Podocyte Proteins Research Articles

Identification of Podocyte Cargo Proteins by Proteomic Analysis of Clathrin-Coated Vesicles.

Clathrin-mediated endocytosis (CME) plays a fundamental role in podocyte health. Genetic ablation of genes implicated in CME has been shown to cause severe proteinuria and foot process effacement in mice. However, little is known about the cargo of clathrin-coated vesicles (CCVs) in podocytes. The goal of this study was to isolate CCVs from podocytes and identify their cargo by proteomic analysis. Glomeruli isolated from Podocin-Cre Rosa-DTRflox mouse kidneys were seeded and treated with diphtheria toxin to obtain pure primary podocyte cultures. CCVs were isolated by differential gradient ultracentrifugation, and enrichment of CCVs was assessed by immunoblotting and electron microscopy (EM). Liquid chromatography-mass spectrometry (LC-MS) was performed for proteomic analysis. Proteins with higher abundance than transferrin receptor protein 1 were evaluated for CCV cargo potential against previously published literature. Immunofluorescence staining of identified cargo proteins and CCVs was performed in podocytes for further verification. Immunoblotting for multiple protein markers of CME revealed enrichment in the CCV fraction. Enrichment of CCVs among other small vesicles was observed via EM. Proteomics yielded a total of >1200 significant proteins. Multiple-step data analysis revealed 36 CCV-associated proteins, of which 10 represent novel, highly abundant cargo proteins in podocytes. Colocalization of cargo proteins and CCVs on immunostaining was observed. Our identification of podocyte CCV cargo proteins helps to elucidate the importance of endocytic trafficking for podocyte health and maintenance of the glomerular environment.

468-P: PDK1 Is a Key Regulator of Podocyte Viability and Maintenance of Glomerular Function

In diabetic states, altered activity of insulin signaling pathway is likely to play key roles in insulin resistance and in kidney dysfunction. Since the 3-phosphoinositide-dependent protein kinase 1 (PDK1) serves as a major signaling molecule in the regulation of cellular responses in multiple organs by mediating the phosphoinositide 3-kinase signaling pathway, we tested the hypothesis that podocyte-specific deletion of PDK1 leads to podocyte injury and proteinuric kidney disease. PDK1 was selectively silenced in podocytes using Cre-mediated recombination controlled by the podocin promoter. Mice with a podocyte-specific deletion of PDK1 (PDK1 KO) developed proteinuria, and died due to end-stage renal disease by 5 weeks after birth. Histologically, features characteristic of glomerulosclerosis, including podocyte foot process effacement, mesangial sclerosis, and protein casts, were observed in PDK1 KO at 3 weeks of age. We also examined podocyte distribution in glomeruli using the podocyte markers synaptopodin and WT-1, and found that their expressions were lost in some glomeruli of PDK1 KO at 3 weeks of age although their expression patterns at 1 weeks of age PDK1 KO were almost comparable with control mice. We next examined a possible role of forkhead transcription factor, forkhead box O1 (FoxO1) in linking PDK1 of podocytes to kidney function. For this, we generated podocyte-specific PDK1 and FoxO1 double knockout mice (DKO) by introducing Foxo1-flox gene into PDK1 KO mice. DKO exhibited a reduced level of proteinuria compared with age-matched PDK1 KO, and the maximal survival time of DKO had extended to 6.6 weeks after birth. Taken together, PDK1-FoxO1 pathway may be one of the critical signal transduction pathways that links podocyte injury and kidney dysfunction in diabetic states. Disclosure W. Iwata: None. H. Unoki-Kubota: None. H. Kato: None. M. Matsumoto: None. M. Nangaku: Consultant; Self; Akebia Therapeutics, Astellas Pharma Inc., Bayer AG, Boehringer Ingelheim International GmbH, Daiichi Sankyo, GlaxoSmithKline plc., Japan Tobacco Inc., Kyowa Hakko Kirin Co., Ltd., Mitsubishi Tanabe Pharma Corporation, Novo Nordisk Inc., Otsuka Pharmaceutical Co., Ltd., Scohia Pharma Inc. Research Support; Self; Takeda Pharmaceutical Company Limited. Speaker’s Bureau; Self; AstraZeneca, Torii Pharmaceutical Co. Ltd. Y. Kaburagi: None.

