Human Podocytes Research Articles

Long Noncoding RNA ENSG00000254693 Promotes Diabetic Kidney Disease via Interacting with HuR.

Diabetic kidney disease (DKD) is one of the most common complications of diabetes mellitus (DM), without suitable therapies, causing end-stage renal diseases (ESRDs) ultimately. Moreover, there is increasing evidence demonstrating that long noncoding RNAs (lncRNAs) play crucial roles in the development of DKD. Our RNA sequencing data revealed a large group of differentially expressed lncRNAs in renal tissues of DKD, of which lncRNA ENSG00000254693 (lncRNA 254693 for short) changed drastically. In this study, we found that the expression of lncRNA 254693 was increased in both DKD patients and high-glucose-induced human podocytes. 5′/3′RACE and Northern blot assays were used to find the full length of lncRNA ENSG00000254693 which is 558 nucleotides and nonisoform that existed in human podocyte. Downregulation of lncRNA 254693 remarkably reversed the elevation of inflammation, apoptosis, and podocyte injury caused by high glucose. Then, we did bioinformatics analysis via RBPDB and found that lncRNA 254693 can combine with HuR, a RNA binding protein. Meanwhile, immunofluorescence and in situ hybridization double staining was used to prove the existence of colocalization between them. Intriguingly, lncRNA 254693 knockdown decreased HuR levels, while HuR knockdown also decreased the level of lncRNA 254693 and its stability. After this, RNA immunoprecipitation assay results confirmed the binding association between them again. In addition, we found that HuR was increased in high glucose-induced podocytes, and the silence of HuR could alleviate podocyte injury, inflammation, and apoptosis. These results together suggested a novel feedback regulation between lncRNA 254693 and HuR which could involve in podocyte injury and may serve as a predicted target for DKD therapies.

Selective Inhibition of Histone Deacetylase Class IIa With MC1568 Ameliorates Podocyte Injury.

Histone deacetylases (HDACs) inhibitors are promising therapeutic agents against proteinuric kidney diseases, here, we investigated the effect of MC1568, a selective inhibitor of HDAC class IIa, on the development and progression of nephrotic syndrome in a murine model induced by Adriamycin (ADR). In kidney tissues of FSGS patients, all four members of HDAC IIa were significantly upregulated in podocytes. In ADR-treated cultured human podocyte, expression of HDAC IIa were induced, meanwhile inhibition of HDAC IIa with MC1568 restored cytoskeleton structure and suppressed expression of desmin and α-SMA. In mice, administration of MC1568 at 14 days after ADR ameliorated proteinuria and podocyte injury, also decreased expression of Fibronectin and α-SMA. Mechanistically, MC1568 inhibited ADR induced β-catenin activation in vitro and in vivo. Together, these finding demonstrate that HDAC IIa inhibition ameliorates podocyte injury and proteinuria, which provide a possibility that MC1568 may be used in nephrotic syndrome.

NUP133 Controls Nuclear Pore Assembly, Transcriptome Composition, and Cytoskeleton Regulation in Podocytes.

Steroid-resistant nephrotic syndrome (SRNS) frequently leads to end-stage renal disease, ultimately requiring kidney replacement therapies. SRNS is often caused by hereditary monogenic mutations, specifically affecting specialized epithelial cells (podocytes) of the glomerular filtration barrier. Mutations in several components of the nuclear pore complex, including NUP133 and NUP107, have been recently identified to cause hereditary SRNS. However, underlying pathomechanisms, eliciting podocyte-specific manifestations of these nucleoporopathies, remained largely elusive. Here, we generated an in vitro model of NUP133-linked nucleoporopathies using CRISPR/Cas9-mediated genome editing in human podocytes. Transcriptome, nuclear pore assembly, and cytoskeleton regulation of NUP133 loss-of-function, mutant, and wild-type podocytes were analyzed. Loss of NUP133 translated into a disruption of the nuclear pore, alterations of the podocyte-specific transcriptome, and impaired cellular protrusion generation. Surprisingly, comparative analysis of the described SRNS-related NUP133 mutations revealed only mild defects. Am impaired protein interaction in the Y-complex and decrease of NUP133 protein levels might be the primary and unifying consequence of mutant variants, leading to a partial loss-of-function phenotype and disease manifestation in susceptible cell types, such as podocytes.

