Podocytes Research Articles

Activation of Hippo Pathway Damages Slit Diaphragm by Deprivation of Ajuba Proteins.

Nuclear exclusion of the cotranscription factor YAP, which is a consequence of activation of the Hippo signaling pathway, leads to FSGS and podocyte apoptosis. Ajuba proteins play an important role in the glomerular filtration barrier by keeping the Hippo pathway inactive. In nephrocytes from Drosophila melanogaster , a well-established model system for podocyte research, Ajuba proteins ensure slit diaphragm (SD) formation and function. Hippo pathway activation leads to mislocalization of Ajuba proteins, decreased SD formation, rearrangement of the actin cytoskeleton, and increased SD permeability. Targeting the kinases of the Hippo pathway with specific inhibitors in the glomerulus could, therefore, be a promising strategy for therapy of FSGS. The highly conserved Hippo pathway, which regulates organ growth and cell proliferation by inhibiting transcriptional cofactors YAP/TAZ, plays a special role in podocytes, where activation of the pathway leads to apoptosis. The Ajuba family proteins (Ajuba, LIM domain-containing protein 1 (LIMD1) and Wilms tumor protein 1-interacting protein [WTIP]) can bind and inactivate large tumor suppressor kinases 1 and 2, (LATS1/2) two of the Hippo pathway key kinases. WTIP, furthermore, connects the slit diaphragm (SD), the specialized cell-cell junction between podocytes, with the actin cytoskeleton. We used garland cell nephrocytes of Drosophila melanogaster to monitor the role of Ajuba proteins in Hippo pathway regulation and structural integrity of the SD. Microscopy and functional assays analyzed the interplay between Ajuba proteins and LATS2 regarding expression, localization, interaction, and effects on the functionality of the SD. In nephrocytes, the Ajuba homolog Djub recruited Warts (LATS2 homolog) to the SD. Knockdown of Djub activated the Hippo pathway. Reciprocally, Hippo activation reduced the Djub level. Both Djub knockdown and Hippo activation led to morphological changes in the SD, rearrangement of the cortical actin cytoskeleton, and increased SD permeability. Knockdown of Warts or overexpression of constitutively active Yki prevented these effects. In podocytes, Hippo pathway activation or knockdown of YAP also decreased the level of Ajuba proteins. Ajuba proteins regulate the structure and function of the SD in nephrocytes, connecting the SD protein complex to the actin cytoskeleton and maintaining the Hippo pathway in an inactive state. Hippo pathway activation directly influencing Djub expression suggests a self-amplifying feedback mechanism.

Identification of circular dorsal ruffles as signal platforms for the AKT pathway in glomerular podocytes.

Circular dorsal ruffles (CDRs) are rounded membrane ruffles induced by growth factors to function as precursors of the large-scale endocytosis called macropinocytosis. In addition to their role in cellular uptake, recent research using cell line systems has shown that CDRs/macropinocytosis regulate the canonical AKT-mTORC1 growth factor signaling pathway. However, as CDRs have not been observed in tissues, their physiological relevance has remained unclear. Here, utilizing ultrahigh-resolution scanning electron microscopy, we first report that CDRs are expressed in glomerular podocytes ex vivo and in vivo, and we visually captured the transformation process to macropinocytosis. Moreover, through biochemical and imaging analyses, we show that AKT phosphorylation localized to CDRs upstream of mTORC1 activation in podocyte cell lines and isolated glomeruli. These results demonstrate the physiological role of CDRs as signal platforms for the AKT-mTORC1 pathway in glomerular podocytes at the tissue level. As mTORC1 plays critical roles in podocyte metabolism, and aberrant activation of mTORC1 triggers podocytopathies, our results strongly suggest that targeting CDR formation could represent a potential therapeutic approach for these diseases.