TM7SF1, an important autophagy regulatory protein in mouse podocytes

The autophagy, which can be regulated by lysosomal membrane proteins, plays a critical role in maintaining normal podocyte function. TM7SF1 is a novel lysosomal membrane protein, but its effect on autophagy is still unknown. This study aimed to identify the role of TM7SF1 in mouse podocyte (MPC5) autophagy. Interestingly, we detected an increase in LC3BII and SQSTM1/P62 in MPC5 through inhibiting TM7SF1, and which can be completely corrected after blocking the autolysosome degradation with chloroquine (CQ). Moreover, inhibition of TM7SF1 expression did not increase the mRNA level of SQSTM1/P62. Theses results suggested that inhibition of TM7SF1 led to impaired degradation of autophagy products, which manifest as an abnormal accumulation of LC3BII and SQSTM1/P62. Further studies showed that the downregulation of TM7SF1 resulted in a significant decrease in the number of acid lysosomes, which directly led to decreases in the number and function of autolysosomes. In conclusion, TM7SF1 is therefore essential for autolysosomes degradation pathway at the end of autophagy flow, and for the maintenance of podocyte function.

Sleeve Gastrectomy Attenuates Diabetic Nephropathy by Upregulating Nephrin Expressions in Diabetic Obese Rats.

Diabetic nephropathy (DN) is the leading cause of end-stage renal disease, and sleeve gastrectomy (SG) is considered to be an effective strategy to improve pre-existing DN. However, the mechanism remains unknown. Animal model of DN was induced by high-fat diet (HFD) and streptozotocin (STZ). SG or sham surgery was performed and rats were sacrificed at 4, 8, and 12weeks after surgery. The basic parameters (blood glucose, body weight, kidney weight), indicators of renal function including serum creatinine (Scr), blood urea nitrogen (BUN), urine microalbumin, urine creatinine (Ucr), microalbumin creatinine ratio (UACR), ultrastructural changes of glomerulus, and the expression of nephrin gene and protein in glomerular podocytes were compared among groups. Blood glucose and body weight of SG rats were significantly lower than those of the sham-operated rats, and renal function of SG groups were also significantly improved within the postoperative period of 12weeks. The results of periodic acid-Schiff staining (PAS) and transmission electron microscopy (TEM) showed that glomerular hypertrophy and accumulation of extracellular matrix proteins were significantly alleviated after SG, and the thickness of basement membrane and the fusion or effacement of foot processes were also significantly improved. The mRNA and protein expression of nephrin in SG groups was significantly higher than that in the sham group. These results suggest that SG attenuates DN by upregulating the expression of nephrin and improving the ultrastructure of glomerular filtration membrane. This study indicates that SG can be used as an available therapeutic intervention for DN.

Structural Disorder and Induced Folding of the WWC Family Proteins

The WWC family consists of three proteins‐ KIBRA/WWC1, WWC2, and WWC3. Each protein contains two WW domains which recognize proline‐rich sequences of the type PPxY (P is proline, Y is tyrosine, x is any amino acid). WW domains are named for two conserved tryptophans spaced 23 residues apart and adopt a three‐stranded antiparallel beta sheet fold. The WWC proteins are “atypical” in that, the second WW domain (WW2) contains an isoleucine rather than a conserved tryptophan. Recent studies of the KIBRA WW domains shows that this substitution leads to unfolding of the WW2 domains. However, the domain undergoes binding‐induced folding when bound to some partners but remains largely unfolded when bound to others. High sequence similarity of the WW domains in the WWC family suggests that unfolding and binding induced folding of the WW2 domain may be conserved in this family, but it is unclear why some partners induce folding of the WW2 domain while others do not. Our working hypothesis is that partners which contain PPxY motifs in close proximity will bind the WWC proteins with relatively high affinity and induce folding of the WW2 domain. In this study, we test this hypothesis with two binding partners, Large Tumor Suppressor 1 (LATS1), a kinase and tumor suppressor, and Synaptopodin (SYNPO), a synaptic and podocyte protein. We demonstrate by isothermal titration calorimetry that wildtype LATS1 and SYNPO which have two PPxY motifs each, separated by 194 and 15 residues respectively, bound KIBRA and WWC3 with modest micromolar binding affinities. However, mutant LATS1 (LATS1‐VA) and SYNPO (SYNPO_AA) constructs in which the PPxY motifs are separated by two amino acids, bound KIBRA and WWC3 with 20 to 180‐fold enhanced affinity. Using solution magnetic resonance spectroscopy, we demonstrated that the WW2 domain of KIBRA and WWC3 remain unfolded when bound to wildtype LATS1 and SYNPO, while the enhanced binding of the mutants LATS1_VA and SYNPO_AA is accompanied by folding of the WW2 domain. The structural heterogeneity of the WW2 domain may be an important for modulating binding to diverse PPxY motif partners.Support or Funding InformationThis work is supported by start‐up funds from Oregon State University (OSU), OSU College of Science Disease Mechanism and Prevention Fund, and a New Investigator Award from the Medical Research Foundation of Oregon. NMR experiments were collected at the Oregon State University NMR Facility funded in part by the National Institutes of Health, HEI Grant 1S10OD018518, and by the M. J. Murdock Charitable Trust grant # 2014162

Intravenous administration of iPS-MSCSPIONs mobilized into CKD parenchyma and effectively preserved residual renal function in CKD rat.