Cytoskeletal protein degradation in brain death donor kidneys associates with adverse posttransplant outcomes

In brain death, cerebral injury contributes to systemic biological dysregulation, causing significant cellular stress in donor kidneys adversely impacting the quality of grafts. Here, we hypothesized that donation after brain death (DBD) kidneys undergo proteolytic processes that may deem grafts susceptible to posttransplant dysfunction. Using mass spectrometry and immunoblotting, we mapped degradation profiles of cytoskeletal proteins in deceased and living donor kidney biopsies. We found that key cytoskeletal proteins in DBD kidneys were proteolytically cleaved, generating peptide fragments, predominantly in grafts with suboptimal posttransplant function. Interestingly, α‐actinin‐4 and talin‐1 proteolytic fragments were detected in brain death but not in circulatory death or living donor kidneys with similar donor characteristics. As talin‐1 is a specific proteolytic target of calpain‐1, we investigated a potential trigger of calpain activation and talin‐1 degradation using human ex vivo precision‐cut kidney slices and in vitro podocytes. Notably, we showed that activation of calpain‐1 by transforming growth factor‐β generated proteolytic fragments of talin‐1 that matched the degradation fragments detected in DBD preimplantation kidneys, also causing dysregulation of the actin cytoskeleton in human podocytes; events that were reversed by calpain‐1 inhibition. Our data provide initial evidence that brain death donor kidneys are more susceptible to cytoskeletal protein degradation. Correlation to posttransplant outcomes may be established by future studies.

IDF21-0319 Novel compounds found to regulate VEGF-A alternative splicing in diabetic podocytes

Background: Alternative splicing (AS) gives rise to multiple proteins from the same gene, by skipping or including exons or parts of them, or by keeping introns as coding sequences. The resulting AS isoforms may function differently from each other. Vascular endothelial growth factor A (VEGF-A) is an angiogenic protein. The alternatively spliced variant, VEGF-A165b, is formed when a distal 3’ splice site in exon 8 is selected. The anti-angiogenic and anti-permeability VEGF-A165b has reno-protective properties and has been shown to rescue kidney function in mouse models of diabetic nephropathy. Aim: The aim of this study was to investigate three novel compounds that regulate VEGF-A AS in podocytes exposed to a diabetic environment. Method: Two compounds trovafloxacin and 10058-F4 (5-[(4-Ethylphenyl) methylene]-2-thioxo-4-thiazolidinone), were identified to switch VEGF-A splicing in a previous screen performed by our lab using a library of pharmacologically active compounds (LOPAC). The third compound, delphinidin, was found to be key compound regulating VEGF-A AS in a natural blueberry and sea buckthorn extract (DIAVIT). Human podocytes were exposed to a diabetic environment (glucose soup [GS]: 25 mM glucose, 1 ng/ml TNF-α, 1 ng/ml IL-6, and 100 nM insulin), in comparison to a normal glucose (5.5 mM glucose) and an osmotic (5.5 mM glucose % 2B 19.5 mM mannitol) control, for 48 hours. RNA and protein were extracted for RT-PCR and Western blotting analysis of splice isoforms. Results: Changes in the VEGF-A165a/VEGF-A165b splicing ratio as well as the activation and expression of splice factors known to regulate this event will be compared between the diabetic environment and controls, in addition to the effects of each compound. Trovafloxacin (10 mM), 10058-F4 (10 mM), and delphinidin (10 mg/ml) significantly increased the anti-angiogenic VEGF-A165b relative to pro-angiogenic VEGF-A165a in podocytes exposed to a normal glucose environment at either the mRNA or protein level. Furthermore, 10058-F4 and delphinidin were also found to increase the VEGF-A165b/VEGF-A165a ratio in podocytes exposed to a diabetic environment. Regarding 10058-F4, pilot data suggests that this compound may influence the expression of Clk-1, a kinase known to regulate VEGF-A splice site selection, in diabetic podocytes. On the other hand, delphinidin was found to increase the phosphorylation of SRSF6, a splice factor known to promote VEGF-A165b splice site selection, in diabetic podocytes. 10058-F4 was found to downregulate SRSF1, which may be through c-myc inhibition. Discussion: We have identified three novel compounds that regulate VEGF-A AS to promote the expression of the reno-protective VEGF-A165b isoform in podocytes. This study will further investigate the mechanism of action of these three compounds regarding VEGF-A splicing regulation in diabetic podocytes. The final goal of this project is to identify whether these novel AS regulatory compounds can be used to develop new therapeutic strategies in diabetic nephropathy.

