Human Glomeruli Research Articles

Identification of natriuretic peptide receptor A-related gene expression signatures in podocytes in vivo reveals baseline control of protective pathways.

Natriuretic peptide receptor-A (NPR-A) is the principal receptor for the natriuretic peptides atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP). Targeted deletion of NPR-A in mouse glomerular podocytes significantly enhances renal injury in vivo in the DOCA-salt experimental model. It was therefore hypothesized that natriuretic peptides exert a direct protective effect on glomerular barrier integrity through activation of NPR-A and modulation of gene expression patterns in podocytes. Green fluorescence-positive podocytes from mice with a conditional deletion of Npr1 encoding NPR-A were isolated by fluorescence-activated cell sorting (FACS). Differentially expressed genes (DEGs) in podocytes were identified by RNA sequencing of podocytes from wild-type and NPR-A-deleted mice. Enrichment analysis was performed on the DEGs using Gene Ontology (GO) terms. Identified transcripts were validated by real-time PCR and ELISA of cultured isolated human and mouse glomeruli. In addition, the effect of natriuretic peptides on podocyte migration was investigated by measuring the outgrowth of podocytes from cultured glomeruli. A total of 158 DEGs were identified with 81 downregulated DEGs and 77 upregulated DEGs in Npr1-deficient podocytes. Among the downregulated genes were protein S and semaphorin 3G, which are known to have protective effects in podocytes. Protein S was also expressed in and secreted from isolated human glomeruli. GO enrichment analysis revealed that the upregulated DEGs in NPR-A deficient podocytes were associated with cell migration and motility. In line, BNP significantly decreased podocyte outgrowth from cultured glomeruli. In conclusion, endogenous levels of natriuretic peptides in mice support baseline protective pathways at glomerular podocytes such as protein S and suppress podocyte migration.NEW & NOTEWORTHY A combination of fluorescence-activated cell sorting and RNA sequencing identified 158 changed gene products in adult mouse kidneys with and without podocyte-specific deletion of the natriuretic peptide receptor A. Downregulated products included protein S and semaphorin 3G, both with proven renoprotective impact, whereas upregulated products were related to mobility of podocytes. Protein S was produced and released from human and murine isolated glomeruli, and atrial natriuretic peptide (ANP) led to decreased migration of podocytes.

#2474 TPPP3 maintains the stability of foot cytoskeleton architecture

Abstract Background and Aims Podocytes have a unique cytoskeleton architecture, and the disturbance of the skeleton architecture can lead to foot processes fusion and then proteinuria. Actin microfilaments dominate the cytoskeleton of the foot process, and microtubules and intermediate filaments dominate the synapses of the foot cells. The structural destruction of foot process actin is closely related to the injury of podocytes. The microtubule and actin often work together in cellular processes through stable connections. As the core structure of the skeleton network, microtubules play a key role in supporting and maintaining the skeleton network. However, the mechanism of microtubule structure destruction and podocyte injury remains unclear. Method Immunofluorescence and western blot were used to confirm the correlation between actin injury and decreased acetylation of tubulin in podocytes in vitro and in vivo. Single-cell RNA sequencing comprehensively characterized the expression of TPPP3 in human glomeruli. Immunofluorescence, immunohistochemical, western blot were used to determine the expression of TPPP3, α-tubulin, acetylated tubulin, actin microfilaments. Results In the case of podocyte injury, the acetylation level of tubulin decreased (P < 0.05). Reducing tubulin acetylation can lead to microfilaments injury (P < 0.05) and weakened podocyte motility (P < 0.05). TPPP3 was specifically expressed in human glomerular podocytes, and its expression level was significantly decreased after podocyte injury (P < 0.05). Knockdown of TPPP3 leaded to the level of microtubule acetylation decreased (P < 0.05) and microfilaments were damaged (P < 0.05) in podocytes. High acetylation levels of tubulin can partially restore actin microfilament damage caused by TPPP3 reduction in vivo and in vitro (P < 0.05). Conclusion TPPP3 is involved in maintaining the normal morphology and function of podocytes by stabilizing the actin microfilament skeleton structure by regulating the acetylation level of microtubules.

#187 Vasorin (VASN) commands podocyte quiescence and glomerular homeostasis. A novel FSGS mechanism?