This study traced intravenously administered induced pluripotent stem cell (iPSC)‐derived mesenchymal stem cells (MSC) and assessed the impact of iPSC‐MSC on preserving renal function in SD rat after 5/6 nephrectomy. The results of in vitro study showed that FeraTrack™Direct contrast particles (ie intracellular magnetic labelling) in the iPSC‐MSC (ie iPS‐MSCSPIONs) were clearly identified by Prussian blue stain. Adult‐male SD rats (n = 40) were categorized into group 1 (SC), group 2 [SC + iPS‐MSCSPIONs (1.0 × 106cells)/intravenous administration post‐day‐14 CKD procedure], group 3 (CKD), group 4 [CKD + iPS‐MSCSPIONs (0.5 × 106cells)] and group 5 [CKD + iPS‐MSCSPIONs (1.0 × 106cells)]. By day‐15 after CKD induction, abdominal MRI demonstrated that iPS‐MSCSPIONs were only in the CKD parenchyma of groups 4 and 5. By day 60, the creatinine level/ratio of urine protein to urine creatinine/kidney injury score (by haematoxylin and eosin stain)/fibrotic area (Masson's trichrome stain)/IF microscopic finding of kidney injury molecule‐1 expression was lowest in groups 1 and 2, highest in group 3, and significantly higher in group 4 than in group 5, whereas IF microscopic findings of podocyte components (ZO‐1/synaptopodin) and protein levels of anti‐apoptosis ((Bad/Bcl‐xL/Bcl‐2) exhibited an opposite pattern to creatinine level among the five groups (all P < .0001). The protein expressions of cell‐proliferation signals (PI3K/p‐Akt/m‐TOR, p‐ERK1/2, FOXO1/GSK3β/p90RSK), apoptotic/DNA‐damage (Bax/caspases8‐10/cytosolic‐mitochondria) and inflammatory (TNF‐α/TNFR1/TRAF2/NF‐κB) biomarkers displayed an identical pattern to creatinine level among the five groups (all P < .0001). The iPS‐MSCSPIONs that were identified only in CKD parenchyma effectively protected the kidney against CKD injury.

Activation of TRPV1 channel antagonizes diabetic nephropathy through inhibiting endoplasmic reticulum-mitochondria contact in podocytes

The impairment of podocyte protein filtration function caused by excessive mitochondrial calcium intake is a critical feature of diabetic nephropathy (DN). Ca2+ channel transient receptor potential cation channel subfamily V member 1 (TRPV1) has been reported to protect against ischemia-reperfusion induced acute renal injury, but there is no report about its role in DN. Here, we report that dietary capsaicin potently inhibits and reverses chronic renal structural and functional damages in db/db or streptozotocin (STZ)-induced diabetic mice in a TRPV1-dependent manner. Activation of TRPV1 by capsaicin alleviated hyperglycemia-induced mitochondrial dysfunction in podocytes, accompanied by reduced mitochondria-associated membranes (MAMs) formation and fewer Ca2+ transport from endoplasmic reticulum (ER) to mitochondria. Mechanistically, TRPV1-mediated transient Ca2+ influx activated 5′ AMP-activated protein kinase (AMPK) that reduced the transcription of Fundc1, a key molecule participating in MAMs formation. Inhibition of AMPK or overexpression of Fundc1 obviously blocked the inhibitory effect of capsaicin on MAMs formation and functional decline in podocytes. These findings emphasize the critical role of mitochondrial Ca2+ homeostasis in the maintenance of normal renal function and suggest an effective intervention method to counteract DN.

Neutralizing Anti-Rituximab Antibodies and Relapse in Membranous Nephropathy Treated With Rituximab.