Angiotensin II type 2 receptor activation preserves megalin in the kidney and prevents proteinuria in high salt diet fed rats

Proteinuria is a risk factor for and consequence of kidney injury. Angiotensin II type 2 receptor (AT2R) is an emerging reno-protective target and is anti-proteinuric under pathological conditions, including high salt-fed obese animals. However, the mechanisms remain unknown, particularly whether the anti-proteinuric activity of AT2R is independent of its anti-hypertensive and anti-inflammatory effects. In the present study, obese Zucker rats were fed high sodium (4%) diet (HSD) for 48 h, a time in which blood pressure does not change. HSD caused proteinuria without affecting glomerular slit diaphragm proteins (nephrin and podocin), glomerular filtration rate, inflammatory and fibrotic markers (TNFα, IL-6, and TGF-β), ruling out glomerular injury, inflammation and fibrosis but indicating tubular mechanisms of proteinuria. At cellular and molecular levels, we observed a glycogen synthase kinase (GSK)-3β-mediated megalin phosphorylation, and its subsequent endocytosis and lysosomal degradation in HSD-fed rat kidneys. Megalin is a major proximal tubular endocytic protein transporter. The AT2R agonist C21 (0.3 mg/kg/day, i.p.) administration prevented proteinuria and rescued megalin surface expression potentially by activating Akt-mediated phosphorylation and inactivation of GSK-3β in HSD-fed rat kidneys. Overall, AT2R has a direct anti-proteinuric activity, potentially via megalin regulation, and is suggested as a novel target to limit kidney injury.

Molecular insight in intrarenal inflammation affecting four main types of cells in nephrons in IgA nephropathy.

Immunoglobulin A nephropathy (IgAN) is the most common primary glomerulonephritis and the leading cause of kidney failure in the world. The current widely accepted framework for its pathogenesis is the "multi-hit hypothesis." In this review, we mainly discussed the intrarenal inflammation in IgAN, which is initiated by immune complex deposition with complement molecule activation, by focusing on four main types of cells in nephrons including mesangial cells, endothelial cells, podocytes, and tubular epithelial cells (TECs). Galactose-deficient IgA1 (Gd-IgA1)-containing immune complexes deposit in the mesangium and activate complement molecules and mesangial cells. Activation of mesangial cells by Gd-IgA1 deposition with enhanced cellular proliferation, extracellular matrix (ECM) expansion, and inflammatory response plays a central role in the pathogenesis of IgAN. Regional immune complex deposition and mesangial-endothelial crosstalk result in hyperpermeability of endothelium with loss of endothelial cells and infiltration barrier proteins, and recruitment of inflammatory cells. Podocyte damage is mainly derived from mesangial-podocyte crosstalk, in which tumor necrosis factor-α (TNF-α), transforming growth factor-β (TGF-β), renin-angiotensin-aldosterone system (RAAS), and micro-RNAs are the major players in podocyte apoptosis and disorganization of slit diaphragm (SD) related to proteinuria in patients with IgAN. In addition to filtrated proteins into tubulointerstitium and mesangial-tubular crosstalk involved in the injury of TECs, retinoic acid has been discovered innovatively participating in TEC injury.

Co-Culture of Glomerular Endothelial Cells and Podocytes in a Custom-Designed Glomerulus-on-a-Chip Model Improves the Filtration Barrier Integrity and Affects the Glomerular Cell Phenotype.

Crosstalk between glomerular endothelial cells and glomerular epithelial cells (podocytes) is increasingly becoming apparent as a crucial mechanism to maintain the integrity of the glomerular filtration barrier. However, in vitro studies directly investigating the effect of this crosstalk on the glomerular filtration barrier are scarce because of the lack of suitable experimental models. Therefore, we developed a custom-made glomerulus-on-a-chip model recapitulating the glomerular filtration barrier, in which we investigated the effects of co-culture of glomerular endothelial cells and podocytes on filtration barrier function and the phenotype of these respective cell types. The custom-made glomerulus-on-a-chip model was designed using soft lithography. The chip consisted of two parallel microfluidic channels separated by a semi-permeable polycarbonate membrane. The glycocalyx was visualized by wheat germ agglutinin staining and the barrier integrity of the glomerulus-on-a-chip model was determined by measuring the transport rate of fluorescently labelled dextran from the top to the bottom channel. The effect of crosstalk on the transcriptome of glomerular endothelial cells and podocytes was investigated via RNA-sequencing. Glomerular endothelial cells and podocytes were successfully cultured on opposite sides of the membrane in our glomerulus-on-a-chip model using a polydopamine and collagen A double coating. Barrier integrity of the chip model was significantly improved when glomerular endothelial cells were co-cultured with podocytes compared to monocultures of either glomerular endothelial cells or podocytes. Co-culture enlarged the surface area of podocyte foot processes and increased the thickness of the glycocalyx. RNA-sequencing analysis revealed the regulation of cellular pathways involved in cellular differentiation and cellular adhesion as a result of the interaction between glomerular endothelial cells and podocytes. We present a novel custom-made glomerulus-on-a-chip co-culture model and demonstrated for the first time using a glomerulus-on-a-chip model that co-culture affects the morphology and transcriptional phenotype of glomerular endothelial cells and podocytes. Moreover, we showed that co-culture improves barrier function as a relevant functional readout for clinical translation. This model can be used in future studies to investigate specific glomerular paracrine pathways and unravel the role of glomerular crosstalk in glomerular (patho) physiology.