Abstract Background and Aims Podocyte dysfunction and loss of quiescence contribute to focal segmental glomerulosclerosis (FSGS) and crescentic glomerulonephritis (CGN). Here, we characterized the role of Vasorin (VASN) in podocytes. Method We combined laser capture microdissection and deep RNA sequencing of human glomeruli, RNAscope-based in situ hybridization, immunostaining and mouse models with Vasn-driven fluorescent reporter, targeted Vasn gene deletion in podocytes and cultures of human podocytes. Results VASN mRNA is strongly expressed in healthy human and mouse glomeruli with a podocyte-specific pattern. Both global and podocyte-specific, constitutive or inducible gene deletion of Vasn in mice induced a nephrotic syndrome and kidney failure after day 14, with podocyte dedifferentiation and loss and severe FSGS lesions, indicating a fundamental and non-developmental role of VASN in podocyte homeostasis. Furthermore, we measured markedly decreased VASN expression in podocytes of patients with FSGS and ANCA-associated CGN but not with Minimal Change Disease (MCD) and Membranous Nephropathy (MN). We thus wondered whether VASN abundance could be a disease modifier for glomerulopathies. Mice with podocyte-specific partial heterozygous deletion of Vasn showed no evident baseline renal abnormalities. Still, they showed significantly more podocyte loss, albuminuria and kidney failure than wild-type mice when challenged by type 1 diabetes with glomerular hyperfiltration or CGN experimental models. Human podocytes transduced with lentiviral VASN-targeting shRNA (shVASN) displayed increased focal adhesion dynamics, resistance to oscillating mechanical stretches, enhanced migratory capacity and epithelial-mesenchymal transition (EMT) markers. Moreover, shVASN podocytes had a higher proliferative capacity, increased cell-cycle reentry and a relative S-phase slow-down. Likewise, transcriptomics analysis found the up-regulation of genes involved in cell metabolism, adhesion and EMT and the down-regulation of genes involved in the S to G2/M phase transition. In vivo, we confirmed that VASN deficiency increased podocyte proliferation in situ and urinary shedding. Conclusion These findings highlight the novel role of VASN as an essential master protein that maintains podocyte quiescence and homeostasis during health and kidney diseases while offering a potential new therapeutic target. VASN control mechanisms driving podocyte cell-cycle reentry and detachment and FSGS. Precise mechanisms of VASN functions in podocytes need further insights into VASN molecular partners.

PODO/TERT256 – A promising human immortalized podocyte cell line and its potential use for in vitro research at different oxygen levels

Podocytes are of key interest for the prediction of nephrotoxicity as they are especially sensitive to toxic insults due to their central role in the glomerular filtration apparatus. However, currently, prediction of nephrotoxicity in humans remains insufficiently reliable, thus highlighting the need for advanced in vitro model systems using human cells with improved prediction capacity. Recent approaches for refining in vitro model systems focus on closely replicating physiological conditions as observed under the in vivo situation typical of the respective nephron section of interest. PODO/TERT256, a human immortalized podocyte cell line, were employed in a semi-static transwell system to evaluate its potential use as a human podocyte in vitro system for modelling potential human glomerular toxicity. Furthermore, the impact of routinely employed excessive oxygen tension (21 % - AtmOx), when compared to the physiological oxygen tensions (10 % - PhysOx) observed in vivo, was analyzed. Generally, cultured PODO/TERT256 formed a stable, contact-inhibited monolayer with typical podocyte morphology (large cell body, apical microvilli, finger-like cytoplasmic projections (reminiscent of foot processes), and interdigitating cell-cell junctions) and developed a size-selective filtration barrier. PhysOx, however, induced a more pronounced in vivo like phenotype, comprised of significantly larger cell bodies, significantly enhanced filtration barrier size-selectivity, and a remarkable re-localization of nephrin to the cell membrane, thus suggesting an improved in vitro replication of in vivo characteristics. Preliminary toxicity characterization with the known glomerulotoxin doxorubicin (DOX) suggested an increasing change in filtration permeability, already at the lowest DOX concentrations tested (0.01 μM) under PhysOx, whereas obvious changes under AtmOx were observed as of 0.16 μM and higher with a near all or nothing effect. The latter findings suggested that PODO/TERT256 could serve as an in vitro human podocyte model for studying glomerulotoxicity, whereby culturing at PhyOx tension appeared critical for an improved in vivo-like phenotype and functionality. Moreover, PODO/TERT256 could be incorporated into advanced human glomerulus systems in vitro, recapitulating microfluidic conditions and multiple cell types (endothelial and mesenchymal cells) that can even better predict human glomerular toxicity.

