated into kidney organoids containing all nephron segments. Podocin localisation was assessed using co-immunofluorescence with known markers of subcellular compartments.
Results: Mutant lines revealed a marked reduction of the Podocin protein compared to control and mutant podocin mislocalisation was often in contradiction with existing literature. G92C and P118L mutant proteins accumulated mainly at the slit diaphragm and the endoplasmic reticulum respectively, as previously described. However, co-staining with GM130 demonstrated a partial Golgi retention for P118L, R168H and R291W. R138Q and R291W were both found to partially accumulate in late endosomes. Finally, all 5 mutants displayed significantly more apoptotic Caspase3+ podocytes than controls.
Conclusion: Discrepancies between previous reports and our findings highlight the need for a more appropriate model to study the pathobiology of NPHS2 mutations. These organoids will allow us to explore approaches to rescue individual Podocin defects guiding the development of new therapeutic strategies. Annotation, most of the down-regulated proteins are cytoskeleton and actin-binding proteins.
In contrast, the up-regulated proteins are more associated in clusters affecting transcription and mRNA processing. To identify rare events such as alternative splicing, we additionally analyzed samples by RNA sequencing.
Moreover, we recultivated podocytes that detached rapidly and the mechanically resistant cells that remained on the stretch membrane. We observed that podocytes showed similar behavior after renewed mechanical stress. Analysis by fluorescence microscopy suggests that this is due to differential expression of focal adhesion proteins.
Conclusions: Our proteomic data of stretched podocytes in vitro identified potential key proteins that may also be involved in the detachment of podocytes in vivo as well playing a role in patients suffering from glomerular hypertension.

Development of an improved differentiation protocol for iPSC-derived podocytes
Sergii and tested additional conditions, including extracellular matrices, media, and length of differentiation.
Results. Both protocols generated iPodos with similar efficiency, as measured by synaptopodin, nephrin, and podocin staining. iPodos generated from protocol-2 were morphologically homogeneous, developed more filopodia and complex cell-cell junctions, and showed extended survival up to 4 weeks post-differentiation, than those of protocol-1. iPodos from protocol-1 remained relatively immature, based on the expression of collagen α1α2α1(IV) and lack of α3α4α5(IV).
Response to Protamine sulfate (PS) or Puromycin Aminonucleoside (PAN) treatment was more consistent in protocol 2. PS treatment induced a significant and reproducible dose-and time-dependent decrease in synaptopodin staining, and a robust accumulation of phalloidin aggregation. Both effects were prevented by cyclosporin A, in a similar manner as in mouse podocytes, that were used as a positive control.

Drug development -Oral
Novel Background: Bardoxolone methyl is an electrophilic agent that induces Nrf2 activation by irreversibly and covalently binding to the cysteine residue of Keap1. Bardoxolone methyl has been shown to improve glomerular filtration rate (GFR) in clinical trials and is attracting attention as a novel agent for chronic kidney disease. However, there is concern about longterm efficacy due to the unknown mechanism of GFR improvement and transient increase in albuminuria. Moreover, irreversible Keap1 inhibitors such as Bardoxolone methyl may covalently bind to other proteins in a non-specific manner and induce side effects due to offtarget activities.

Development of Keap1-Nrf2 PPI inhibitor was performed by fluorescence polarization and
Nqo1 induction test. The obtained novel compound UBE-1099 (30 mg/kg/day) and CDDO-Im (3, 10 mg/kg/day; rodent tolerable Bardoxolone methyl analogue) were orally administrated to Alport mice and efficacy was evaluated.
Results: UBE-1099 showed higher Nqo1 induction efficiency compared with CDDO-Im in mouse renal tissue. While CDDO-Im only improved inflammation pathology in Alport mice, UBE-1099 uniformly improved renal function (GFR and Plasma creatinine, but not albuminuria), podocyte injury, glomerulosclerosis, inflammation and fibrosis. Moreover, UBE-1099 treatment significantly prolonged the lifespan of Alport mice.
Conclusions: This study firstly revealed the efficacy of Keap1-Nrf2 PPI inhibitor for glomerulosclerosis. We will elucidate next the mechanism of renal pathology improvement, which may provide useful information for Nrf2 activators including bardoxolone methyl for clinical application.