Membranous Nephropathy (MN) is an autoimmune disease associated with antibodies against podocyte proteins: M-type phospholipase A2 receptor (PLA2R1) or thrombospondin type-1 domain-containing 7A (THSD7A) in 70 and 3% of patients, respectively. Antibody titer is correlated with disease activity: rising during active disease and decreasing before remission. Therefore, decreasing PLA2R1-Antibodies titer has become an important goal of therapy. Rituximab a chimeric monoclonal antibody induces remission in 60–80% of primary MN patients. All monoclonal antibodies such as rituximab can elicit antidrug antibodies, which may interfere with therapeutic response. We aim to analyze the relevance of anti-rituximab antibodies on the outcome of MN after a first course of rituximab. Forty-four MN patients were included and treated with two 1 g infusions of rituximab at 2-weeks interval. Anti-rituximab antibodies, CD19 count, and clinical response were analyzed. Then, we (i) analyzed the association of anti-rituximab antibodies at month-6 with response to treatment: remission, relapse and the need for another rituximab course; (ii) confirmed if anti-rituximab antibodies could neutralize rituximab B-cells depletion; and (iii) tested whether anti-rituximab antibodies could cross-inhibit new humanized anti-CD20 therapies. Anti-rituximab antibodies were detected in 10 patients (23%). Seventeen patients received a second rituximab course after a median time of 12 months (7–12), following nine cases of resistance and eight relapses. Anti-rituximab antibodies were significantly associated with faster B-cell reconstitution at month-6 (75 [57–89] vs. 2 [0–41] cells/μl, p = 0.006), higher proteinuria 12 months after rituximab infusion (1.7 [0.7; 5.8] vs. 0.6 [0.2; 3.4], p = 0.03) and before treatment modification (3.5 [1.6; 7.1] vs. 1.7 [0.2; 1.7] p = 0.0004). Remission rate 6 months after rituximab was not different according to anti-rituximab status (p > 0.99) but the rate of relapse was significantly higher for patients with anti-rituximab antibodies (p < 0.001). These patients required more frequently a second course of rituximab infusions (7/10 vs. 10/34, p = 0.03). Anti-rituximab antibodies neutralized rituximab activity in 8/10 patients and cross-reacted with other humanized monoclonal antibodies in only two patients. Three patients with anti-rituximab antibodies were successfully treated with ofatumumab. Anti-rituximab antibodies could neutralize rituximab B cells cytotoxicity and impact clinical outcome of MN patients. Humanized anti-CD20 seems to be a satisfying therapeutic alternative for patients with anti-rituximab antibodies and resistant or relapsing MN.

Tetrandrine Suppresses Transient Receptor Potential Cation Channel Protein 6 Overexpression- Induced Podocyte Damage via Blockage of RhoA/ROCK1 Signaling.

ObjectivePodocyte damage is common in many renal diseases characterized by proteinuria. Transient receptor potential cation channel protein 6 (TRPC6) plays an important role in renal function through its regulation of intracellular Ca2+ influx and RhoA/ROCK pathways. Chinese herb Stephania tetrandra, with the main active component being tetrandrine, has been used for the treatment of various kidney diseases for several years and has shown a positive effect. This study aimed at investigating the effect and mechanism of tetrandrine in podocyte damage induced by high expression of TRPC6.MethodsImmortalized, differentiated murine podocytes, MPC5 were treated with valsartan (0–800 μM) and tetrandrine (0–40 μM) for 48 h. The maximum safe concentrations of valsartan and tetrandrine were selected using a cell viability assay. MPC5 podocytes stably expressing TRPC6 were constructed using a lentivirus packaging system, followed by treatment with valsartan, tetrandrine, and Y-27632 for 48 h and U73122 (10 μM) for 10 min. The RhoA/ROCK pathway and podocyte-specific proteins (nephrin and synaptopodin) levels were quantified. Podocyte apoptosis and intracellular Ca2+ concentration were measured.ResultsMaximum safe concentrations of 100 μM valsartan and 10 μM tetrandrine showed no observable toxicity in podocytes. MPC5 podocytes stably expressing TRPC6 had higher intracellular Ca2+ influx, apoptotic percentages, and expression of RhoA/ROCK proteins, but lower expression of nephrin and synaptopodin proteins. U73122 treatment for 10 min did not inhibit TRPC6, but suppressed RhoA/ROCK protein. Y-27632 decreased ROCK1 expression, but did not influence the expression of TRPC6 protein. Both 100 μM valsartan and 10 μM tetrandrine for 48 h significantly inhibited intracellular Ca2+ influx, apoptosis, and RhoA/ROCK pathway, and increased nephrin and synaptopodin proteins in podocytes stably expressing TRPC6.ConclusionElevated TRPC6 expression can lead to podocyte injury by inducing intracellular Ca2+ influx and apoptosis of podocytes, and this effect may be mediated by activation of the RhoA/ROCK1 pathway. Tetrandrine can alleviate podocyte injury induced by TRPC6 expression through inhibition of the RhoA/ROCK pathway, suggesting a protective role in podocyte damage.

Intrinsic disorder and amino acid specificity modulate binding of the WW2 domain in kidney and brain protein (KIBRA) to synaptopodin