Protein tyrosine phosphatase 1B is a regulator of alpha-actinin4 in the glomerular podocyte

Glomerular podocytes are instrumental for the barrier function of the kidney, and podocyte injury contributes to proteinuria and the deterioration of renal function. Protein tyrosine phosphatase 1B (PTP1B) is an established metabolic regulator, and the inactivation of this phosphatase mitigates podocyte injury. However, there is a paucity of data regarding the substrates that mediate PTP1B actions in podocytes. This study aims to uncover novel substrates of PTP1B in podocytes and validate a leading candidate. To this end, using substrate-trapping and mass spectroscopy, we identified putative substrates of this phosphatase and investigated the actin cross-linking cytoskeletal protein alpha-actinin4. PTP1B and alpha-actinin4 co-localized in murine and human glomeruli and transiently transfected E11 podocyte cells. Additionally, podocyte PTP1B deficiency in vivo and culture was associated with elevated tyrosine phosphorylation of alpha-actinin4. Conversely, reconstitution of the knockdown cells with PTP1B attenuated alpha-actinin4 tyrosine phosphorylation. We demonstrated co-association between alpha-actinin4 and the PTP1B substrate-trapping mutant, which was enhanced upon insulin stimulation and disrupted by vanadate, consistent with an enzyme-substrate interaction. Moreover, we identified alpha-actinin4 tandem tyrosine residues 486/487 as mediators of its interaction with PTP1B. Furthermore, knockdown studies in E11 cells suggest that PTP1B and alpha-actinin4 are modulators of podocyte motility. These observations indicate that PTP1B and alpha-actinin4 are likely interacting partners in a signaling node that modulates podocyte function. Targeting PTP1B and plausibly this one of its substrates may represent a new therapeutic approach for podocyte injury that warrants additional investigation.

Inhibiting NLRP3 signaling in aging podocytes improves their life- and health-span.

The decrease in the podocyte's lifespan and health-span that typify healthy kidney aging cause a decrease in their normal structure, physiology and function. The ability to halt and even reverse these changes becomes clinically relevant when disease is superimposed on an aged kidney. RNA-sequencing of podocytes from middle-aged mice showed an inflammatory phenotype with increases in the NLRP3 inflammasome, signaling for IL2/Stat5, IL6 and TNF, interferon gamma response, allograft rejection and complement, consistent with inflammaging. Furthermore, injury-induced NLRP3 signaling in podocytes was further augmented in aged mice compared to young ones. The NLRP3 inflammasome (NLRP3, Caspase-1, IL1β IL-18) was also increased in podocytes of middle-aged humans. Higher transcript expression for NLRP3 in human glomeruli was accompanied by reduced podocyte density and increased global glomerulosclerosis and glomerular volume. Pharmacological inhibition of NLRP3 with MCC950, or gene deletion, reduced podocyte senescence and the genes typifying aging in middle-aged mice, which was accompanied by an improved podocyte lifespan and health-span. Moreover, modeling the injury-dependent increase in NLRP3 signaling in human kidney organoids confirmed the anti-senescence effect of MC9950. Finally, NLRP3 also impacted liver aging. Together, these results suggest a critical role for the NLRP3 inflammasome in podocyte and liver aging.

#4996 TPPP3 REGULATES THE MICROTUBULE ACETYLATION AND PROMOTING YAP NUCLEAR RECRUITMENT IN HUMAN PODOCYTES

Abstract Background and Aims Minimal change disease (MCD) is a podocytopathy more commonly seen in children, but it also accounts for 10%–25% of adult NS. Patients with MCD manifest with abundant albuminuria, which is related to the damage of the glomerular filtration barrier. Podocytes are major components of the glomerular filtration barrier and play a crucial role in maintaining the integrity of the glomerular filtration barrier. Alterations in podocyte actin critically affect podocyte function. Tubulin polymerization-promoting protein family member 3 (TPPP3) is regulator of microtubule dynamic that has microtubule bundling activity. However, the mechanism of TPPP3 involved in podocyte apoptosis is remains unknown. Method Single-cell RNA sequencing comprehensively characterized the expression of TPPP3 in human glomeruli. We cultured human immortalized podocyte cells (HPC) in vitro, and constructed the HPC injury model. Immunofluorescence, western blot were used to determine the expression of TPPP3, α-tubulin, acetylated tubulin, phosopho-YAP1, Podocyte apoptosis was determined by Annexin V APC Apoptosis Detection Kit. Results TPPP3 was specifically expressed in human glomerular podocytes, and its expression level was significantly decreased in the cases of MCD and ADR induced podocyte injury(P<0.05). Knockdown of TPPP3 leaded to decreasing expression of acetylated tubulin in podocytes(P<0.05). Phosopho-YAP1 was also observed in the TPPP3 knockdown HPCs(P<0.05). Apoptosis was increased in the TPPP3 knockdown HPCs, suggesting TPPP3 may be related to the podocytes injury and apoptosis. Conclusion TPPP3 is involved with the apoptosis of podocytes by regulates nuclear recruitment of YAP which was promoted by microtubule acetylation.