Drug development -Poster 1 A Precision Medicine Approach to Developing Novel Medicines for Podocytopathies:
The Our understanding of mechanisms driving podocyte injury increasingly support development of targeted therapies for proteinuric kidney diseases. We describe our approach to developing a targeted therapy for podocytopathies driven by TRPC5-Rac1 pathway overactivation and using urinary Rac1 (uRac1) as a novel predictive and target-engagement biomarker.
GFB-887, a podocyte-targeting, subtype selective and potent small molecule TRPC5-inhibitor, was identified as a potential targeted therapy for patients with podocytopathies. In rodent models of focal segmental glomerulosclerosis (FSGS), minimal change disease (MCD) and diabetic nephropathy (DN), GFB-887 reduced both uRac1 and albuminuria. Rac1 was detected in extracellular vesicles isolated from podocyte-conditioned culture media and in human urine, consistent with kidney origin. uRac1 concentrations were increased in patients with FSGS and DN compared to healthy adults indicating a possible predictive biomarker.
In a first-in-human, randomized, double-blind, placebo-controlled, single-ascending dose trial (GFB-887 5 to 900mg), GFB-887 was found to be safe and well tolerated, with a once daily harmacokinetic profile. Statistically significant area under the curve (AUC) reductions in uRac1 concentrations were observed from baseline with increasing doses of GFB-887, indicative of TRPC5-Rac1 pathway engagement and inhibition. To efficiently explore the safety and efficacy of GFB-887, and utility of uRac1 as a predictive and target-engagement biomarker, we utilize a basket trial and adaptive study design approach for our phase 2 program. We are currently enrolling adult patients with FSGS, treatment resistance-MCD, and DN in TRACTION-2: a phase 2, randomized, double-blind, placebo-controlled, multiple-ascending dose study of GFB-887 for 12 weeks (NCT04387448).

Developing a small-molecule therapeutic to treat diabetic kidney disease
PhD Injury and loss of podocytes underly the development and progression of DKD. We previously showed that accumulation of lipid droplets (LDs) in podocytes is associated with cell death, and that reducing LD accumulation combats DKD. We hypothesize that a targeted small molecule that directly blocks LD accumulation in podocytes can halt or reverse the progression of DKD.
Methods: A robust phenotypic assay with human podocytes to identify compounds that reduce LD accumulation in these cells was developed. We screened a chemical library from the Torrey Pines Institute for Molecular Studies (TPIMS) containing >45 million molecules.
Structure-activity-relationship (SAR) studies from identified hits were performed. RNAseq libraries from podocytes treated with one of the identified hits in the presence of sera from subjects with or without DKD were generated.
Results: The assay identified several hits from the TPIMS library. SAR studies showed strong feasibility for hit-to-lead optimization of one series. A representative compound reduced the expression of genes related to TNF-signaling, a pathway associated with LD accumulation and podocyte injury, and the expression of lysosome-associated membrane glycoprotein (LAMP), which correlates with improved autophagy/lipophagy. This compound is currently being tested in a mouse model of DKD.
Conclusions: Our screen identified a promising molecular series that strongly reverses features of podocyte toxicity and death in DKD. These compounds can now help with the investigation of DKD pathology, identification of novel drug targets, and development of a therapeutic candidate. Podocyte dedifferentiation with the associated morphological rearrangements is a major cause of chronic kidney disease. Since there is no causal therapy, end-stage renal disease makes renal replacement therapy inevitable. Vitamin D3 (VitD) is a promising candidate to overcome this, but it is controversially discussed. In this study, we used a semi-automated screening model to investigated the influence of VitD on podocyte differentiation in situ and in vitro and to identify downstream target genes.