The second WW domain (WW2) of the kidney and brain scaffolding protein, KIBRA, has an isoleucine (Ile-81) rather than a second conserved tryptophan and is primarily unstructured. However, it adopts the canonical triple-stranded antiparallel β-sheet structure of WW domains when bound to a two-PPXY motif peptide of the synaptic protein Dendrin. Here, using a series of biophysical experiments, we demonstrate that the WW2 domain remains largely disordered when bound to a 69-residue two-PPXY motif polypeptide of the synaptic and podocyte protein synaptopodin (SYNPO). Isothermal titration calorimetry and CD experiments revealed that the interactions of the disordered WW2 domain with SYNPO are significantly weaker than SYNPO's interactions with the well-folded WW1 domain and that an I81W substitution in the WW2 domain neither enhances binding affinity nor induces substantial WW2 domain folding. In the tandem polypeptide, the two WW domains synergized, enhancing the overall binding affinity with the I81W variant tandem polypeptide 2-fold compared with the WT polypeptide. Solution NMR results showed that SYNPO binding induces small but definite chemical shift perturbations in the WW2 domain, confirming the disordered state of the WW2 domain in this complex. These analyses also disclosed that SYNPO binds the tandem WW domain polypeptide in an antiparallel manner, that is, the WW1 domain binds the second PPXY motif of SYNPO. We propose a binding model consisting of a bipartite interaction mode in which the largely disordered WW2 forms a "fuzzy" complex with SYNPO. This binding mode may be important for specific cellular functions.

Role of phospholipase A2 receptor 1 antibody level at diagnosis for long-term renal outcome in membranous nephropathy.

BackgroundMembranous nephropathy (MN) is an autoimmune disease induced by circulating antibodies against the podocyte protein phospholipase A2 receptor 1 (PLA2R1-ab) in 80% of patients and represents the leading cause of nephrotic syndrome in adults. PLA2R1-ab levels correlate with disease activity and treatment response. However, their predictive role for long-term renal outcome is not clear.MethodsThe aim of this prospective observational multicenter study was to investigate the predictive role of PLA2R1-ab levels at the time of diagnosis for long-term outcome in a cohort of 243 patients with newly diagnosed biopsy-proven PLA2R1-associated MN. Statistical analyses included Cox proportional hazard models. The primary study endpoint was defined prior to data collection as doubling of serum creatinine or development of end-stage renal disease.ResultsDuring the median follow-up time of 48 months, 36 (15%) patients reached the study endpoint. Independent predictors for reaching the study endpoint were baseline PLA2R1-ab levels (HR = 1.36, 95%CI 1.11–1.66, p = 0.01), percentage of tubular atrophy and interstitial fibrosis (HR = 1.32, 95%CI 1.03–1.68, p = 0.03), PLA2R1-ab relapse during follow-up (HR = 3.22, 95%CI 1.36–7.60, p = 0.01), and relapse of proteinuria (HR = 2.60, 95%CI 1.17–5.79, p = 0.02). Fifty-four (22%) patients received no immunosuppressive treatment during the study, in 41 (76%) of them PLA2R1-ab spontaneously disappeared during follow-up, 29 (54%) patients had a complete remission of proteinuria, and 19 (35%) had a partial remission. Patients not treated with immunosuppression were more often females and had lower PLA2R1-ab levels, proteinuria, and serum creatinine at baseline compared to patients receiving immunosuppression. However, no conclusion on the efficacy of immunosuppressive therapies can be made, since this was not a randomized controlled study and treatment decisions were not made per-protocol.ConclusionsPLA2R1-ab levels are, in addition to pre-existing renal damage, predictive factors for long-term outcome and should therefore be considered when deciding the treatment of patients with MN.

PHOSPHATIDYLINOSITOL-3 KINASE (PI3-KINASE) HAS A PROTECTIVE ROLE IN ANGIOTENSIN II-INDUCED PODOCYTE INJURY

Objective: PI3-kinase has distinct roles in cellular processes spanning from metabolism to cell motility and survival. Angiotensin II (Ang II) promotes the development and progression of proteinuria and renal diseases and induces podocyte apoptosis. CD2-associated protein (CD2AP) in podocytes, anchoring slit diaphragm proteins to actin filaments of podocyte cytoskeleton, facilitates the nephrin-induced phosphatidylinositol-3 kinase (PI3-kinase)/AKT signaling, which protects podocytes from apoptosis. Autophagy and apoptosis constitute the two processes through which injured/aged cells or organelles are eliminated. Autophagy is a highly regulated catabolic process that is involved in the turnover of unwanted cellular materials, on the other hand, apoptosis removes damaged or unwanted cells. We investigated the role of PI3-kinase in angiotensin II-induced podocyte injury. Design and method: Mouse podocytes were incubated in media containing various concentrations of Ang II and at different incubation times. Cell survival/death-modifying reagents and Atg5 siRNA were applied. The changes of podocyte autophagy and apoptosis were observed by confocal imaging, western blotting, FACS and TUNEL assay according to the presence of Ang II. Results: Ang II-treated podocytes showed an increase in autophagosomes compared with control cells at early phase in a dose-dependent manner. This pro-autophagic effect of Ang II was inhibited by pretreatment with 3-methyladenine (3-MA), an inhibitor of PI3-kinase class III. Atg5 siRNA reduced LC3 puncta levels and increased the number of apoptotic podocytes over that observed with Ang II treatment at 12 hours. Thereafter, Ang II induced podocyte apoptosis at later stages, 12 and 24 hours in concentration- and time-dependent manners in FACS and TUNEL assays. PI3-kinase inhibitors, 3-MA and LY294002, and Atg5 siRNA further increased Ang II-induced podocyte apoptosis. Thereafter, Ang II induced podocyte apoptosis significantly in concentration- and time-dependent manners in FACS and TUNEL assays. Therefore, autophagy precedes apoptosis for cytoprotection in angiotensin II-induced podocyte injury. Conclusions: We suggest that PI3-kinase have a protective role in Ang II-induced podocyte injury by promoting autophagy and suppressing apoptosis.