Growth associated protein 43 deficiency promotes podocyte injury by activating the calmodulin/calcineurin pathway under hyperglycemia

Podocyte injury is a crucial factor in the pathogenesis of diabetic kidney disease (DKD), and finding potential therapeutic interventions that can mitigate podocyte injury holds significant clinical relevance. This study was to elucidate the role of growth associated protein-43(Gap43) in podocyte injury of high glucose (HG). We confirmed the expression of Gap43 in human glomerulus and found that Gap43 expression was downregulated in podocytes of patients with DKD and HG-treated podocytes in vitro. Gap43 knockdown in podocytes promoted podocyte apoptosis, increased migration ability and decreased nephrin expression, while overexpression of Gap43 markedly suppressed HG-induced injury. Moreover, the increased expression and activity of calcineurin (CaN) were also abrogated by overexpression Gap43 in HG. Pretreatment with a typical CaN inhibitor FK506 in Gap43 knockdown podocytes restored the injury. Mechanistically, co-immunoprecipitation experiments suggested that Gap43 could bind to calmodulin (CaM). Pull-down assay further demonstrated that Gap43 and CaM directly interacts with each other via amino acids 30–52 of Gap43 and amino acids 133–197 of CaM. In addition, we also identified Pax5 as potential transcription inhibitor factor mediating Gap43 expression. In conclusion, the study indicated that the Gap43/CaM-CaN pathway may be exploited as a promising therapeutic target for protecting against podocyte injury in high glucose.

Upregulated PD-1 signaling antagonizes glomerular health in aged kidneys and disease.

With an aging population, kidney health becomes an important medical and socioeconomic factor. Kidney aging mechanisms are not well understood. We previously showed that podocytes isolated from aged mice exhibit increased expression of programmed cell death protein 1 (PD-1) surface receptor and its 2 ligands (PD-L1 and PD-L2). PDCD1 transcript increased with age in microdissected human glomeruli, which correlated with lower estimated glomerular filtration rate and higher segmental glomerulosclerosis and vascular arterial intima-to-lumen ratio. In vitro studies in podocytes demonstrated a critical role for PD-1 signaling in cell survival and in the induction of a senescence-associated secretory phenotype. To prove PD-1 signaling was critical to podocyte aging, aged mice were injected with anti–PD-1 antibody. Treatment significantly improved the aging phenotype in both kidney and liver. In the glomerulus, it increased the life span of podocytes, but not that of parietal epithelial, mesangial, or endothelial cells. Transcriptomic and immunohistochemistry studies demonstrated that anti–PD-1 antibody treatment improved the health span of podocytes. Administering the same anti–PD-1 antibody to young mice with experimental focal segmental glomerulosclerosis (FSGS) lowered proteinuria and improved podocyte number. These results suggest a critical contribution of increased PD-1 signaling toward both kidney and liver aging and in FSGS.

FC017: Adaptive Remodelling of Mesangial Extracellular Matrix Proteoglycan Composition during IGA Nephropathy

Abstract BACKGROUND AND AIMS IgA nephropathy (IgAN) is characterized by the deposition of galactose deficient IgA (gd-IgA1) containing immune complexes in the mesangium leading to activation of the mesangial cells and local inflammation. Hallmarks of the disease are increased by mesangial proliferation and matrix expansion. The exact composition of the mesangial matrix is not known and nor is the full role of the expansion in disease progression. One important group of proteins found in extracellular matrixes is proteoglycans (PGs). PGs are composed of a core protein to which one or several negatively charged glycosaminoglycan (GAG) side chains are attached. Both the core protein and GAGs determine the properties of PGs and changes in the amount and structure of PGs could influence disease progression. In this study, we aimed to elucidate changes in the PG expression in the glomerulus in IgAN as well as how gd-IgA1 affects the mesangial cells’ PG expression. METHOD Using previously published glomerular transcriptomic data from patients with IgAN compared to healthy controls we investigated PG expression as well as changes in expression on a glomerular level [1]. Secondly, we investigated how gd-IgA1 affects the PG production of the cells in vitro using data from a previously published proteomic data set [1] as well as new in vivo experiments using real-time PCR as well as immunohistochemistry. To further explore the effects on PGs in IgAN, we employed a novel glycoproteomic approach that provides the combined characterization of GAG linkage regions and core protein identities in complex sample matrices. RESULTS We found 31 PGs to be expressed in the human glomeruli and of those 6 PGs were found to be significantly regulated in patients with IgAN. SDC1 (syndecan-1), was downregulated, while MXRA5 (matrix-remodeling associated 5), CD44 (CD44), TNC (tenascin C), FMOD (fibromodulin) and PRELP (PRELP) were up-regulated. The proteomic data set on mesangial cells treated with gd-IgA1 revealed that mesangial cells in vitro expressed 19 PGs and of those 10 were significantly regulated. Chondroitin sulfate proteoglycan 4, glypican-1, agrin, aggrecan, versican, lumican, protein AMBP, tenascin, collagen alpha-1(VII) chain and laminin subunit alpha-4. All of the significantly regulated PGs had an increased expression, except aggrecan, which was down-regulated. An interesting finding from the glycoproteomic experiments was the identification of collagen VII as a novel PG carrying chondroitin GAG in mesangial cells. In addition, immunohistochemistry revealed that gd-IgA1 treatment of mesangial cells does not lead to a total increase in GAGs, but rather to a switch from heparan sulfate to chondroitin sulfate GAGs. CONCLUSION In conclusion, this study gives a comprehensive view of the PGs expressed by mesangial cells and their alterations in response to gd-IgA1 as part of the expansion of the mesangium in IgAN. The switch of heparan sulfate GAGs to chondroitin sulfate GAGs is an interesting finding but the role of this switch needs further investigation.