Preterm birth leads to a decreased number of differentiated podocytes and accelerated podocyte differentiation.

Preterm birth was previously identified as a high-risk factor for the long-term development of chronic kidney disease. However, the detailed pattern of podocyte (PD) changes caused by preterm birth and the potential mechanism underlying this process have not been well clarified. In present study, a rat model of preterm birth was established by delivery of pups 2days early and podometric methods were applied to identify the changes in PDs number caused by preterm birth. In addition, single-cell RNA sequencing (scRNA-seq) and subsequent bioinformatic analysis were performed in the preterm rat kidney to explore the possible mechanism caused by preterm birth. As results, when the kidney completely finished nephrogenesis at the age of 3weeks, a reduction in the total number of differentiated PDs in kidney sections was detected. In addition, 20 distinct clusters and 12 different cell types were identified after scRNA-seq in preterm rats (postnatal day 2) and full-term rats (postnatal day 0). The numbers of PDs and most types of inherent kidney cells were decreased in the preterm birth model. In addition, 177 genes were upregulated while 82 genes were downregulated in the PDs of full-term rats compared with those of preterm rats. Further functional GO analysis revealed that ribosome-related genes were enriched in PDs from full-term rats, and kidney development-related genes were enriched in PDs from preterm rats. Moreover, known PD-specific and PD precursor genes were highly expressed in PDs from preterm rats, and pseudotemporal analysis showed that PDs were present earlier in preterm rats than in full-term rats. In conclusion, the present study showed that preterm birth could cause a reduction in the number of differentiated PDs and accelerate the differentiation of PDs.

ATP/ADP biosensor organoids for drug nephrotoxicity assessment.

Drug nephrotoxicity is a common healthcare problem in hospitalized patients and a major limitation during drug development. Multi-segmented kidney organoids derived from human pluripotent stem cells may complement traditional cell culture and animal experiments for nephrotoxicity assessment. Here we evaluate the capability of kidney organoids to investigate drug toxicity in vitro. Kidney organoids express renal drug transporters, OAT1, OAT3, and OCT2, while a human proximal tubular cell line shows the absence of OAT1 and OAT3. Tenofovir and aristolochic acid (AA) induce proximal tubular injury in organoids which is ameliorated by an OAT inhibitor, probenecid, without damage to podocytes. Similarly, cisplatin causes proximal tubular damage that can be relieved by an OCT inhibitor, cimetidine, collectively suggesting the presence of functional OATs and OCTs in organoid proximal tubules. Puromycin aminonucleoside (PAN) induced segment-specific injury in glomerular podocytes in kidney organoids in the absence of tubular injury. Reporter organoids were generated with an ATP/ADP biosensor, which may be applicable to high-throughput screening in the future. In conclusion, the kidney organoid is a useful tool for toxicity assessment in the multicellular context and may contribute to nephrotoxicity assessment during drug development.