MON-205 THE PROTECTIVE ROLE OF PHOSPHATIDYLINOSITOL-3 KINASE (PI3-KINASE) IN ANGIOTENSIN II-INDUCED PODOCYTE INJURY

Angiotensin II (Ang II) promotes the development and progression of proteinuria and renal diseases and induces podocyte apoptosis. CD2-associated protein (CD2AP) in podocytes, anchoring slit diaphragm proteins to actin filaments of podocyte cytoskeleton, facilitates the nephrin-induced phosphatidylinositol-3 kinase (PI3-kinase)/AKT signaling, which protects podocytes from apoptosis. Autophagy and apoptosis constitute the two processes through which injured/aged cells or organelles are eliminated.

Coro2b, a podocyte protein downregulated in human diabetic nephropathy, is involved in the development of protamine sulphate-induced foot process effacement

Podocytes have an important role in the pathogenesis of diabetic nephropathy (DN). Podocyte foot process effacement, mediated largely by the actin-based cytoskeleton of foot processes, is commonly detected in DN and is believed to be a key pathogenic event in the development of proteinuria. In this study, we identified coronin 2b (Coro2b), a member of known actin-regulating proteins, the coronins, as a highly podocyte-enriched molecule located at the cytoplasmic side of the apical plasma membrane. Studies in human renal biopsies show that glomerular Coro2b expression is significantly down-regulated in patients with DN. Studies in knockout mice indicate that Coro2b is not required for the development or maintenance of the glomerular filtration barrier. Moreover, inactivation of Coro2b specifically in podocytes does not affect the outcome of nephropathy in a streptozotocin-induced diabetes model. However, Coro2b seems to modulate the reorganization of foot processes under pathological conditions as Coro2b knockout podocytes are partially protected from protamine sulfate perfusion-induced foot process effacement. Taken together, our study suggests a role for Coro2b in the pathogenesis of glomerulopathies. Further studies regarding the involvement of Coro2b in podocyte health and diseases are warranted.

Markers of renal fibrosis: How do they correlate with podocyte damage in glomerular diseases?

BackgroundRenal fibrosis is the result of the interaction of cellular and molecular pathways, which is induced by sustained glomerular injury and involves the podocytes and multiple profibrotic factors. In this study, we investigated the correlation of the mRNA expression of podocyte proteins and profibrotic factors with renal fibrosis measured in renal biopsies of patients with primary and secondary glomerulopathies.MethodsEighty-four adult patients with primary or secondary glomerular diseases and 12 controls were included. Demographic and clinical data were collected. Seventy-two percent of the renal biopsies were done less than one year from clinical disease manifestation. The quantification of the podocyte-associated mRNAs of alpha-actinin-4, podocin, and podocalyxin, as well as of the profibrotic factors TGF-β1, CTGF, and VEGF-A were quantified by real-time polymerase chain reaction. The percent positive area of renal fibrosis was measured by immunohistochemistry staining, using anti-CTGF and anti-HHF35 antibodies and unpolarized Sirius Red. Correlations between the expression of tissue mRNAs and the positive area of fibrosis for the measured markers were made by Spearman’s rank correlation coefficient.ResultsIn relation to control biopsies, podocyte-specific proteins were downregulated in podocytopathies, in proliferative nephritis, in diabetic kidney disease (DRD), and in IgA nephropathy (IgAN). Messenger RNA of TGF-β1, CTGF, and VEGF-A was upregulated in patients with podocytopathies and in DRD but not in proliferative nephritis and IgAN. Tissue mRNA expression of TGF-β1, CTGF, and VEGF-A were strongly correlated with renal fibrosis, as measured by HHF35; however, the correlation, albeit significant, was moderate for Sirius Red and weak for CTGF. The percent positive area of renal fibrosis measured by Sirius Red was similar between podocytopathies and DRD and significantly higher in podocytopathies compared to IgAN or proliferative nephritis.ConclusionsIn patients with glomerular diseases, the mRNA of TGF-β1, CTGF, and VEGF-A correlated positively with the extent of renal fibrosis, and the positive area of fibrosis was larger in the podocytopathies and in DRD as measured by Sirius Red. The pathways connecting podocyte damage and activation of profibrotic factors to kidney tissue fibrosis need to be better investigated.