Application of Scanning Ion Conductance Microscopy in the Studies of Podocytes Morphological Changes

High‐resolution imaging of glomerular podocytes morphology is becoming an essential and helpful tool for studying their role in renal disease progression. Electron microscopy (EM) is commonly used to analyze the effacement of the podocytes’ cell foot processes and the glomerular filtration barrier integrity loss. However, EM does not allow studying living samples and requires multiple complex procedures for sample preparation. Here, we propose and demonstrate the successful application of Scanning Ion Conductance Microscopy (SICM) for studying morphological changes of cultured podocytes and freshly isolated glomeruli.SICM provides a high‐resolution (comparable to EM) non‐optical imaging of cell topography using hopping probe microscopy principles and new dynamic reconstruction software. This technique can be used for analysis of cell membrane morphology and different functional characteristics, such as cell volume, membrane potentials, single ion‐channel currents, and other parameters. The major advantage of the current method is that it can be applied to live samples with complex morphology, such as intact glomeruli, under physiologically relevant liquid conditions.We have established and demonstrated a successful application of the SICM method in the live human glomerulus and provided a proof of principle for future dynamic analysis of membrane morphology and varied functional parameters in the living glomerulus. As a proof of principle, we applied this method for estimation the pathological structural changes in podocyte foot processes in type 2 diabetic nephropathy (T2DN) rats. Using topographical quantification of the length and irregular shape of the podocyte foot processes, we showed an absence of interdigitated foot processes on the selected region of the glomerular slit diaphragm in isolated T2DN glomeruli.In addition to the application of SICM with freshly isolated glomeruli, we used this approach to visualize and quantify foot process dynamics in cultured human podocytes during pharmacological applications. Thus, we showed that destroying the F‐actin with cytochalasin D completely stopped the migration of the podocyte filopodium through the remodeling of the actin cytoskeleton. Here, we further applied SICM to detect morphological changes in human podocytes after activation of Protease‐activated receptor‐1 (PAR1) by acute application of agonist TFLLR‐NH2. A series of experiments revealed that PAR1 activation led to retraction of the lamellipodium and a significant decrease in cell surface area (+51±46 vs. ‐15±8 µm2, 30 min after application of vehicle or TFLLR, respectively; n=5, p<0.05).In summary, SICM has numerous advantages and has excellent potential for precise quantitative analyses of the morphological changes in the glomerulus and cultured podocytes. Moreover, it opens the possibility of performing patch‐clamp recordings on the specific parts of the podocyte cell body and advances our understanding of the function and mechanism of the glomerulus filtration barrier.

Alterations in Protein Translation and Carboxylic Acid Catabolic Processes in Diabetic Kidney Disease.

Diabetic kidney disease (DKD) remains the leading cause of end-stage kidney disease despite decades of study. Alterations in the glomerulus and kidney tubules both contribute to the pathogenesis of DKD although the majority of investigative efforts have focused on the glomerulus. We sought to examine the differential expression signature of human DKD in the glomerulus and proximal tubule and corroborate our findings in the db/db mouse model of diabetes. A transcriptogram network analysis of RNAseq data from laser microdissected (LMD) human glomerulus and proximal tubule of DKD and reference nephrectomy samples revealed enriched pathways including rhodopsin-like receptors, olfactory signaling, and ribosome (protein translation) in the proximal tubule of human DKD biopsy samples. The translation pathway was also enriched in the glomerulus. Increased translation in diabetic kidneys was validated using polyribosomal profiling in the db/db mouse model of diabetes. Using single nuclear RNA sequencing (snRNAseq) of kidneys from db/db mice, we prioritized additional pathways identified in human DKD. The top overlapping pathway identified in the murine snRNAseq proximal tubule clusters and the human LMD proximal tubule compartment was carboxylic acid catabolism. Using ultra-performance liquid chromatography–mass spectrometry, the fatty acid catabolism pathway was also found to be dysregulated in the db/db mouse model. The Acetyl-CoA metabolite was down-regulated in db/db mice, aligning with the human differential expression of the genes ACOX1 and ACACB. In summary, our findings demonstrate that proximal tubular alterations in protein translation and carboxylic acid catabolism are key features in both human and murine DKD.