Macrophage inflammatory protein-1β as a novel therapeutic target for renal protection in diabetic kidney disease

Diabetic kidney disease (DKD) is the leading cause of end-stage renal disease worldwide and the prevalence of DKD has increased over recent decades. Inflammation is involved in the development and progression of DKD. In this study, we explored the potential role of macrophage inflammatory protein-1β (MIP-1β) in DKD. Clinical non-diabetic subjects and DKD patients with different levels of urine albumin-to-creatinine ratio (ACR) were enrolled in the study. Leprdb/db mice and MIP-1β knockout mice were also used as mouse models for DKD. We found that serum MIP-1β levels were elevated in the DKD patients, especially those with ACRs that were less than or equal to 300, suggesting that MIP-1β is activated in clinical DKD. The administration of anti-MIP-1β antibodies attenuated DKD severity in the Leprdb/db mice, which also showed reduced glomerular hypertrophy and podocyte injury, as well as decreased inflammation and fibrosis, suggesting that MIP-1β plays a role in the development of DKD. The MIP-1β knockout mice showed improved renal function and decreased renal glomerulosclerosis and fibrosis in DKD. Furthermore, podocytes from the MIP-1β knockout mice showed less high glucose-induced inflammation and fibrosis compared to those from wild-type mice. In conclusion, the inhibition or deletion of MIP-1β protected podocytes, modulated renal inflammation, and ameliorated experimental DKD, suggesting that novel anti-MIP-1β strategies could potentially be used to treat DKD.

Long non-coding RNA DLX6-AS1 is the key mediator of glomerular podocyte injury and albuminuria in diabetic nephropathy by targeting the miR-346/GSK-3β signaling pathway

Progressive albuminuria is the primary clinical symptom of diabetic nephropathy (DN), leading to a gradual decline in kidney function. DLX6-AS1 was the first reported long non-coding RNA (lncRNA) to participate in organogenesis and play crucial roles in the brain or neural cell development. Herein, we investigated the DLX6-AS1 (Dlx6-os1 in mice) role in DN pathogenesis. We found that DLX6-AS1 expression in DN patients correlated with the extent of albuminuria. Dlx6-os1 overexpression induced cellular damage and inflammatory responses in cultured podocytes through miR-346-mediated regulation of the GSK-3β pathway. In various established diabetic and newly developed knockout mouse models, Dlx6-os1 knockdown/knockout significantly reduced podocyte injury and albuminuria. The Dlx6-os1 effects were remarkably modulated by miR-346 mimics or mutants and significantly diminished in podocyte-specific GSK-3β-knockout mice. Thus, DLX6-AS1 (Dlx6-os1) promotes DN development by accelerating podocyte injury and inflammation through the upregulation of the GSK-3β pathway, providing a novel molecular target for DN therapy.

Loss of S1P Lyase Expression in Human Podocytes Causes a Reduction in Nephrin Expression That Involves PKCδ Activation.

Sphingosine 1-phosphate (S1P) lyase (SPL, Sgpl1) is an ER-associated enzyme that irreversibly degrades the bioactive lipid, S1P, and thereby regulates multiple cellular functions attributed to S1P. Biallelic mutations in the human Sglp1 gene lead to a severe form of a particular steroid-resistant nephrotic syndrome, suggesting that the SPL is critically involved in maintaining the glomerular ultrafiltration barrier, which is mainly built by glomerular podocytes. In this study, we have investigated the molecular effects of SPL knockdown (kd) in human podocytes to better understand the mechanism underlying nephrotic syndrome in patients. A stable SPL-kd cell line of human podocytes was generated by the lentiviral shRNA transduction method and was characterized for reduced SPL mRNA and protein levels and increased S1P levels. This cell line was further studied for changes in those podocyte-specific proteins that are known to regulate the ultrafiltration barrier. We show here that SPL-kd leads to the downregulation of the nephrin protein and mRNA expression, as well as the Wilms tumor suppressor gene 1 (WT1), which is a key transcription factor regulating nephrin expression. Mechanistically, SPL-kd resulted in increased total cellular protein kinase C (PKC) activity, while the stable downregulation of PKCδ revealed increased nephrin expression. Furthermore, the pro-inflammatory cytokine, interleukin 6 (IL-6), also reduced WT1 and nephrin expression. In addition, IL-6 caused increased PKCδ Thr505 phosphorylation, suggesting enzyme activation. Altogether, these data demonstrate that nephrin is a critical factor downregulated by the loss of SPL, which may directly cause podocyte foot process effacement as observed in mice and humans, leading to albuminuria, a hallmark of nephrotic syndrome. Furthermore, our in vitro data suggest that PKCδ could represent a new possible pharmacological target for the treatment of a nephrotic syndrome induced by SPL mutations.