Epigallocatechin Gallate Induces the Demethylation of Actinin Alpha 4 to Inhibit Diabetic Nephropathy Renal Fibrosis via the NF-KB Signaling Pathway In Vitro.

Actinin alpha 4 (ACTN4) is expressed in the kidney podocytes. ACTN4 gene methylation in patients with diabetic nephropathy (DN) remains high. Underlying mechanism of epigallocatechin-3-gallate (EGCG) inducing ACTN4 demethylation, and its inhibitory effect on DN renal fibrosis remains unclear.Methods:Human podocyte cell line, HPC, was treated with high glucose to establish model of DN. The levels of cytokines, vascular endothelial growth factor (VEGF) and interleukin (IL)-8, and fibrosis markers, alpha smooth muscle actin (α-SMA) and fibronectin (FN), were determined using enzyme-linked immunosorbent assay. HPC cells were treated with EGCG, and cell viability was determined by MTT assay, ACTN4 gene methylation was analyzed by MSP. mRNA and protein expression levels were measured using RT-qPCR and Western blotting, respectively.Results:Actinin alpha 4 gene promoter was hypermethylated in the high glucose-treated groups. EGCG reversed the hypermethylated status of ACTN4, along with the upregulation of ACTN4 levels and downregulation of DNA methyltransferase 1 (DNMT1), NF-κB p65, p-NF-κB p65, IκB-α, VEGF, IL-8, α-SMA, and FN levels (P<.05).Conclusion:Epigallocatechin-3-gallate reduced hypermethylation of ACTN4 in HPC cells by downregulating DNMT1 expression and restoring ACTN4 expression, contributing to the upregulation of the NF-KB p65, p-NF-KB p65, IKB-α, VEGF, IL-8, α-SMA, and FN levels (P<.05).

IDF21-0092 Influence of the NLRP3 inflammasome in podocytes on the pathogenesis of diabetes dependent hyperfiltration-induced CKD

Background: Diabetic nephropathy (DN) is one of the most severe complications of Type 1 diabetes mellitus (T1) that is accounting for about 40% of end-stage renal disease (ESRD). The pathology is seen to correlate with inflammation. Amongst others, pattern recognition receptors are important mediators of disease progression, e.g. nucleotide-binding oligomerization domain receptors, together with inflammasomes. Especially the Nod-like receptor protein 3 (NLRP3) inflammasome multiprotein complex seems to be a key mediator in DN. The assembly of this promotes the maturation of proinflammatory cytokines (canonical pathway) such as IL-1β and IL-18. It is assumed that there are non-canonical pathways, that trigger innate inflammatory response. Whereas the role of NLRP3 in specialized immune cells is well understood, its contribution to processes of disease and homeostasis in renal parenchymal cells is controversially discussed in literature. Aim: With this project we expect to get a better understanding of the role of canonical and non-canonical NLRP3 signaling in podocytes. Furthermore, we aim to investigate if an inhibition of the NLRP3 signaling may lead to an ameliorated or even absent hyperfiltration-induced CKD. Method: To verify, if the NLRP3 inflammasome is involved in canonical, proinflammatory signaling, primary human podocytes derived from renal progenitor cells were stimulated in vitro. By using different concentrations of LPS, ATP, glucose and nigericin, metabolic stress was imitated. IL-1β and IL-18 ELISAs were used to verify canonical pathway activation. We further introduced podocyte-specific NLRP3 inflammasome modulations and Streptozotocin dependent diabetes in mice with a NLRP3 overactive variant (Nlrp3A350V fl/wt) to investigate, if NLRP3 overactivation leads to an aggravated phenotype of hyperfiltration-induced CKD. By using pharmaceuticals such as Oridonine and beta-Hydroxybutyrate we examined, if the inhibition of NLRP3 signaling in C57BL/6J wildtype mice with diabetes leads to an ameliorated outcome. To investigate if it is possible to reverse diabetes dependent hyperfiltration-induced CKD in NLRP3 overactive mice, we treated them with beta-Hydroxybutyrate. The primary endpoint of all in vivo experiments was defined as urinary albumin-creatinine-ratio. Furthermore, relevant histopathological alterations were observed. Results: Our data suggest that human derived podocytes are not susceptible for metabolic stress dependent canonical NLRP3 inflammasome signaling compared with THP-1 + PMA cells. Even though mice presented with severe diabetes and polyuria, we did not observe a significant difference in mice with NLRP3 overactivation regarding urinary albumin excretion and histological changes. So far, also the treatment with NLRP3 inhibitors did not ameliorate hyperfiltration-induced CKD. Discussion: Investigation of the NLRP3 functionality in podocytes provided a deeper insight and better understanding of the pathogenesis of diabetes dependent hyperfiltration-induced CKD. Even though it might have been possible that the specific inhibition of the inflammasome associated proteins should have synergistic effects in inflammation in ESRD and might be a future therapeutic target beyond IL-1β inhibition, our data do not support this hypothesis.