A glomerular transcriptomic landscape of apolipoprotein L1 in Black patients with focal segmental glomerulosclerosis

Apolipoprotein L1 (APOL1)-associated focal segmental glomerulosclerosis (FSGS) is the dominant form of FSGS in Black individuals. There are no targeted therapies for this condition, in part because the molecular mechanisms underlying APOL1's pathogenic contribution to FSGS are incompletely understood. Studying the transcriptomic landscape of APOL1 FSGS in patient kidneys is an important way to discover genes and molecular behaviors that are unique or most relevant to the human disease. With the hypothesis that the pathology driven by the high-risk APOL1 genotype is reflected in alteration of gene expression across the glomerular transcriptome, we compared expression and co-expression profiles of 15,703 genes in 16 Black patients with FSGS at high-risk vs 14 Black patients with a low-risk APOL1 genotype. Expression data from APOL1-inducible HEK293 cells and normal human glomeruli were used to pursue genes and molecular pathways uncovered in these studies. We discovered increased expression of APOL1 and nine other significant differentially expressed genes in high-risk patients. This included stanniocalcin, which has a role in mitochondrial and calcium-related processes along with differential correlations between high- and low-risk APOL1 and metabolism pathway genes. There were similar correlations with extracellular matrix- and immune-related genes, but significant loss of co-expression of mitochondrial genes in high-risk FSGS, and an NF-κB-down regulating gene, NKIRAS1, as the most significant hub gene with strong differential correlations with NDUF family (mitochondrial respiratory genes) and immune-related (JAK-STAT) genes. Thus, differences in mitochondrial gene regulation appear to underlie many differences observed between high- and low-risk Black patients with FSGS.

Three-dimensional morphometric analysis of human kidney nephron structures in health and disease

Background and Hypothesis: Chronic kidney disease (CKD) is very common and affects as many as 37 million people in the United States. Diabetes and hypertension are the most common causes of CKD. The pathogenesis of CKD is not fully elucidated. Morphological changes such as glomerulosclerosis and tubular atrophy are commonly observed with advanced disease. However, it is unclear if such changes occur at earlier stages of disease, a time when therapeutic interventions are likely to have the most benefit. Measurements of glomerular, vascular and tubular dimensions have been typically derived from thin section, but a pipeline for acquiring these morphometric measurements in 3-dimentions have not been performed. Using enhanced tissue clearing, confocal 3D imaging and volumetric segmentation and analysis, we aimed at developing an approach to measure the dimensions of various structures within the human kidney. Our overarching hypothesis is that such approach could allow the detection of morphometric changes in early disease. 
 Experimental Design or Project Methods: Kidney tissue were used either from donor nephrectomies or stored frozen biopsies from Biobank Biopsy Cohort of Indiana (BBCI). Enhanced tissue clearing and staining for nuclei, endothelium and proximal tubules were performed. Optical sectioning and 3D Imaging was done using confocal microscopy. Volume rendering, segmentation and analysis were done using the Imaris Software (Bitplane). Various built in and customized segmentation approaches were used.
 Results: Volumes spanning 200 µm in thickness encompassing entire glomeruli and tubules were obtained. 3D Rendering of these volumes allowed visualization and enabled 3D segmentation. Dimensions of entire human glomeruli (maximum and minimum diameters, glomerular volumes and cellular densities), tubules and vessels were defined in control and samples with diabetic kidney disease.
 Conclusion and Potential Impact: Our studies established an approach for accurate measurements of the dimensions of nephronal structures preserved in their 3D environment within tissue. We also established the feasibility of this approach in comparing changes with disease vs. control. Such methodology will open up a new line of investigations to better understand the pathogenesis of CKD, particularly at its early stages.

AIM2 Suppresses Inflammation and Epithelial Cell Proliferation during Glomerulonephritis.