The classical pathway triggers pathogenic complement activation in membranous nephropathy

Membranous nephropathy (MN) is an antibody-mediated autoimmune disease characterized by glomerular immune complexes containing complement components. However, both the initiation pathways and the pathogenic significance of complement activation in MN are poorly understood. Here, we show that components from all three complement pathways (alternative, classical and lectin) are found in renal biopsies from patients with MN. Proximity ligation assays to directly visualize complement assembly in the tissue reveal dominant activation via the classical pathway, with a close correlation to the degree of glomerular C1q-binding IgG subclasses. In an antigen-specific autoimmune mouse model of MN, glomerular damage and proteinuria are reduced in complement-deficient mice compared with wild-type littermates. Severe disease with progressive ascites, accompanied by extensive loss of the integral podocyte slit diaphragm proteins, nephrin and neph1, only occur in wild-type animals. Finally, targeted silencing of C3 using RNA interference after the onset of proteinuria significantly attenuates disease. Our study shows that, in MN, complement is primarily activated via the classical pathway and targeting complement components such as C3 may represent a promising therapeutic strategy.

Periglomerular afferent innervation of the mouse renal cortex.

Recent studies using a novel method for targeted ablation of afferent renal nerves have demonstrated their importance in the development and maintenance of some animal models of hypertension. However, relatively little is known about the anatomy of renal afferent nerves distal to the renal pelvis. Here, we investigated the anatomical relationship between renal glomeruli and afferent axons identified based on transient receptor potential vanilloid 1 channel (TRPV1) lineage or calcitonin gene related peptide (CGRP) immunolabeling. Analysis of over 6,000 (10,000 was accurate prior to the removal of the TH data during the review process) glomeruli from wildtype C57BL/6J mice and transgenic mice expressing tdTomato in TRPV1 lineage cells indicated that approximately half of all glomeruli sampled were closely apposed to tdTomato+ or CGRP+ afferent axons. Glomeruli were categorized as superficial, midcortical, or juxtamedullary based on their depth within the cortex. Juxtamedullary glomeruli were more likely to be closely apposed by afferent axon subtypes than more superficial glomeruli. High-resolution imaging of thick, cleared renal slices and subsequent distance transformations revealed that CGRP+ axons closely apposed to glomeruli were often found within 2 microns of nephrin+ labeling of glomerular podocytes. Furthermore, imaging of thick slices suggested that CGRP+ axon bundles can closely appose multiple glomeruli that share the same interlobular artery. Based on their expression of CGRP or tdTomato, prevalence near glomeruli, proximity to glomerular structures, and close apposition to multiple glomeruli within a module, we hypothesize that periglomerular afferent axons may function as mechanoreceptors monitoring glomerular pressure. These anatomical findings highlight the importance of further studies investigating the physiological role of periglomerular afferent axons in neural control of renal function in health and disease.

Podocyte Geranylgeranyl Transferase Type-I Is Essential for Maintenance of the Glomerular Filtration Barrier.