Protective effects of tadalafil on damaged podocytes in an adriamycin-induced nephrotic syndrome model

Podocyte injury is responsible for nephrotic syndrome. Previously, we found that tadalafil, a phosphodiesterase 5 inhibitor, might have protective effects on podocytes. Here, we investigated the effects of tadalafil in a nephrotic syndrome model and human podocyte cells.We divided adriamycin (ADR)-induced nephrotic syndrome model rats into the following groups: control + vehicle, control + tadalafil, ADR + vehicle, and ADR + tadalafil. The tadalafil-treated groups were orally administered 10 mg/kg tadalafil for 2 weeks. Renal parameters were measured. Immunohistology and immunofluorescence assays of glomerular injury were performed. Human primary podocytes were treated with or without tadalafil, and ADR. Cell viability and permeability assays were performed.ADR + vehicle exhibited severe proteinuria compared with control + vehicle and control + tadalafil. ADR + tadalafil attenuated proteinuria compared with ADR + vehicle. Wilms’ tumor 1 (WT1) immunostaining revealed that the number of WT1-positive cells was decreased by ADR; however, this decrease was prevented by ADR + tadalafil. In human podocytes, tadalafil increased the viability of ADR-treated cells, which was abrogated by KT5823, a cGMP-dependent protein kinase (PKG) inhibitor. Moreover, tadalafil prevented albumin permeability in ADR-treated cells. ADR treatment alone increased the permeability of albumin compared with the control.Tadalafil might inhibit kidney injury progression by preventing damage to podocytes and dysfunction of the glomerular filtration barrier.

Fungus-Derived 3-Hydroxyterphenyllin and Candidusin A Ameliorate Palmitic Acid-Induced Human Podocyte Injury via Anti-Oxidative and Anti-Apoptotic Mechanisms.

Diabetic nephropathy (DN) is a leading cause of end-stage renal disease. An elevated fatty acid plasma concentration leads to podocyte injury and DN progression. This study aimed to identify and characterize cellular mechanisms of natural compounds that inhibit palmitic acid (PA)–induced human podocyte injury. By screening 355 natural compounds using a cell viability assay, 3-hydroxyterphenyllin (3-HT) and candidusin A (CDA), isolated from the marine-derived fungus Aspergillus candidus PSU-AMF169, were found to protect against PA-induced podocyte injury, with half-maximal inhibitory concentrations (IC50) of ~16 and ~18 µM, respectively. Flow cytometry revealed that 3-HT and CDA suppressed PA-induced podocyte apoptosis. Importantly, CDA significantly prevented PA-induced podocyte barrier impairment as determined by 70 kDa dextran flux. Reactive oxygen species (ROS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) direct scavenging assays indicated that both compounds exerted an anti-oxidative effect via direct free radical–scavenging activity. Moreover, 3-HT and CDA upregulated the anti-apoptotic Bcl2 protein. In conclusion, 3-HT and CDA represent fungus-derived bioactive compounds that have a novel protective effect on PA-induced human podocyte apoptosis via mechanisms involving free radical scavenging and Bcl2 upregulation.

Anti-phospholipase A2 receptor antibodies directly induced podocyte damage in vitro

