Role of the SLC22A17/lipocalin-2 receptor in renal endocytosis of proteins/metalloproteins: a focus on iron- and cadmium-binding proteins.

The transmembrane protein SLC22A17 [or the neutrophil gelatinase-associated lipocalin/lipocalin-2 (LCN2)/24p3 receptor] is an atypical member of the SLC22 family of organic anion and cation transporters: it does not carry typical substrates of SLC22 transporters but mediates receptor-mediated endocytosis (RME) of LCN2. One important task of the kidney is the prevention of urinary loss of proteins filtered by the glomerulus by bulk reabsorption of multiple ligands via megalin:cubilin:amnionless-mediated endocytosis in the proximal tubule (PT). Accordingly, overflow, glomerular, or PT damage, as in Fanconi syndrome, results in proteinuria. Strikingly, up to 20% of filtered proteins escape the PT under physiological conditions and are reabsorbed by the distal nephron. The renal distal tubule and collecting duct express SLC22A17, which mediates RME of filtered proteins that evade the PT but with limited capacity to prevent proteinuria under pathological conditions. The kidney also prevents excretion of filtered essential and nonessential transition metals, such as iron or cadmium, respectively, that are largely bound to proteins with high affinity, e.g., LCN2, transferrin, or metallothionein, or low affinity, e.g., microglobulins or albumin. Hence, increased uptake of transition metals may cause nephrotoxicity. Here, we assess the literature on SLC22A17 structure, topology, tissue distribution, regulation, and assumed functions, emphasizing renal SLC22A17, which has relevance for physiology, pathology, and nephrotoxicity due to the accumulation of proteins complexed with transition metals, e.g., cadmium or iron. Other putative renal functions of SLC22A17, such as its contribution to osmotic stress adaptation, protection against urinary tract infection, or renal carcinogenesis, are discussed.

Cytoprotective remedies for ameliorating nephrotoxicity induced by renal oxidative stress.

AIMS: Nephrotoxicity is the hallmark of anti-neoplastic drug metabolism that causes oxidative stress. External chemical agents and prescription drugs release copious amounts of free radicals originating from molecular oxidation and unless sustainably scavenged, they stimulate membrane lipid peroxidation and disruption of the host antioxidant mechanisms. This review aims to provide a comprehensive collection of potential cytoprotective remedies in surmounting the most difficult aspect of cancer therapy as well as preventing renal oxidative stress by other means. MATERIALS AND METHODS: Over 400 published research and review articles spanning several decades were scrutinised to obtain the relevant data which is presented in 3 categories; sources, mechanisms, and mitigation of renal oxidative stress. KEY-FINDINGS: Drug and chemical-induced nephrotoxicity commonly manifests as chronic or acute kidney disease, nephritis, nephrotic syndrome, and nephrosis. Renal replacement therapy requirements and mortalities from end-stage renal disease are set to rapidly increase in the next decade for which 43 different cytoprotective compounds which have the capability to suppress experimental nephrotoxicity are described. SIGNIFICANCE: The renal system performs essential homeostatic functions that play a significant role in eliminating toxicants, and its accumulation and recurrence in nephric tissues results in tubular degeneration and subsequent renal impairment. Global statistics of the latest chronic kidney disease prevalence is 13.4 % while the end-stage kidney disease requiring renal replacement therapy is 4-7 million per annum. The remedial compounds discussed herein had proven efficacy against nephrotoxicity manifested consequent to impaired antioxidant mechanisms in preclinical models produced by renal oxidative stress activators.

Mutational signatures in GATA3 transcription factor and its DNA binding domain that stimulate breast cancer and HDR syndrome.