A tightly regulated actin cytoskeleton attained through balanced activity of RhoGTPases is crucial to maintaining podocyte function. However, how RhoGTPases are regulated by geranylgeranylation, a post-translational modification, has been unexplored. The authors found that loss of the geranylgeranylation enzyme geranylgeranyl transferase type-I (GGTase-I) in podocytes led to progressive albuminuria and foot process effacement in podocyte-specific GGTase-I knockout mice. In cultured podocytes, the absence of geranylgeranylation resulted in altered activity of its downstream substrates Rac1, RhoA, Cdc42, and Rap1, leading to alterations of β1-integrins and actin cytoskeleton structural changes. These findings highlight the importance of geranylgeranylation in the dynamic management of RhoGTPases and Rap1 to control podocyte function, providing new knowledge about podocyte biology and glomerular filtration barrier function. Impairment of the glomerular filtration barrier is in part attributed to podocyte foot process effacement (FPE), entailing disruption of the actin cytoskeleton and the slit diaphragm. Maintenance of the actin cytoskeleton, which contains a complex signaling network through its connections to slit diaphragm and focal adhesion proteins, is thus considered crucial to preserving podocyte structure and function. A dynamic yet tightly regulated cytoskeleton is attained through balanced activity of RhoGTPases. Most RhoGTPases are post-translationally modified by the enzyme geranylgeranyl transferase type-I (GGTase-I). Although geranylgeranylation has been shown to regulate activities of RhoGTPases and RasGTPase Rap1, its significance in podocytes is unknown. We used immunofluorescence to localize GGTase-I, which was expressed mainly by podocytes in the glomeruli. To define geranylgeranylation's role in podocytes, we generated podocyte-specific GGTase-I knockout mice. We used transmission electron microscopy to evaluate FPE and measurements of urinary albumin excretion to analyze filtration barrier function. Geranylgeranylation's effects on RhoGTPases and Rap1 function were studied in vitro by knockdown or inhibition of GGTase-I. We used immunocytochemistry to study structural modifications of the actin cytoskeleton and β1 integrins. Depletion of GGTase-I in podocytes in vivo resulted in FPE and concomitant early-onset progressive albuminuria. A reduction of GGTase-I activity in cultured podocytes disrupted RhoGTPase balance by markedly increasing activity of RhoA, Rac1, and Cdc42 together with Rap1, resulting in dysregulation of the actin cytoskeleton and altered distribution of β1 integrins. These findings indicate that geranylgeranylation is of crucial importance for the maintenance of the delicate equilibrium of RhoGTPases and Rap1 in podocytes and consequently for the maintenance of glomerular integrity and function.

Coaxial cell printing of a human glomerular model: an in vitro glomerular filtration barrier and its pathophysiology

Much effort has been expended in emulating the kidney’s glomerular unit because of its limitless potential in the field of drug screening and nephrotoxicity testing in clinics. Herein, we fabricate a functional bilayer glomerular microvessel-on-a-chip that recapitulates the specific arrangement of the glomerular endothelial cell, podocyte layers, and the intervening glomerular basement membrane (GBM) in a single step. Our perfusable chip allows for the co-culture of monolayer glomerular endothelium and podocyte epithelium, which display mature functional markers of glomerular cells, and their proper interactions produce GBM proteins, which are the major components of the GBM in vivo. Furthermore, we test the selective permeability capacity, a representative hallmark function of the glomerular filtration barrier. Lastly, we evaluate the response of our glomerular model to Adriamycin- and hyperglycemia-induced injury to evaluate its applicability for drug screening and glomerular disease modeling.

Low-Dose Erythropoietin Amplifies Beneficial Effects of Angiotensin II Blockade on Glomerulosclerosis

Exogenous erythropoietin (EPO) is used to treat anemia in patients with chronic kidney disease (CKD). Concerns about the possible adverse effect of EPO on the progression of CKD have been raised owing to nonerythroid cell effects. We investigated the effects of low-dose EPO, independent of correcting anemia, on existing glomerulosclerosis. Adult mice underwent 5/6 nephrectomy and were randomized into the following 4 groups at week 8 after surgery: vehicle (VEH), losartan (angiotensin II type 1 receptor blocker [ARB]), darbepoetin-α (DA), or combination (DA+ARB). Four weeks later, mice were euthanized, followed by evaluation of renal structure and function. Glomerular endothelial cells and podocytes were cultured to evaluate the effects of DA on cell migration, apoptosis, and Akt signaling. ARB reduced blood pressure, albuminuria, and the level of serum creatinine and increased hematocrit compared with VEH, whereas low-dose DA only reduced the level of serum creatinine. Combination treatment showed a trend to increase hematocrit and survival compared with ARB alone. Combination treatment but not ARB alone significantly reduced the progression of glomerulosclerosis compared with VEH. Low-dose DA resulted in more preserved glomerular and peritubular capillary endothelial cells with increased p-Akt and even further endothelial cell preservation in combination with ARB. In cultured glomerular endothelial cells, angiotensin II induced more apoptosis, reduced migration, and decreased p-Flk1, a receptor for the proangiogenic vascular endothelial growth factor. DA counteracted these injuries and increased p-Akt, a key factor in angiogenesis and cell survival. DA also protected cultured podocytes against transforming growth factor β–induced apoptosis and synaptopodin loss. Low-dose EPO directly protects glomerular and peritubular endothelial cells via Akt phosphorylation. Therefore, treatment using a combination of low-dose EPO and ARB results in less progression of glomerulosclerosis in an experimental CKD model.