Background The pathogenesis of primary membranous nephropathy (MN) involves the antibodies against antigens on the cell surface of podocytes, with the majority of M-type phospholipase A2 receptor (PLA2R), and a profound podocyte dysfunction. The effects of anti-PLA2R antibodies directly to the podocytes remain unclear. Methods Anti-PLA2R antibodies from patients with PLA2R-associated MN were affinity-purified using a column coupled with recombinant human PLA2R protein. Their effects on conditionally immortalized human podocytes were assessed by apoptosis assays, cellular calcium detection, wound healing assay, and immunofluorescent staining. Proteomics analysis was performed by LC-MS/MS and on PANTHER database. Results The stimulation by anti-PLA2R antibodies could induce early-stage apoptosis of podocytes (MFI of Annexin V = 104.3 ± 19.2 vs. 36.7 ± 7.6, p = 0.004). The increase of calcium concentration in podocytes (MFI = 3309.3 ± 363.6 vs. 1776.3 ± 212.7, p = 0.015) might attribute to the endoplasmic reticulum calcium efflux. The expression of calcium/calmodulin-dependent protein kinase IV (CaMK4) was also increased (MFI = 134.4 ± 9.8 vs. 105.3 ± 10.1, p = 0.011). Proteomics results suggested that anti-PLA2R antibody treatment led to damage on cellular structure, and produced functional disorders on protein binding, actin filament binding, and microtubule motor activity. The staining of F-actin on foot process was reduced (MFI = 27.3 ± 2.8 vs. 47.5 ± 1.0, p = 0.001) and the motility and adherence capacity of podocytes were reduced (number of migrated cells = 44.7 ± 3.1 vs. 53.3 ± 4.9, p = 0.001) after incubation with anti-PLA2R antibodies. Conclusion These data indicate that anti-PLA2R antibodies may directly induce podocyte damage independent of the complement system, which expands the mechanism of anti-PLA2R antibodies on MN.

Activation of the Renin–Angiotensin System Disrupts the Cytoskeletal Architecture of Human Urine-Derived Podocytes

High blood pressure is one of the major public health problems that causes severe disorders in several tissues including the human kidney. One of the most important signaling pathways associated with the regulation of blood pressure is the renin–angiotensin system (RAS), with its main mediator angiotensin II (ANGII). Elevated levels of circulating and intracellular ANGII and aldosterone lead to pro-fibrotic, -inflammatory, and -hypertrophic milieu that causes remodeling and dysfunction in cardiovascular and renal tissues. Furthermore, ANGII has been recognized as a major risk factor for the induction of apoptosis in podocytes, ultimately leading to chronic kidney disease (CKD). In the past, disease modeling of kidney-associated diseases was extremely difficult, as the derivation of kidney originated cells is very challenging. Here we describe a differentiation protocol for reproducible differentiation of sine oculis homeobox homolog 2 (SIX2)-positive urine-derived renal progenitor cells (UdRPCs) into podocytes bearing typical cellular processes. The UdRPCs-derived podocytes show the activation of the renin–angiotensin system by being responsive to ANGII stimulation. Our data reveal the ANGII-dependent downregulation of nephrin (NPHS1) and synaptopodin (SYNPO), resulting in the disruption of the podocyte cytoskeletal architecture, as shown by immunofluorescence-based detection of α-Actinin. Furthermore, we show that the cytoskeletal disruption is mainly mediated through angiotensin II receptor type 1 (AGTR1) signaling and can be rescued by AGTR1 inhibition with the selective, competitive angiotensin II receptor type 1 antagonist, losartan. In the present manuscript we confirm and propose UdRPCs differentiated to podocytes as a unique cell type useful for studying nephrogenesis and associated diseases. Furthermore, the responsiveness of UdRPCs-derived podocytes to ANGII implies potential applications in nephrotoxicity studies and drug screening.

Rap1 Activity Is Essential for Focal Adhesion and Slit Diaphragm Integrity.

Glomerular podocytes build, with their intercellular junctions, part of the kidney filter. The podocyte cell adhesion protein, nephrin, is essential for developing and maintaining slit diaphragms as functional loss in humans results in heavy proteinuria. Nephrin expression and function are also altered in many adult-onset glomerulopathies. Nephrin signals from the slit diaphragm to the actin cytoskeleton and integrin β1 at focal adhesions by recruiting Crk family proteins, which can interact with the Rap guanine nucleotide exchange factor 1 C3G. As Rap1 activity affects focal adhesion formation, we hypothesize that nephrin signals via Rap1 to integrin β. To address this issue, we combined Drosophila in vivo and mammalian cell culture experiments. We find that Rap1 is necessary for correct targeting of integrin β to focal adhesions in Drosophila nephrocytes, which also form slit diaphragm-like structures. In the fly, the Rap1 activity is important for signaling of the nephrin ortholog to integrin β, as well as for nephrin-dependent slit diaphragm integrity. We show by genetic interaction experiments that Rap1 functions downstream of nephrin signaling to integrin β and downstream of nephrin signaling necessary for slit diaphragm integrity. Similarly, in human podocyte culture, nephrin activation results in increased activation of Rap1. Thus, Rap1 is necessary for downstream signal transduction of nephrin to integrin β.