Transcription factors (TFs) play important roles in many biochemical processes. Many human genetic disorders have been associated with mutations in the genes encoding these transcription factors, and so those mutations became targets for medications and drug design. In parallel, since many transcription factors act either as tumor suppressors or oncogenes, their mutations are mostly associated with cancer. In this perspective, we studied the GATA3 transcription factor when bound to DNA in a crystal structure and assessed the effect of different mutations encountered in patients with different diseases and phenotypes. We generated all missense mutants of GATA3 protein and DNA within the adjacent and the opposite GATA3:DNA complex models. We mutated every amino acid and studied the new binding of the complex after each mutation. Similarly, we did for every DNA base. We applied Poisson-Boltzmann electrostatic calculations feeding into free energy calculations. After analyzing our data, we identified amino acids and DNA bases keys for binding. Furthermore, we validated those findings against experimental genetic data. Our results are the first to propose in silico modeling for GATA:DNA bound complexes that could be used to score effects of missense mutations in other classes of transcription factors involved in common and genetic diseases.

The structural and functional workings of KEOPS.

KEOPS (Kinase, Endopeptidase and Other Proteins of Small size) is a five-subunit protein complex that is highly conserved in eukaryotes and archaea and is essential for the fitness of cells and for animal development. In humans, mutations in KEOPS genes underlie Galloway-Mowat syndrome, which manifests in severe microcephaly and renal dysfunction that lead to childhood death. The Kae1 subunit of KEOPS catalyzes the universal and essential tRNA modification N6-threonylcarbamoyl adenosine (t6A), while the auxiliary subunits Cgi121, the kinase/ATPase Bud32, Pcc1 and Gon7 play a supporting role. Kae1 orthologs are also present in bacteria and mitochondria but function in distinct complexes with proteins that are not related in structure or function to the auxiliary subunits of KEOPS. Over the past 15 years since its discovery, extensive study in the KEOPS field has provided many answers towards understanding the roles that KEOPS plays in cells and in human disease and how KEOPS carries out these functions. In this review, we provide an overview into recent advances in the study of KEOPS and illuminate exciting future directions.

Osmotic Nephrosis and Acute Kidney Injury Associated With SGLT2 Inhibitor Use: A Case Report.

We report a case of a patient who developed dialysis-requiring acute kidney injury (AKI) after the use of canagliflozin. A 66-year-old man with type 2 diabetes who was recovering from left knee septic arthritis at a rehabilitation facility was admitted with oliguric AKI 5 days after starting treatment with canagliflozin, an inhibitor of sodium/glucose cotransporter 2 (SGLT2). The patient presented with hematuria, non-nephrotic-range proteinuria, and serum creatinine level of 6.8 (baseline, 1.1-1.3) mg/dL. There was no recent use of radiocontrast agents or exposure to other nephrotoxins. The patient subsequently required hemodialysis. Due to recent antibiotic use (ampicillin-sulbactam), acute interstitial nephritis was considered in the differential diagnosis. Kidney biopsy was performed, which showed the presence of osmotic nephropathy. The patient's kidney function returned to baseline after 2 weeks of hemodialysis. This case provides evidence of an association of osmotic nephropathy with the use of canagliflozin and discusses potential mechanisms. We recommend kidney biopsy for cases of severe AKI associated with SGLT2 inhibitors to better understand the relationship of this complication with the use of this class of medications.

Estrogen-related receptor-α mediates puromycin aminonucleoside-induced mesangial cell apoptosis and inflammatory injury.