Genetic screening in glomerular diseases -Oral
Cultured glomeruli from transgenic mice expressing CFP nephrin promoter-dependently (GlomAssay) were treated with VitD and its analogue calcipotriol and were analyzed by a semi-automated imaging procedure. Additionally, VitD-, calcipotriol-and VitD receptor (VDR) inhibitor (PS121912) -treated glomeruli were investigated by RNA-Seq to reveal the molecular biological effect of VitD on podocyte differentiation. Furthermore, we treated cultured murine podocytes with VitD and calcipotriol to elucidate the morphological and molecular changes by Immunofluorescence, RT-qPCR and Western blot.
VitD-/ calcipotriol-treated glomeruli showed a significantly higher CFP intensity compared to controls, indicating elevated nephrin levels, which was verified by RT-qPCR and Western blot.
We found an up-regulation of VDR in VitD-and calcipotriol-treated glomeruli compared to controls. By RNA-Seq, we identified treatment specific molecular patterns in different signaling pathways. Cultured podocytes showed a significant up-regulation of nephrin, VDR and CYP24A1 due to the treatment of VitD. This is accompanied by an altered podocyte morphology due to reorganization of the actin cytoskeleton.
Our results show that VitD influences podocyte differentiation in situ and in vitro based on specific expression patterns. Methods: We performed genome-wide and conditional regional analysis to identify variants associated with haematuria in the UK Biobank. To identify independent COL4A4/4A3 variants, conditional regional analysis was performed in sequential rounds, incorporating previous signals as covariates. If regional variants are in LD, statistical significance for association will be lost. We used both Haplotype Reference Consortium (HRC) imputed genome-wide data and regional TOPMed-imputed data. is crucial in the regulation of T Lymphocyte activation and has been shown to mediate antiinflammatory effects in other T cell-mediated disease models.
Methods: NTN was induced in BTLA knock out mice (BTLA-KO) and littermate controls.
Regular urine analysis (ACR) was performed throughout the course of the disease. 14 days after NTN induction, blood, kidneys and spleens were harvested for further analysis.
Histological assessment of the kidneys was used to evaluate the severity of NTN. Local immune response in the kidney and systemic immunity was analyzed via flow cytometry and qPCR. Conclusions: Collectively, these results suggest that the Rab-Rabphilin system could be involved in the alterations observed in injured podocytes under hypertension and diabetes conditions, and that a mechanism may be activated to reduce damage through the vesicular transport enhancement directed by this system.  Conclusions: Three-dimensional optical imaging combined with deep learning segmentation can be used to perform detailed and sensitive morphometric analysis of foot processes with high throughput. This more complete and multi-parametric analysis reveals that the foot process effacement patterns differ between different types of kidney disease. We believe that this might provide an important tool not only as an additional readout in diagnostics, but also to advance our understanding regarding the connection between foot process morphology and albumin filtration. We established that human immortalized podocytes express mRNA and protein of phosphate transporters -NaPi 2c (SLC34A3), Pit 1 (SLC20A1) and Pit 2 (SLC20A2), which are sodiumdependent and mediate intracellular Pi transport, as well as XPR1, which is responsible for extracellular transport. We found that cells grown in a medium with high glucose concentration (30 mM, HG) expressed less Pit 1 mRNA (real-time PCR) and protein (western blot) than podocytes cultured in a medium with a standard glucose concentration (11 mM, SG). The amount of Pit 2 protein is also decreasing in HG. We determined that the localization of Pit 1

Renal progenitor-derived extracellular vesicles ameliorate podocyte function in a human in vitro model of glomerular permeability
and Pit 2 in cell membrane declined in HG (cell surface biotinylation). We also found that in HG the activity of tissue nonspecific alkaline phosphatase increases, causing a rise in Pi generation.
The above data suggest that hyperglycemic conditions enhance the production of Pi in the podocytes and its retention in the extracellular space, which may induce glomerular calcification.

Spatial Transcriptomic Profiling of Collapsing Glomerulopathy
Dr Conclusion: Spatial transcriptional profiling represents a powerful new method to dissect transcriptional programs of pathologically discernible kidney lesions.  Conclusion: This study represents the first time that OSBPL7 has been implicated in CKD.

Decreased urinary levels of Sirt1 in podocytes as non-invasive biomarker of early renal damage in hypertension
OSBPL7 deficiency in podocytes leads to ER stress and ultimately apoptosis suggesting that OSBPL7 levels are beneficial to podocyte function.

Can Podocyte number and density predict the response to therapy in patients with
Primary FSGS?
Ms. Results: CTNS -/podocytes show peculiar metabolic alterations, with increased levels of intracellular glucose and impaired glycolytic pathway. In addition, they display altered oxidative stress response and impaired mitochondrial function, resulting in decreased abundances of TCA cycle and energy metabolites.
Conclusions: Cystinosis podocytes show signs of altered energy metabolism and increased oxidative stress. Understanding these mechanisms may pave the way for developing novel therapies in cystinosis.