Ceria-Zirconia nanoparticles reduce intracellular globotriaosylceramide accumulation and attenuate kidney injury by enhancing the autophagy flux in cellular and animal models of Fabry disease

BackgroundFabry disease (FD) is a lysosome storage disease (LSD) characterized by significantly reduced intracellular autophagy function. This contributes to the progression of intracellular pathologic signaling and can lead to organ injury. Phospholipid–polyethyleneglycol-capped Ceria-Zirconia antioxidant nanoparticles (PEG-CZNPs) have been reported to enhance autophagy flux. We analyzed whether they suppress globotriaosylceramide (Gb3) accumulation by enhancing autophagy flux and thereby attenuate kidney injury in both cellular and animal models of FD.ResultsGb3 was significantly increased in cultured human renal proximal tubular epithelial cells (HK-2) and human podocytes following the siRNA silencing of α galactosidase A (α-GLA). PEG-CZNPs effectively reduced the intracellular accumulation of Gb3 in both cell models of FD and improved both intracellular inflammation and apoptosis in the HK-2 cell model of FD. Moreover these particles attenuated pro fibrotic cytokines in the human podocyte model of FD. This effect was revealed through an improvement of the intracellular autophagy flux function and a reduction in reactive oxygen species (ROS). An FD animal model was generated in which 4-week-old male B6;129-Glatm1Kul/J mice were treated for 8 weeks with 10 mg/kg of PEG-CZNPs (twice weekly via intraperitoneal injection). Gb3 levels were reduced in the kidney tissues of these animals, and their podocyte characteristics and autophagy flux functions were preserved.ConclusionsPEG-CZNPs alleviate FD associated kidney injury by enhancing autophagy function and thus provide a foundation for the development of new drugs to treat of storage disease.Graphical

POS-395 APOL1 RENAL RISK VARIANTS COMPROMISED THE PODOCYTE DIFFERENTIATION BY DOWNREGULATION OF VITAMIN D RECEPTOR

APOL1 is endogenously expressed by human podocytes. The risk variant (RV) of APOL1 which are present in American African population shown to be associated with podocyte injury and kidney disease. The expression of APOL1 RV in podocyte cause their apoptosis by altering membrane ion flux, lysosomal swelling and involving the dysregulation of mitochondrial or endosomal functioning. But the molecular mechanism though which these APOL1 RV cause podocyte disintegration still undefined. Recently, we have reported that the APOL1 RV lost their ability to downregulate miR193a expression (a podocyte fate defining microRNA), resulting in dedifferentiation of podocytes.

POS-337 ASPARAGINYL ENDOPEPTIDASE PROTECTS AGAINST PODOCYTE INJURY IN DIABETIC NEPHROPATHY THROUGH CLEAVING COFILIN-1

Asparaginyl endopeptidase (AEP) is a lysosomal cysteine protease that specifically cleaves its substrates after asparagine residues. Physiologically, AEP is most abundant in kidney proximal tubule and is required for normal kidney physiology and homeostasis. Recent studies reported that AEP exerts anti-fibrotic effect through degrading of matrix fibronection during unilateral ureteral obstruction (UUO) animal model. However, its role and mechanism in podocyte injury during diabetic nephropathy (DN) remains unclear. Diabetic mice model was established by intraperitoneal injection of streptozotocin (STZ) and oral administration of AEP inhibitor was adopted to inhibit AEP activity, and also, AEP knockout mice were employed. In vitro study, human podocyte cell line was employed, and subjected to different treatments after differentiation. RNA interfere and adenovirus expression vector were utilized to knockdown AEP or overexpression of AEP and cofilin 1 fragments respectively. Moreover, AEP recombinase was used to detect its substrate---cofilin 1 cleavage activity in vitro. In the present study, AEP was highly upregulated in diabetic glomeruli and hyperglycemic stimuli treated-podocytes; however, AEP gene knockout and its compound inhibitor treatment accelerated DN in streptozotocin-induced diabetic mice, whereas specific induction of AEP in glomerular cells attenuated podocyte injury and renal function deterioration. In vitro, elevated AEP was involved in actin cytoskeleton maintenance and anti-apoptosis effects. Mechanistically, we found that AEP directly cleaved the actin-binding protein cofilin-1 after the asparagine 138 (N138) site. The protein levels of endogenous cofilin-1 1-138 fragments were upregulated in diabetic podocytes, consistent with the changes in AEP levels. Importantly, we found that cofilin-1 1-138 fragments were remarkably unphosphorylated than full-length cofilin-1, indicating the enhanced cytoskeleton maintenance activity of cofilin-1 1-138. Then we validated cofilin-1 1-138 could rescue podocytes from cytoskeleton disarrangement and injury in diabetic conditions. Taken together, our data suggests a protective role of elevated AEP in podocyte injury during DN progression through cleaving cofilin-1 to maintain podocyte cytoskeleton dynamics and defend damage.