Glomerular diseases are the leading cause of chronic kidney disease, and mesangial cells (MCs) have been demonstrated to be involved in the pathogenesis. Puromycin aminonucleoside (PAN) is a nephrotoxic drug that induces glomerular injury with elusive mechanisms. The present study was undertaken to investigate the role of PAN in MC apoptosis, as well as the underlying mechanism. Here we found that PAN induced MC apoptosis accompanied by declined cell viability and enhanced inflammatory response. The apoptosis was further evidenced by increments of apoptosis regulator BAX (BAX) and caspase-3 expression. In line with the apoptotic response in MCs following PAN treatment, we also found a remarkable induction of estrogen-related receptor-α (ERRα), an orphan nuclear receptor, at both mRNA and protein levels. Interestingly, ERRα silencing by an siRNA approach resulted in an attenuation of the apoptosis and inflammatory response caused by PAN. More importantly, overexpression of ERRα in MCs significantly triggered MC apoptosis in line with increased BAX and caspase-3 expression. In PAN-treated MCs, ERRα overexpression further aggravated PAN-induced apoptosis. In agreement with the in vitro study, we also observed increased ERRα expression in line with enhanced apoptotic response in renal cortex from PAN-treated rats. These data suggest a detrimental effect of ERRα on PAN-induced MC apoptosis and inflammatory response, which could help us to better understand the pathogenic mechanism of MC injury in PAN nephropathy.

NIX-mediated mitophagy protects against proteinuria-induced tubular cell apoptosis and renal injury.

Proteinuria, the most common symptom of renal injury, is an independent factor for renal tubular injury. However, the underlying mechanism remains to be fully elucidated. Mitochondrion is an important target for proteinuria-induced renal tubular cell injury. Insufficient mitophagy exacerbates cell injury by initiating mitochondrial dysfunction-related cell apoptosis. In the experiment, the role of NIP3-like protein X (NIX)-mediated mitophagy was investigated in proteinuria-induced renal injury. In this study, we demonstrated that NIX expression was reduced in renal tubules and correlated with the decline of estimated glomerular filtration rate and increase of the proteinuria in patients. In proteinuric mice, NIX-mediated mitophagy was significantly suppressed. Meanwhile, the proteinuric mice exhibited renal dysfunction, increased mitochondrial fragmentation, and tubular cell apoptosis. Overexpression of NIX attenuated those disruptions in proteinuric mice. In cultured renal tubular epithelial cells, albumin induced a decrease in NIX-mediated mitophagy and an increase in cell apoptosis. Overexpression of NIX attenuated albumin-induced cell apoptosis, whereas NIX siRNA aggravated these perturbations. These results indicate that proteinuria suppresses NIX-mediated mitophagy in the renal tubular epithelial cell, which triggers the cell undergoing mitochondria-dependent cell apoptosis. Collectively, our finding suggests that restoration of NIX-mediated mitophagy might be a novel therapeutic target for alleviating proteinuria-induced kidney injury.

Re-expression of Sall1 in podocytes protects against adriamycin-induced nephrosis.

The highly conserved spalt (sal) gene family members encode proteins characterized by multiple double zinc finger motifs of the C2H2 type. Humans and mice each have four known Sal-like genes (SALL1-4 in humans and Sall1-4 in mice). Sall1 is known to have a crucial role in kidney development. To explore the significance of Sall1 in differentiated podocytes, we investigated podocyte-specific Sall1-deficient mice (Sall1 KOp°d°/p°d°) using a podocin-Cre/loxP system and siRNA Sall1 knockdown (Sall1 KD) podocytes. Under physiological conditions, Sall1 KOp°d°/p°d° mice exhibited no proteinuria during their lifetime, but foot-process effacement was detected in some of the podocytes. To elucidate the role of Sall1 in injured podocytes, we used an adriamycin (ADR)-induced model of nephrosis and glomerulosclerosis. Surprisingly, the expression of Sall1 was elevated in control mice on day 14 after ADR injection. On day 28 after ADR injection, Sall1 KOp°d°/p°d° mice exhibited significantly higher levels of proteinuria and higher numbers of sclerotic glomeruli. Differentiated Sall1 KD podocytes showed a loss of synaptopodin, suppressed stress fiber formation, and, ultimately, impaired directed cell migration. In addition, the loss of Sall1 increased the number of apoptotic podocytes following ADR treatment. These results indicated that Sall1 has a protective role in podocytes; thus, we investigated the endoplasmic reticulum stress marker GRP78. GRP78 expression was higher in ADR-treated Sall1 KOp°d°/p°d° mice than in control mice. Sall1 appeared to influence the expression of GRP78 in injured podocytes. These results suggest that Sall1 is associated with actin reorganization, endoplasmic reticulum stress, and apoptosis in injured podocytes. These protective aspects of Sall1 re-expression in injured podocytes may have the potential to reduce apoptosis and possibly glomerulosclerosis.