Absent in melanoma-2 (AIM2) is an inflammasome-forming innate immune sensor for dsDNA but also exhibits inflammasome-independent functions such as restricting cellular proliferation. AIM2 is expressed in the kidney, but its localization and function are not fully characterized. In normal human glomeruli, AIM2 localized to podocytes. In patients with glomerulonephritis, AIM2 expression increased in CD44+-activated parietal epithelial cells within glomerular crescents. To explore AIM2 effects in glomerular disease, studies in Aim2 -/- mice were performed. Aim2-/- glomeruli showed reduced expression of Wilm tumor gene-1 (WT1), WT1-driven podocyte genes, and increased proliferation in outgrowth assays. In a nephrotoxic serum (NTS)-induced glomerulonephritis model, Aim2-/- (B6) mice exhibited more severe glomerular crescent formation, tubular injury, inflammation, and proteinuria compared with wild-type controls. Inflammasome activation markers were absent in both Aim2 -/- and wild-type kidneys, despite an increased inflammatory transcriptomic signature in Aim2 -/- mice. Aim2 -/- mice also demonstrated dysregulated cellular proliferation and an increase in CD44+ parietal epithelial cells during glomerulonephritis. The augmented inflammation and epithelial cell proliferation in Aim2 -/- (B6) mice was not due to genetic background, as Aim2 -/- (B6.129) mice demonstrated a similar phenotype during NTS glomerulonephritis. The AIM2-like receptor (ALR) locus was necessary for the inflammatory glomerulonephritis phenotype observed in Aim2 -/- mice, as NTS-treated ALR -/- mice displayed equal levels of injury as wild-type controls. Podocyte outgrowth from ALR -/- glomeruli was still increased, however, confirming that the ALR locus is dispensable for AIM2 effects on epithelial cell proliferation. These results identify a noncanonical role for AIM2 in suppressing inflammation and epithelial cell proliferation during glomerulonephritis.

Recreating Renal Function in a Human Glomerulus and Proximal Tubule Microphysiological System

Recreating Renal Function in a Human Glomerulus and Proximal Tubule Microphysiological System

Abstract P245: Activation Of Protease-activated Receptors 1 Leads To Structural Changes In Immortalized Cultured Human Podocytes.

Previous studies have suggested that activation of the protease-activated receptors (PAR) exacerbates the development of diabetic nephropathy. PARs are transmembrane proteins activated by extracellular serine proteases, such as thrombin and trypsin, which are frequently elevated in disease states. We hypothesize that serine protease activation of the PAR1 signaling cascade upregulates calcium channel activity and promotes excessive intracellular Ca 2+ ([Ca 2+ ] i ), leading to podocyte foot retraction, apoptosis, and glomerular filtration barrier (GFB) damage. Immunofluorescent labeling revealed the co-localization of PAR1 and podocyte marker nephrin in freshly isolated human glomeruli. To explore the functional role of PAR1 mediated signaling, we used an immortalized cultured human podocyte (hPod) cell line. Live confocal imaging revealed rapid elevation of [Ca 2+ ] i in response to a PAR1 specific agonist (TFLLR-NH 2 ). Moreover, preincubation with a PAR1 antagonist (RWJ 56100) completely blocked this effect (1616±48 vs. 206±135 a.u., for TFLLR vs. TFLLR+RJW, n<100 cells, p<0.05). Furthermore, we tested potential downstream signaling proteins in hPod cells after PAR1 activation using Western blot analysis. Application of TFLLR-NH 2 led to a decrease in PAR1 expression due to internalization and lysosomal degradation, accompanied by an increase in TRPC6 expression and time-dependent changes in p-ERK1/2 and PLC-γ1 cascades. We further used scanning ion-conductance microscopy (SICM) to detect morphological changes in podocytes, which measures real-time surface topography in hPod cells. This imaging revealed normal protrusion of lamellipodium under control conditions. In contrast, PAR1 activation led to retraction of the lamellipodium, and a significant decrease in surface area (+51±46 vs. -15±8 μm 2 , 30 min after application of vehicle or TFLLR, respectively; n=5, p<0.05). Our studies suggested that PAR1 is involved in GPCR-induced calcium influx in podocytes. Furthermore, these data demonstrate that PAR1-mediated signaling is involved in podocyte structural alterations and may increase [Ca 2+ ] i , potentially leading to loss of GFB integrity and podocyte apoptosis.

Modelling Renal Filtration and Reabsorption Processes in a Human Glomerulus and Proximal Tubule Microphysiological System.