POS-397 PROBUCOL AMELIORATES PODOCYTE INJURY IN D-GALACTOSE-INDUCED AGING MICE BY REGULATING MDM2/ERK1/2 SIGNALING PATHWAY

Probucol is widely used in clinical treatment as a lipid-reducing and anti-oxidant drug. However, it is unclear whether probucol exerts a renoprotective effect on aging mice. In the present study, we aimed to investigate the effect of probucol on kidneys of D-galactose-induced aging mice and explored the potential mechanisms. The aging mice model was established by subcutaneous injection of D-galactose (100 mg/kg, once daily for 6 weeks). At the end of the 6th week, the mice were administered with probucol or vehicle once daily for 2 weeks. Urine and blood samples as well as kidney tissues were collected for analysis. D-galactose-treated human podocyte cells (HPC) were employed for experiments in vitro. Probucol could improve renal function in D-galactose-induced aging mice, as indicated by reduced blood creatinine and urea nitrogen. Meanwhile, glomerulonephritis progression and podocyte injury were alleviated in probucol-treated mice, as showed by decreased glomerular sclerosis index as well as increased podocyte markers. Besides, probucol ameliorated renal fibrosis and podocyte apoptosis in aging mice. Furthermore, probucol suppressed murine double minute 2 (MDM2) expression, accompanied by the decreased protein level of ERK1/2. Pretreatment with probucol in D-galactose-induced HPC blocked MDM2/ERK1/2 signaling pathway. Thus, probucol ameliorates podocyte injury in D-galactose-induced aging mice by regulating MDM2/ERK1/2 signaling pathway.

CAMP-response element binding protein mediates podocyte injury in diabetic nephropathy by targeting lncRNA DLX6-AS1

BackgroundProgressive proteinuria is one of the earliest clinical features of diabetic nephropathy (DN). In our previous study, lncRNA DLX6-AS1 (DLX6-AS1, Dlx6os1 in the mouse) was found to be associated with the extent of albuminuria in DN patients. Furthermore, the lack of Dlx6os1 was pivotal in switching off the inflammatory response in db/db mouse model. However, the regulatory factors responsible for elevated DLX6-AS1 in DN remains unknown. MethodsTo identify potential regulatory factors for DLX6-AS1, JASPAR database and DNA pull down combined subsequent liquid chromatography-tandem mass spectrometry were used. Dual-luciferase reporter assay and chromatin immunoprecipitation were then performed to confirm binding sites. We also investigated the effects of the regulatory factors on DN progression in db/db mouse model and cultured human podocytes. ResultsOur analyses demonstrated that cAMP-response element binding protein (CREB) was highly expressed and closely associated with DLX6-AS1 in DN. In db/db mouse and in cultured podocytes, CREB silencing significantly reduced the level of DLX6-AS1 or Dlx6os1 and attenuated renal damage. Mechanistically, CREB overexpression aggravated renal inflammation and destroyed the structure of podocytes by targeting DLX6-AS1. The damaging role of CREB in podocyte injury was also inhibited by 666–15, a selective inhibitor, in a dose-dependent manner. In vivo, the inhibition of CREB by 666–15 significantly attenuated albuminuria and ameliorated inflammatory infiltration in podocytes. ConclusionsOur findings indicated that CREB is a key mediator of podocyte injury and acts by regulating DLX6-AS1. Thus, CREB may be an effective and potential therapeutic target for the treatment of DN.

Steroid-sensitive nephrotic syndrome candidate gene CLVS1 regulates podocyte oxidative stress and endocytosis.