Thomas Benzing University Hospital of Cologne, Cologne, Germany
Background: It has been debated for many decades whether foot processes display a preferred orientation with respect to the orientation of glomerular capillaries. To date, this question has been left unanswered, probably due to limitations of previously available imaging techniques.
Methods: We applied a recently published imaging protocol, which allows for super-resolved 3D imaging of whole glomerular capillaries. In this way, the angular distribution of several hundreds of primary processes and several thousands of secondary foot processes with respect to the capillary orientation was determined.
Results: Strikingly, we found a clear preference for primary processes to be aligned perpendicularly to the capillary orientation, whereas foot processes aligned preferably in parallel with the capillary orientation in wild-type mice. The result was highly reproduced between several animals. This orientation preference was less prominent in newborn mice, where glomerular vascularization is not fully developed, and totally lost in both genetic and non-genetic disease models with foot process effacement.
Conclusions: Using novel imaging tools, we could, for the first time, establish that foot processes are not randomly distributed on glomerular capillaries. We have in previous studies suggested a role for foot processes to provide a counter-acting force to the capillary wall stress resulting from capillary blood pressure. If the capillary is approximated to have a cylindrical shape, the circumferential wall stress component is larger than the longitudinal wall stress component. Thus, the preferred orientation of foot processes in parallel with capillaries might be needed to withstand the higher wall stress component in this direction. Methods: Using mass spectrometry, we found that ACTN4 is phosphorylated at S159. We used phosphomimetic and nonphosphorylatable ACTN4 to comprehensively study the effects of this phosphorylation in vitro and in vivo. We conducted x-ray crystallography, F-actin binding and bundling assays, and immunofluorescence staining to evaluate F-actin alignment.
Microfluidic organ-on-a-chip technology was used to assess for podocyte detachment when simultaneously exposed to fluid flow and cyclic strain. We then used CRISPR/Cas9 to generate mouse models and assessed for renal injury by measuring albuminuria and examining kidney histology. We performed targeted mass spectrometry to determine whether high extracellular glucose and TGF-b increase phosphorylation of ACTN4.
Results: Compared to wild type (WT), phosphomimetic ACTN4 demonstrated increased binding and bundling activity with F-actin in vitro. Phosphomimetic Actn4 mouse podocytes exhibited more spatially correlated F-actin alignment and a higher rate of detachment under mechanical stress. Phosphomimetic Actn4 mice developed proteinuria and glomerulosclerosis after subtotal nephrectomy. Moreover, we found that exposure to high extracellular glucose and TGF-b stimulates phosphorylation of ACTN4 at S159 in podocytes.
Conclusions: These findings suggest that increased phosphorylation of ACTN4 at S159 leads to biochemical, cellular, and renal pathology that is similar to pathology resulting from human disease-causing mutations in ACTN4. ACTN4 may mediate podocyte injury as a consequence of both genetic mutations and signaling events that modulate phosphorylation. Sphingosine-1-phosphate lyase insufficiency causes accumulation of sphingosine-1phosphate (S1P) and is associated with focal segmental glomerulosclerosis, a renal disease that is often characterized by the recurrence of proteinuria after kidney transplantation. We previously reported a role of sphingomyelin phosphodiesterase acid-like 3b (SMPDL3b), which regulates lipid composition, in podocyte response to injury. In this study we tested the hypothesis that dysregulation of renal SMPDL3b expression affects the availability of S1P, thus contributing to proteinuria and chronic kidney disease (CKD) progression.
A murine model of Alport syndrome (AS), a surrogate model for proteinuric CKD, was used.
AS mice were crossed to podocyte-specific Smpdl3b-deficient mice (DKO) and kidneys from 5-months-old animals were harvested and processed for in-depth phenotypical analysis, including urinary albumin-to-creatinine ratio (ACR), histological analysis, transmission electron microscopy analysis, glomeruli isolation and serum analysis. Lipidomic analysis was performed using LC-MS analysis. All animal studies were performed in accordance with the NIH IACUC Guidelines. For statistical analysis One-Way ANOVA or Student t-test were used.
SMPDL3b expression was increased in kidneys of AS mice when compared to controls. We found decreased ACR in DKO mice compared to AS mice in association with increased S1P.
Improved ACR was associated with less foot processes effacement, but was not associated with improved body weight, serum creatinine, glomerular filtration rate or renal fibrosis. DKO mice demonstrated significantly lower S1P levels in kidney cortexes compared to AS mice.
Our data indicate that SMPDL3b expression may affect availability of S1P and regulate proteinuria without affecting disease progression in experimental proteinuric CKD.