Disruption of the exocyst induces podocyte loss and dysfunction

Although the slit diaphragm proteins in podocytes are uniquely organized to maintain glomerular filtration assembly and function, little is known about the underlying mechanisms that participate in trafficking these proteins to the correct location for development and homeostasis. Identifying these mechanisms will likely provide novel targets for therapeutic intervention to preserve podocyte function following glomerular injury. Analysis of structural variation in cases of human nephrotic syndrome identified rare heterozygous deletions of EXOC4 in two patients. This suggested that disruption of the highly-conserved eight-protein exocyst trafficking complex could have a role in podocyte dysfunction. Indeed, mRNA profiling of injured podocytes identified significant exocyst down-regulation. To test the hypothesis that the exocyst is centrally involved in podocyte development/function, we generated homozygous podocyte-specific Exoc5 (a central exocyst component that interacts with Exoc4) knockout mice that showed massive proteinuria and died within 4 weeks of birth. Histological and ultrastructural analysis of these mice showed severe glomerular defects with increased fibrosis, proteinaceous casts, effaced podocytes, and loss of the slit diaphragm. Immunofluorescence analysis revealed that Neph1 and Nephrin, major slit diaphragm constituents, were mislocalized and/or lost. mRNA profiling of Exoc5 knockdown podocytes showed that vesicular trafficking was the most affected cellular event. Mapping of signaling pathways and Western blot analysis revealed significant up-regulation of the mitogen-activated protein kinase and transforming growth factor-β pathways in Exoc5 knockdown podocytes and in the glomeruli of podocyte-specific Exoc5 KO mice. Based on these data, we propose that exocyst-based mechanisms regulate Neph1 and Nephrin signaling and trafficking, and thus podocyte development and function.

Inhibitory Effects of Eucalyptol on Diabetes-associated Dysfunction of Actin Cytoskeleton and Focal Adhesion Formation in Kidney Podocytes (P06-011-19)

ObjectivesChronic hyperglycemia causes glomerular podocyte damage that can result in glomerular focal adhesion and cytoskeleton rearrangement. Eucalyptol (1,8-cineole) is a natural organic essential oil and a monoterpenoid with anti-inflammatory and antioxidant properties. MethodsImmotalized mouse podocytes were incubated in media containing 33 mM glucose for 4 days in the presence of 1–20 μM eucalyptol. Antibodies of F-actin, ezrin, Arp2/3, cortactin, paxillin, vinculin, talin, and FAK were used for Western blot analysis. Podocytes were stained with rhodamine-phalloidin to stain actin filaments. The db/db mice were orally administrated with 10 mg/kg eucalyptol. Kidney tissue extracts were prepared for Western blotting and immunohistochemically stained. ResultsGlucose suppressed the induction of the cytoskeletal proteins responsible for the maintenance of podocyte cytoskeleton structural integrity. However, eucalyptol prompted such reduction in diabetic podocytes. Eucalyptol enhanced rhodamine-phalloidin-red staining of podocyte F-actin diminished in glucose-exposed podocytes. In addition, the tissue levels of the cytoskeletal proteins were reduced in diabetic kidneys. Oral administration of eucalyptol to db/db mice augmented the kidney tissue levels of cytoskeletal proteins and boosted glomerular level of F-actin. On the other hand, the induction of focal adhesion proteins was dampened in glucose-loaded podocytes, while the activation of these proteins inductions were highly elevated. The presence of eucalyptol reversed the aforementioned effects of focal adhesion proteins in diabetic podocytes. Furthermore, eucalyptol enhanced the decreased levels of the focal adhesion proteins in diabetic kidneys. ConclusionsThese results demonstrated that eucalyptol ameliorated actin cytoskeleton integrity and focal adhesion formation in diabetic kidneys. Therefore, eucalyptol may be a potent renoprotective agent counteracting diabetes-associated podocyte detachment and disruption of focal adhesion proteins. Funding SourcesThis work was supported by the National Research Foundation of Korea (NRF) grants funded by the Korea government (MEST) (NRF-2017R1A6A3A04011473).