Kidney microphysiological systems (MPS) serve as potentially valuable preclinical instruments in probing mechanisms of renal clearance and osmoregulation. Current kidney MPS models target regions of the nephron, such as the glomerulus and proximal tubule (PCT), but fail to incorporate multiple filtration and absorption interfaces. Here, we describe a novel, partially open glomerulus and PCT microdevice that integrates filtration and absorption in a single MPS. The system equalizes pressure on each side of the PCT that operates with one side “closed” by recirculating into the bloodstream, and the other “opened” by exiting as primary filtrate. This design precisely controls the internal fluid dynamics and prevents loss of all fluid to the open side. Through this feature, an in vitro human glomerulus and proximal tubule MPS was constructed to filter human serum albumin and reabsorb glucose for seven days of operation. For proof-of-concept experiments, three human-derived cell types—conditionally immortalized human podocytes (CIHP-1), human umbilical vein endothelial cells (HUVECs), and human proximal tubule cells (HK-2)—were adapted into a common serum-free medium prior to being seeded into the three-component MPS (T-junction splitter, glomerular housing unit, and parallel proximal tubule barrier model). This system was optimized geometrically (tubing length, tubing internal diameter, and inlet flow rate) using in silico computational modeling. The prototype tri-culture MPS successfully filtered blood serum protein and generated albumin filtration in a physiologically realistic manner, while the device cultured only with proximal tubule cells did not. This glomerulus and proximal convoluted tubule MPS is a potential prototype for the human kidney used in both human-relevant testing and examining pharmacokinetic interactions.

Super-Enhancer-Associated Transcription Factors Maintain Transcriptional Regulation in Mature Podocytes.

Transcriptional programs control cell fate, and identifying their components is critical for understanding diseases caused by cell lesion, such as podocytopathy. Although many transcription factors (TFs) are necessary for cell-state maintenance in glomeruli, their roles in transcriptional regulation are not well understood. The distribution of H3K27ac histones in human glomerulus cells was analyzed to identify superenhancer-associated TFs, and ChIP-seq and transcriptomics were performed to elucidate the regulatory roles of the TFs. Transgenic animal models of disease were further investigated to confirm the roles of specific TFs in podocyte maintenance. Superenhancer distribution revealed a group of potential TFs in core regulatory circuits in human glomerulus cells, including FOXC1/2, WT1, and LMX1B. Integration of transcriptome and cistrome data of FOXC1/2 in mice resolved transcriptional regulation in podocyte maintenance. FOXC1/2 regulated differentiation-associated transcription in mature podocytes. In both humans and animal models, mature podocyte injury was accompanied by deregulation of FOXC1/2 expression, and FOXC1/2 overexpression could protect podocytes in zebrafish. FOXC1/2 maintain podocyte differentiation through transcriptional stabilization. The genome-wide chromatin resources support further investigation of TFs' regulatory roles in glomeruli transcription programs.

MO625INHIBITION OF SGLT-2 CANNOT RESCUE NEPHRIN EXPRESSION IN DIABETIC NEPHROPATHY

Abstract Background and Aims Early glomerular damage in diabetes is induced by high blood glucose level (hyperglycemia) which affects the glomerular filtration barrier and leads to proteinuria. Podocyte-specific proteins like the transmembrane protein nephrin form the slit diaphragm that is important for proper function of the glomerular filtration barrier. Sodium-glucose co-transporter 2 (SGLT2) specific proteins are involved in glucose reabsorption in the kidney and maintain the normal glucose level in the blood. Recent studies showed remarkable success of SGLT2-inhibition in patients with diabetic nephropathy. Therefore we wanted to study if hyperglycemia induced reduction of nephrin expression is affected bySGLT-2 inhibition. Therefore we induced hyperglycemia in zebrafish larvae by knockdown of Pancreatic duodenal homeobox 1 (Pdx1) transcription factor and treated zebrafish larvae with Empagliflozin. In parallel we treated Bl/6 mice with streptozotozin and treated them with Empagliflozin. We then analyzed nephrin expression in both model systems. Method Zebrafish is an ideal model to study glomerular diseases, because the zebrafish larvae develops a pronephros with high homology to the human glomerulus. In order to inhibit SLGT-2 after Pdx1-knockdown, we treated both control and diabetic zebrafish larvae with 10µM Empagliflozin from 1dpf to 5dpf. We used a zebrafish line that expresses a fluorescent Vitamin D binding plasma protein Tg(l-fabp:DBP:eGFP) to measure proteinuria by measuring the GFP signal in both retinal and glomerular vessels of 120 hpf larvae. Immunohistochemistry against nephrin was performed using a specific zebrafish antibody. Results Conclusion Despite the promising effects of SLGT-2 inhibitor treatment in patients with diabetic nephropathy the early effects on nephrin expression are not addressed and remain an unchanged problem by this novel treatment option.