Characterization of Purinergic Receptor 2 Signaling in Diabetic Podocytes
Dr

Campinas, Brazil
Background: Proteinuria is considered a prognostic marker for kidney disease and one of the main symptoms of diseases affecting podocytes, the so called podocytopathies. Nevertheless, there is no conclusive understanding on the secondary effects of the protein flow on podocytes and how podocytes respond to this injury. Therefore, this study aimed to evaluate the expression of PODXL and TRPC6 genes after supplementation of albumin in podocytes in vitro. Methods: We exposed conditionally immortalized human podocytes to progressive concentrations of albumin (0, 3,6,9,12,15,18,20,30 and 40 mg/ml) for 24 hours under two conditions: 1) without treatment and 2) after exposure to puromycin aminoglycoside (PAN). We evaluated PODXL and TRPC6 relative expression through qPCR. Statistical analyzes were performed with ANOVA and Tukey's a posteriori test (p≤0.05). Results: We observed a higher rate of PODXL transcript in both groups (p<0,0001). On the other hand, TRPC6 presented a decrease in the transcript level after supplementation in the non-PAN group (p<0,0001) and an increase in the rate in the 40 mg/mL/PAN treatment group (p<0,0001). We suggest that PODXL overexpression in both groups may represent a defence mechanism of podocytes to maintain slit diaphragms intact. Conversely, TRPC6's expression diverged in each group.
When considering the PAN group, the higher Ca+2 influx caused by TRPC6 increased expression may increase apoptosis, aggravating podocyte damage after proteinuria insult.
Conclusions: This is a pilot study for future studies on molecular mechanisms in vivo that could be associated with podocyte damage caused by proteinuria. METHODS: Proteomics, Cloning, protein-expression and purification (using baculovirussystems), SPR (surface-plasma-resonance), Co-Immunoprecipitation, ELISA, Immunofluorescence.
RESULTS: Since NEPHRIN and NEPH1 interacted with tyrosine phosphatase SHP-2 in a phosphorylation-dependent manner, we investigated whether ligands that induce SHP-2 stimulation also induced NEPHRIN and NEPH1 activation. Screening of multiple ligands identified HGF as a prominent inducer of NEPHRIN and NEPH1 phosphorylation. To establish HGF as a ligand, we used baculovirus expression system to generate purified NEPHRIN and NEPH1 proteins and confirmed a direct interaction between HGF and the extracellular domains of NEPHRIN and NEPH1. We also demonstrate ligand-induced phosphorylation of these proteins. Additionally, we demonstrate that SHP-2 directly dephosphorylates these proteins, thus presenting for the first time an activation and deactivation mechanism for these proteins. We further demonstrate that HGF-induced activation of these proteins is critical for podocytes recovery from injury, where addition of purified HGF to injured podocytes significantly enhanced their recovery in a NEPHRIN and NEPH1-dependent fashion.
Conclusions: We provide compelling evidence that ligand-induced activation of NEPHRIN and NEPH1 by HGF is a component of podocyte recovery mechanism that restores lost podocyte function.
Podocyte hypertrophy also returned to normal with Pio and GQ-16. While both GQ-16 and Pio restored glomerular Nphs1 and hepatic Pcsk9 expression and reduced hypercholesterolemia, GQ-16 also restored glomerular Nrf2, and reduced disease-associated hypoalbuminemia and hypercoagulopathy. Furthermore, RNA-seq analysis identified both common and distinct glomerular genes altered by Pio and GQ-16 treatments. Moreover, Pio but not GQ-16 significantly induced aP2 (fatty acid binding protein) in adipocytes and in WAT. Both, Pio and GQ-16 induced insulin sensitizing adipokines in WAT with varying degrees.
Conclusions: Selective modulation of PPARg by a partial agonist may be a more beneficial approach than a full PPARg agonist in reducing proteinuria, hypoalbuminemia, and hypercoagulopathy in NS and in decreasing drug-associated side effects such as adipogenesis and weight gain.