Emerging Perspectives on the Rare Tubulopathy Dent Disease: Is Glomerular Damage a Direct Consequence of ClC-5 Dysfunction?

Dent disease (DD1) is a rare tubulopathy caused by mutations in the CLCN5 gene. Glomerulosclerosis was recently reported in DD1 patients and ClC-5 protein was shown to be expressed in human podocytes. Nephrin and actin cytoskeleton play a key role for podocyte functions and podocyte endocytosis seems to be crucial for slit diaphragm regulation. The aim of this study was to analyze whether ClC-5 loss in podocytes might be a direct consequence of the glomerular damage in DD1 patients. Three DD1 kidney biopsies presenting focal global glomerulosclerosis and four control biopsies were analyzed by immunofluorescence (IF) for nephrin and podocalyxin, and by immunohistochemistry (IHC) for ClC-5. ClC-5 resulted as down-regulated in DD1 vs. control (CTRL) biopsies in both tubular and glomerular compartments (p < 0.01). A significant down-regulation of nephrin (p < 0.01) in DD1 vs. CTRL was demonstrated. CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/Caspase9) gene editing of CLCN5 in conditionally immortalized human podocytes was used to obtain clones with the stop codon mutation p.(R34Efs*14). We showed that ClC-5 and nephrin expression, analyzed by quantitative Reverse Transcription/Polymerase Chain Reaction (qRT/PCR) and In-Cell Western (ICW), was significantly downregulated in mutant clones compared to the wild type ones. In addition, F-actin staining with fluorescent phalloidin revealed actin derangements. Our results indicate that ClC-5 loss might alter podocyte function either through cytoskeleton disorganization or through impairment of nephrin recycling.

Glomerulonephritis and impact of vitamin D; a short look to the recent evidence

Implication for health policy/practice/research/medical education Mechanistic impact of vitamin D therapy in glomerulopathies consists of preservation of morphological integrity of slit diaphragm, which leads to restoration of tight junction proteins. Vitamin D also avoids the loss of nephrin component of the glomerular basement membrane.

Identification of Novel Genetic Risk Factors for Focal Segmental Glomerulosclerosis in Children: Results From the Chronic Kidney Disease in Children (CKiD) Cohort

Focal segmental glomerulosclerosis (FSGS) is a major cause of pediatric nephrotic syndrome, and African Americans exhibit an increased risk for developing FSGS compared with other populations. Predisposing genetic factors have previously been described in adults. Here we performed genomic screening of primary FSGS in a pediatric African American population. Prospective cohort with case-control genetic association study design. 140 African American children with chronic kidney disease from the Chronic Kidney Disease in Children (CKiD) cohort, including 32 cases with FSGS. Over 680,000 common single-nucleotide polymorphisms (SNPs) were tested for association. We also ran a pathway enrichment analysis and a human leucocyte antigen (HLA)-focused association study. Primary biopsy-proven pediatric FSGS. Multivariate logistic regression models. The genome-wide association study revealed 169 SNPs from 14 independent loci significantly associated with FSGS (false discovery rate [FDR]<5%). We observed notable signals for genetic variants within the APOL1 (P=8.6×10-7; OR, 25.8 [95% CI, 7.1-94.0]), ALMS1 (P=1.3×10-7; 13.0% in FSGS cases vs 0% in controls), and FGFR4 (P=4.3×10-6; OR, 24.8 [95% CI, 6.3-97.7]) genes, all of which had previously been associated with adult FSGS, kidney function, or chronic kidney disease. We also highlighted novel, functionally relevant genes, including GRB2 (which encodes a slit diaphragm protein promoting podocyte structure through actin polymerization) and ITGB1 (which is linked to renal injuries). Our results suggest a major role for immune responses and antigen presentation in pediatric FSGS through (1) associations with SNPs in PTPRJ (or CD148, P=3.5×10-7), which plays a role in T-cell receptor signaling, (2) HLA-DRB1∗11:01 association (P=6.1×10-3; OR, 4.5 [95% CI, 1.5-13.0]), and (3) signaling pathway enrichment (P=1.3×10-6). Sample size and no independent replication cohort with genomic data readily available. Our genetic study has identified functionally relevant risk factors and the importance of immune regulation for pediatric primary FSGS, which contributes to a better description of its molecular pathophysiological mechanisms. We assessed the genetic risk factors for primary focal segmental glomerulosclerosis (FSGS) by simultaneously testing over 680,000 genetic markers spread across the genome in 140 children, including 32 with FSGS lesions. Fourteen independent genetic regions were significantly associated with pediatric FSGS, including APOL1 and ALMS1-NAT8, which were previously found to be associated with FSGS and chronic kidney diseases in adults. Novel genes with relevant biological functions were also highlighted, such as GRB2 and FGFR4, which play a role in the kidney filtration barrier and in kidney cell differentiation, respectively. Finally, we revealed the importance of immune regulation in pediatric FSGS through associations involving cell surface proteins presenting antigens to the immune system and interacting with T-cell receptors.