Addition of cyclic angiotensin-(1-7) to angiotensin-converting enzyme inhibitor therapy has a positive add-on effect in experimental diabetic nephropathy

The Renin-Angiotensin System (RAS) possesses a counter-regulatory axis composed of angiotensin converting enzyme (ACE)2, angiotensin-(1-7) [Ang-(1-7)] and the Mas receptor, which opposes many AT1-receptor-mediated effects of ligand angiotensin II. Ang-(1-7), as a ligand of the Mas receptor, has inhibitory effects on renal inflammation and fibrosis in experimental diabetes. However, Ang-(1-7) has a short half-life in plasma, which may render it unsuitable for use in clinics. Here, we investigated the effects of the lanthionine-stabilized Ang-(1-7), cyclic (c)Ang-(1-7), a lanthipeptide that is more peptidase-resistant than the linear peptide, in BTBR ob/ob mice with type 2 diabetic nephropathy. BTBR ob/ob mice received vehicle, cAng-(1-7), or the ACE inhibitor lisinopril. The treatment started at ten weeks of age, when the animals had already developed albuminuria, and ended at 19-20 weeks of age. cAng-(1-7) limited albuminuria progression, and limited podocyte dysfunction similarly to lisinopril. cAng-(1-7), unlike lisinopril, reduced glomerular fibrosis and inflammation, and counteracted glomerular capillary rarefaction. Furthermore, when cAng-(1-7) was combined with lisinopril, a superior antiproteinuric effect than with lisinopril alone was found, in association with better preservation of podocyte proteins and amelioration of capillary density. Thus, adding cAng-(1-7) to ACE-inhibitor therapy could benefit those diabetic patients who do not respond completely to ACE-inhibitor therapy.

Anemoside B4 Protects Rat Kidney from Adenine-Induced Injury by Attenuating Inflammation and Fibrosis and Enhancing Podocin and Nephrin Expression.

Anemoside B4 (B4) isolated from Radix Pulsatilla has anti-inflammatory activities in the colon and antitumor effects. However, its role in the prevention and treatment of kidney injury has not been reported. Here, we reported the effects of B4 on chronic kidney injury (CKI) and studied its related mechanism based on an adenine-induced kidney injury model in rats. The results showed that serum BUN (blood urea nitrogen), Crea (creatinine), and urinary proteins increased significantly after oral administration of adenine. Meanwhile, the adenine contents in both renal tissue and urine increased markedly compared with those of normal rats. Moreover, IL-1β, IL-6, TNFα, and NFκB expression was upregulated in the kidney. Simultaneously, the expression of NLRP3 (the nucleotide-binding and oligomerization domain–like receptor, leucine-rich repeat and pyrin domain–containing 3) in the inflammasome, which consists of Caspase 1, ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain), and IL-18, was significantly upregulated. B4 could significantly decrease BUN and Crea; reduce urinary proteins in rats; suppress the expression of IL-6, IL-1β, NFκB, NLRP3, Caspase 1, ASC, and IL-18; and increase urinary adenine contents and promote its excretion. In addition, B4 also upregulated the expression of podocin and nephrin, two major podocyte proteins, and reduced the fiber collagen in the renal interstitial, suggesting that B4 could protect the glomerular matrix from adenine injury in addition to its anti-inflammatory effects. The results of this study show new perspective of B4 as a potential drug against adenine-induced renal injury.

Tripterygium glycoside protects diabetic kidney disease mouse serum-induced podocyte injury by upregulating autophagy and downregulating β-arrestin-1.

Diabetic kidney disease (DKD), one of the most common causes of end-stage renal disease(ESRD), remains prevalent in many populations. Podocyte loss and apoptosis play a crucial role in the progression of DKD. Tripterygium glycoside (TG), a widely used Chinese herb, exerted comprehensive protective effects on preventing DKD progression. This study was performed to assess the podocyte protective effect of tripterygium glycoside on DKD by the potential role of activation of autophagy and downregulating β-arrestin-1. Tripterygium glycoside and small interfering RNA (siRNA) of β-arrestin-1 were added to 10% db/db mice high-glucose serum induced podocytes in vitro. Autophagic activity was evaluated by transmission electronic microscopy, immunofluorescence staining and western blot analysis. Apoptotic activity was evaluated by Annexin V-FITC/PI flow cytometric analysis. The levels of nephrin and podocin, a marker protein of podocytes, were examined using western blot analysis. Significantly ameliorated podocyte apoptosis, increased nephrin and podocin levels and inhibited expression of β-arrestin-1 were observed after pretreatment of tripterygium glycoside in DKD mouse serum treated podocytes. Significantly higher levels of autophagic activity were also observed. Silencing β-arrestin-1 upregulated autophagic activity and ameliorated podocyte apoptosis. Silencing β-arrestin-1 in combination with tripterygium glycoside enhanced the levels of LC3-II and LC3-II/LC3-I ratios and reduced the expression of p62. Finally, we observed a notable reduction in podocyte apoptotic rate in DKD serum + siRNA-β-arrestin-1 + TG group compared to DKD serum + siRNA-β-arrestin-1 group, and upregulated protein levels of nephrin and podocin compared to treatment with siRNA-β-arrestin-1 only. This study demonstrated that tripterygium glycoside provided protection against podocyte injury induced by high-glucose serum, and that this effect was mediated by the concomitant activation of autophagy and downregulation of β-arrestin-1.