Podocyte signalling pathways -Poster 1 The Effect of Insulin on Lysosome Activity in Cultured Rat Podocytes
MSc We observed a 19% decrease in podocin expression in PINK1-depleted podocytes, with comparable levels of nephrin. However, membrane fractions of both proteins were significantly lower compared with control cells: by 47% for podocin and by 42% for nephrin. This was accompanied with 36% increase in albumin permeability in shPINK1 expressing podocytes.
PINK1 inhibition had considerable effect on the membrane expression of insulin receptor and glucose transporter type 4 (GLUT4), which were reduced by 75% and 50%, respectively.
These findings confirm an important role of mitophagy in the podocytes biology and provide a novel insight in the molecular mechanism which may underlie the development of podocyte insulin resistance. Methods: To investigate this, we utilized Drosophila nephrocytes and studied the functional role of the mechanosensor Cheerio (dFilamin). FilaminB is upregulated in podocyte injury models and upon increased biomechanical stress, therefore serving as an ideal candidate for mediating mechano-protection in response to injury. Nephronectin (NPNT), which is mainly expressed by podocytes, is one important ECM protein for GBM integrity and has been shown to be altered in different kidney diseases. While NPNT was reported to be increased in diabetic nephropathy, NPNT levels in membranous glomerulonephropathy and FSGS were reduced. Furthermore, NPNT has been shown to be down-regulated by transforming growth factor beta (TGFβ), on both mRNA and protein level. Nck2KO-STZ mice exhibited greater proteinuria than diabetic WT-and Nck1KO-STZ mice.

Results
Histologically, podocyte density was equivalently reduced in all three diabetic groups, and surprisingly, glomerulosclerosis and glomerular hypertrophy were increased in WT-STZ, but not in either of the NckKO-STZ mice. Serum creatinine, cortical vimentin mRNA, and total nephrin protein (markers for glomerular filtration rate, epithelial-mesenchymal transition, and SD structure, respectively), were significantly elevated in Nck2KO-STZ, but not in WTs-STZs and Nck1KO-STZs, reflecting our findings regarding proteinuria.
In agreement with human datasets, this study shows that a loss of Nck2, but not Nck1, is associated with increased DP/DN susceptibility.

Dok1/2 adaptor proteins regulate adhesion in kidney podocytes
Ms Background: Post-transplant recurrence of steroid-resistant nephrotic syndrome (SRNS) is thought to be due to an unknown "circulating factor", the identity of which has so far remained elusive. Our previous work suggests a signalling role for protease-activated receptor-1 (PAR-1), leading to impaired podocyte function. The signalling pathways downstream of PAR-1 in podocytes are unknown and could reveal novel mechanistic insights into the disease.
Methods: Conditionally immortalized human podocytes (ciPods) were treated with PAR-1 agonist peptide or nephrotic plasma (NP), in the absence or presence of four different PAR-1 antagonists.
Results: PAR-1 agonist and patient relapse NP, but not paired remission plasma, induced the phosphorylation of VASP, JNK, and proteins involved in pro-fibrotic pathways. These changes were inhibited by PAR-1 inhibitors, but not by TGF-β1 inhibition. Four PAR-1 inhibitors demonstrated specific antagonistic properties. The phosphorylation of VASP and JNK in a new 3D spheroid model and from stem cell-derived kidney organoids corroborated the finding from the 2D model. Functionally, relapse NP induced podocyte motility and podocyte loss from spheroids both of which were also selectively rescued by PAR-1 inhibitors. Treatment of kidney organoids with relapse NP induced the same VASP and pro-fibrotic phosphorylation in podocytes and the loss of podocyte-specific markers.
Conclusions: We propose that the circulating factor acts as a pro-fibrotic effector by activating PAR-1. A greater understanding of these signalling pathways will lead to the identification of novel therapeutic targets for this disease. Results: SARA podo mice showed less glomerulosclerosis histologically and less proteinuria than Ctrl podo mice after Adriamycin treatment. Expression levels of markers specific for podocytes are being measured to confirm that SARA maintained cellular phenotype.