PS-C33-3: THE EFFECT OF AGE AND HYPERTENSION ON PODOMETRICS IN DIFFERENT CORTICAL ZONES OF THE HUMAN KIDNEY

Objective: Podocyte loss and resultant nephron loss are common processes in the progression of glomerulosclerosis and CKD. Aging and hypertension are the main causes of benign nephrosclerosis, which is histologically characterized by concomitant appearances of glomerular collapse and enlargement. Glomerular hypertrophy and glomerulosclerosis often preferentially occur in specific cortical zones of the kidney, possibly due to the difference in kidney artery perfusion. However, it is unknown if podometrics, such as podocyte number per glomerulus, podocyte density and podocyte volume, differ among different cortical zones in the human kidneys. The aim of the present study is to determine the effect of aging and hypertension on podometrics in different cortical zones of the human kidneys. Design and method: Autopsy kidneys from 50 Japanese adults without apparent glomerular disease were cross-sectionally analyzed. Podometrics in different cortical zones (superficial/middle/juxtamedullary) were estimated on a single section using a model-based stereology. Results: Median age was 68 years and median eGFR was 73.5 mL/min/1.73m2. Hypertension was identified in 21 (42%) subjects. While podocyte number per glomerulus was similar in the three zones, superficial glomeruli were the smallest and had the highest podocyte density and smallest podocytes. Increasing age and hypertension were associated with lower podocyte number, with age mostly affecting superficial glomeruli, and hypertension mostly affecting juxtamedullary glomeruli. There was no significant interaction between age and hypertension (Figure). Neither podocyte density, podocyte volume nor volume density of podocytes in glomeruli was associated with age or hypertension. Conclusions: Podometric changes, especially decreased podocyte number per glomerulus, may also contribute to the development and different distribution of glomerular morphological phenotypes in aging and hypertension-related benign nephrosclerosis.

Targeting DNA Methylation in Podocytes to Overcome Chronic Kidney Disease.

The number of patients with chronic kidney disease (CKD) is on the rise worldwide, and there is urgent need for the development of effective plans against the increasing incidence of CKD. Podocytes, glomerular epithelial cells, are an integral part of the primary filtration unit of the kidney and form a slit membrane as a barrier to prevent proteinuria. The role of podocytes in the pathogenesis and progression of CKD is now recognized. Podocyte function depends on a specialized morphology with the arranged foot processes, which is directly related to their function. Epigenetic changes responsible for the regulation of gene expression related to podocyte morphology have been shown to be important in the pathogenesis of CKD. Although epigenetic mechanisms include DNA methylation, histone modifications, and RNA-based regulation, we have focused on DNA methylation changes because they are more stable than other epigenetic modifications. This review summarizes recent literature about the role of altered DNA methylation in the kidney, especially in glomerular podocytes, focusing on transcription factors and DNA damage responses that are closely associated with the formation of DNA methylation changes.