We performed next-generation sequencing in patients with familial steroid-sensitive nephrotic syndrome (SSNS) and identified a homozygous segregating variant (p.H310Y) in the gene encoding clavesin-1 (CLVS1) in a consanguineous family with 3 affected individuals. Knockdown of the clavesin gene in zebrafish (clvs2) produced edema phenotypes due to disruption of podocyte structure and loss of glomerular filtration barrier integrity that could be rescued by WT CLVS1 but not the p.H310Y variant. Analysis of cultured human podocytes with CRISPR/Cas9-mediated CLVS1 knockout or homozygous H310Y knockin revealed deficits in clathrin-mediated endocytosis and increased susceptibility to apoptosis that could be rescued with corticosteroid treatment, mimicking the steroid responsiveness observed in patients with SSNS. The p.H310Y variant also disrupted binding of clavesin-1 to α-tocopherol transfer protein, resulting in increased reactive oxygen species (ROS) accumulation in CLVS1-deficient podocytes. Treatment of CLVS1-knockout or homozygous H310Y-knockin podocytes with pharmacological ROS inhibitors restored viability to control levels. Taken together, these data identify CLVS1 as a candidate gene for SSNS, provide insight into therapeutic effects of corticosteroids on podocyte cellular dynamics, and add to the growing evidence of the importance of endocytosis and oxidative stress regulation to podocyte function.

Mechanism of Rhubarb for Diabetic Kidney Disease through the AMPK/NF‐κB Signaling Pathway Based on Network Pharmacology

AbstractRhubarb (Da‐huang in pinyin, Radix Rhei Et Rhizome in pharmaceutical name) is a heat‐clearing and laxative herbal medicine, which is widely used to treat diabetic kidney disease in China. The study aims to decrypt the underlying mechanisms of the treatment of diabetic kidney disease using a systems pharmacology approach. Active compounds and corresponding potential therapeutic targets were harvested by utilizing the database of TCMSP. DKD targets were adopted from DrugBank, OMIM, TTD, and GAD databases. Then a network of Rhubarb‐ compounds ‐ target ‐ DKD network was harvested. The PPI network was then generated by the String database and subjected to Cytoscape software to harvest major and core targets network by topological analysis. Genes from the core targets network were further subjected to GO and KEGG enrichment analysis to figure out potential targeting pathways. Human podocyte cells (HPC) were used to further investigate the mechanism of against DKD. A total of 6 active ingredients and 271 targets of Rhubarb were picked out. 11 cellular biological processes and 18 pathways of mostly associated with inflammatory response, apoptosis, fibrosis, and peripheral circulation. Moreover, experimental validation suggests that Rhubarb increased cell viability. Rhubarb extract significantly suppressed the expression of NF‐kappa B, Bcl‐2, Caspase‐3, and Cleaved Caspase‐3, promoted the expression of AMPK and Bax, and decreased the release of cytokines. Rhubarb could alleviate DKD via the molecular mechanisms predicted by network pharmacology.

Differentially expressed proteins in genetically edited human podocytes: contributing to the understanding of early molecular events in Fabry Disease / Proteínas diferencialmente expressas em podócitos humanos geneticamente editados: contribuições para elucidação dos eventos moleculares iniciais na doença de Fabry

Background: Podocyte damage and subsequent proteinuric chronic nephropathy are prominent features of Fabry Disease (FD), a multisystemic X-linked inherited lysosomal storage disorder caused by deficient activity of the alpha-galactosidase A (α-GAL A) enzyme following mutations in the GLA gene. Methods: A proteomic approach based on two-dimensional gel electrophoresis coupled with mass spectrometry was used to explore differentially expressed proteins in podocytes with α-GAL A deficiency. This deficiency was developed through GLA gene deletion using CRISPR/Cas9 genome-editing technology in an immortalized human podocyte culture cell line. To further validate our proteomic findings, we compared the expression of autophagy-specific biomarkers (LC3B and p62) using western blotting, as well as evaluated apoptosis using propidium iodide fluorescence microscopy. Results: Our results showed that protein levels of ubiquitin carboxyl-terminal esterase L1, alpha-enolase, and heat shock protein 60 were reduced FD podocytes. Functional analysis using gene ontology found these proteins were predominately involved in biological processes, including autophagy regulation and apoptosis. Additionally, we found that autophagy-specific biomarkers LC3B and p62 were overexpressed, confirming impaired autophagy regulation and greater apoptosis in FD podocytes. Conclusions Our findings suggest impaired proteostasis in FD podocytes due to concomitant dysfunction of the ubiquitin–proteasome system and the autophagy pathway; both of which are potentially implicated in the pathogenesis of FD nephropathy.