Bone marrow-derived mesenchymal stem cells ameliorate nephrosis through repair of impaired podocytes.

PURPOSE: The purpose of this study was to investigate the effects of bone marrow-derived mesenchymal stem cells (BMSC) on podocytes of puromycin amino nuclear glucoside (PAN) -induced nephrosis in mice. METHODS: Mice were randomly divided into Control, PAN and BMSC groups. Mice were injected with PAN (0.5 mg/g weight) via the tail vein. The 24-h urinary protein was obtained after modelling, and urinary protein excretion was determined. The blood and kidney specimens were isolated after the tenth day of modelling. Blood samples were collected for measuring serum creatinine (SCr) and blood urea nitrogen (BUN). A sample of kidney was taken for observing pathological changes through hematoxylin-eosin staining and electron microscopy, and the rest of the kidney was used for detecting the protein and mRNA expression of nephrin, CD2AP, synaptopodin, TRPC6 by real-time quantitative PCR, Western-blot and immunohistochemistry. RESULTS: After PAN injection, podocyte foot process fusion was detected by electron microscopy, and the 24 h urinary protein excretion increased compared with control mice on days 3, 7 and 10 post-PAN injection (P.

Syndecan-4 ectodomain evokes mobilization of podocyte TRPC6 channels and their associated pathways: An essential role for integrin signaling.

PodocyteTRPC6 channels have been implicated in glomerular diseases. Syndecan-4 (Sdc4) is a membrane proteoglycan that can be cleaved to release a soluble ectodomain capable of paracrine and autocrine signaling. We have confirmed that overexpression of Sdc4 core protein increases surface abundance of TRPC6 channels in cultured podocytes, whereas Sdc4 knockdown has the opposite effect. Exposure to soluble Sdc4 ectodomain also increased the surface abundance of TRPC6, and increased cationic currents evoked by a diacylglycerol analog in podocytes. Sdc4 ectodomain increased generation of reactive oxygen species (ROS), reduced activation of RhoA, increased activation of Rac1, increased nuclear abundance of NFATc1, and increased total ß3-integrin. The effects of Sdc4 ectodomain on cell-surface TRPC6 were blocked by the ROS quencher TEMPOL, and by the Rac1 inhibitor NSC-23766, but were not blocked by inhibition of calcineurin-NFATc1 signaling. The Sdc4 core protein co-immunoprecipitates with ß3-integrin in cultured podocytes. Moreover, effects of Sdc4 ectodomain on TRPC6, ROS generation, Rac1 and RhoA modulation, and NFATc1 activation were blocked by cilengitide, a selective inhibitor of outside-in signaling through αv-containing integrins. Exposure to TNF, or serum from three patients with recurrent FSGS in relapse, increased shedding of podocyte Sdc4 ectodomains into the surrounding medium. This was also observed after treating podocytes with the metalloproteinase ADAM17 or after overexpression of the Sdc4 core protein. Increased concentrations of Sdc4 ectodomain were detected in urine of rats during acute puromycin aminonucleoside nephrosis. Locally generated Sdc4 may play a role in regulating TRPC6 channels, and may contribute to glomerular pathology.

Cyclic AMP prevents decrease of phosphorylated ezrin/radixin/moesin and chloride intracellular channel 5 expressions in injured podocytes.