SARA in
Gene expression profiles of podocytes isolated from SARA/Ctrl podo mice by flow cytometry are being analyzed by RNA sequencing. This high throughput analysis will provide insight into the mechanisms by which SARA maintains podocyte phenotype and protects against glomerulosclerosis.
Elucidating the mechanisms by which SARA functions will help unearth new molecular targets for therapies directed at glomerular diseases. increase throughout gestation in pregnant women. During pregnancy, renal glomerular filtration rates increase by 50% which implicated the possible unregulated function of renal filtration barrier. The aim of this study is to evaluate whether STBEVs can be internalized by human podocytes and influence their functions.
Methods: Small STBEVs (known as STB exosomes, STBEXs) were isolated from dual-lobe placental perfusion of normal pregnancy women (n=3) by ultracentrifuge and characterized by immunoblotting and nanoparticle tracking analysis. STBEXs at two different concentrations (20mg/ml and 40mg/ml) were stained with PKH26 and incubated with podocyte over a time course (15mins, 30mins, 2h, 4h, 6h and 24h). STBEXs internalization was assessed by flow cytometry and confocal microscopy. The nephrin expression and migration ability of podocytes influenced by STBEXs were assessed by western blot and migration assay.
Results: The internalization of STBEXs by podocyte can be observed from 30mins and increased throughout the time course, which yield the highest signal at 24h incubation.
Conclusions: STBEXs can be taken up by podocytes and alter podocytes' functions through alternating nephrin expression and enhancing podocyte migration ability. We speculate one of the functions STBEXs on podocyte is enhancing the slit diaphragm structure and may contribute to the higher filtration rate seen in pregnancy. Methods: We performed transcriptome analyses on glomeruli isolated from GC (immunosuppressive)-and Pio (non-immunosuppressive)-treated rats 11 days after NS induction with PAN (n=4/group).

Glucocorticoid-and Pioglitazone-Induced Proteinuria Reduction Correlates with
Results: Unsupervised clustering revealed partial reversibility of PAN-associated mRNA changes after either GC or Pio. IPA analyses + web-based bioinformatic platforms identified 29 genes-of-interest common to GC-and Pio-induced proteinuria reduction, which included ECM remodeling, lipid metabolism, DNA-binding, and cytoskeletal organization. Using realtime PCR expression differences, 29 genes-of-interest were selected for further analysis, Conclusions: Our set of 5 podocyte enriched genes (with a causal link to BP) include known glomerular genes, those with unknown function and those that highlight the limitations of single-cell transcriptomics in renal tissue.
Sex differences in podocyte molecular signatures and homeostasis might explain clinically observed sex differences in kidney diseases and could provide a rationale for novel approaches in personalized therapies.
Thus, male and female ROSA mT/mG-NHPS2Cre mice with GFP-expression restricted to podocytes were treated with rapamycin as a general metabolic stress model for three weeks to investigate intrinsic and stress-related sexual dimorphism of podocytes on a genomic and proteomic level. Metabolomics of renal cortex tissues were included to investigate functional consequences of rapamycin.
Kidney function and morphology remained normal in all experimental groups, yet RNA sequencing revealed strong intrinsic sex-differences with more than 1700 mRNA transcripts significantly sex-differently expressed. Furthermore, stress responses due to mTOR inhibition displayed sexual dimorphism in transcriptomes and metabolomes and signaling pathways as assessed by western blots. Gene set enrichment analyses pointed to sex differences in enriched GOs related to transcription, cytoskeleton, focal adhesion, mitochondria and translation. Additionally, proteomics displayed an increased abundance of cytoskeleton and focal adhesion molecules in females. Rapamycin treatment abolished the significant transcriptional sex differences, mainly due to increased changes in the male podocyte transcriptome. Metabolomics further supported the finding that females were less susceptible towards stress induced by mTOR inhibition showing less accumulation of amino acids and glycolysis metabolites after rapamycin.
In conclusion, for the first time podocyte-specific sexual dimorphism was identified in gene regulation and protein expression patterns in addition to sex-different metabolic changes in stress responses, which might explain and contribute significantly to sex differences in renal disease susceptibilities and progression. Results: We identify 78 BM proteins expressed by kidney organoids during differentiation in 3D culture. Overall, we observed increasing levels of BM proteins along the differentiation time-course, and distinct pattern of temporal expression for key BM proteins as such early components (COL4A1, COL4A2, LAMB1, LAMC1) and glomerular BM laminins (LAMA5, LAMB2), altogether indicating gradual BM assembly and remodeling over time. We also detected increased levels of EPB41L5 (implicated in incorporating laminin-511 and -521 into specialized kidney BMs) concomitant with the upregulation of deposited laminin alpha-5, suggesting a recapitulation of BM specialization in vitro. Integrated proteomic analyses between kidney organoid, human and mouse kidney datasets indicated that kidney organoids recapitulate a kidney BM signature seen in vivo.
Conclusion: we demonstrated that kidney organoids are a high-fidelity system to study BM assembly in vertebrates.