BACKGROUND: Our previous in vitro studies suggested that cyclin AMP (cAMP) signaling protects against podocyte injury. However, the molecular mechanisms remain unknown. The aim of the present study was to explore the role of forskolin, an agonist for adenylate cyclase, on ezrin/radixin/moesin (ERM) phosphorylation and chloride intracellular channel 5 (CLIC5) expressions in injured podocytes. METHODS: ADR nephrosis model were induced by adriamycin (ADR) injection in BalB/C mice. Parts of ADR nephrosis mice were pretreated with forskolin. Albuminuria was estimated by urine Coomassie blue stain. Nephrin, synaptopodin, CLIC5, phosphorylated ERM and podocalyxin were measured by confocal microscopy. CLIC5 and phosphorylated ERM also were studied using western blotting. RhoA and Rac1 were estimated by G-Lisa kit. RESULTS: We found that forskolin partially alleviated albuminuria and width of foot processes. Nephrin, synaptopodin, phosphorylated-ERM (p-ERM) and CLIC5 expression were decreased in ADR mice, which were improved by forskolin pretreatment. In vitro studies, pretreatment of podocytes with pCPT-cAMP(PKA-selective cAMP analogue)prevented puromycin aminonucleoside (PAN)-induced CLIC5 downregulation. 8-pCPT-2'-O-Me-cAMP (2Me-cAMP, an Epac-selective cAMP analogue) blocked PAN-induced p-ERM downregulation. PAN inhibited RhoA activation in podocytes, which could be prevented by pCPT-cAMP pretreatment. Y-27632, a Rho inhibitor, decreased CLIC5 expression in podocytes. CONCLUSION: Activation cAMP signaling might attenuate albuminuria in ADR-induced nephrosis mice. Different downstream signaling pathway might mediate cAMP protection on CLIC5 and p-ERM expression, respectively.

Podocin is translocated to cytoplasm in puromycin aminonucleoside nephrosis rats and in poor-prognosis patients with IgA nephropathy.

Podocytes serve as the final barrier to urinary protein loss through a highly specialized structure called a slit membrane and maintain foot process and glomerular basement membranes. Podocyte injury results in progressive glomerular damage and accelerates sclerotic changes, although the exact mechanism of podocyte injury is still obscure. We focus on the staining gap (podocin gap) defined as the staining difference between podocin and synaptopodin, which are normally located in the foot process. In puromycin aminonucleoside nephrosis rats, the podocin gap is significantly increased (p < 0.05) and podocin is translocated to the cytoplasm on days 7 and 14 but not on day 28. Surprisingly, the gap is also significantly increased (p < 0.05) in human kidney biopsy specimens of poor-prognosis IgA nephropathy patients. This suggests that the podocin gap could be a useful marker for classifying the prognosis of IgA nephropathy and indicating the translocation of podocin to the cytoplasm. Next, we find more evidence of podocin trafficking in podocytes where podocin merges with Rab5 in puromycin aminonucleoside nephrosis rats at day 14. In immunoelectron microscopy, the podocin positive area was significantly translocated from the foot process areas to the cytoplasm (p< 0.05) on days 7 and 14 in puromycin aminonucleoside nephrosis rats. Interestingly, podocin is also translocated to the cytoplasm in poor-prognosis human IgA nephropathy. In this paper, we demonstrate that the translocation of podocin by endocytosis could be a key traffic event of critical podocyte injury and that the podocin gap could indicate the prognosis of IgA nephropathy.

CXCL10 induces the recruitment of monocyte-derived macrophages into kidney, which aggravate puromycin aminonucleoside nephrosis.

The mechanism responsible for trafficking of monocyte-derived macrophages into kidney in the puromycin aminonucleoside model of nephrotic syndrome in rats (PAN-NS), and the significance of this infiltration, remain largely unknown. CXCL10, a chemokine secreted in many T helper type 1 (Th1) inflammatory diseases, exhibits important roles in trafficking of monocytes and activated T cells. We hypothesized that induction of circulating interferon (IFN)-γ and glomerular tumour necrosis factor (TNF)-α during PAN-NS would stimulate the release of CXCL10 by podocytes, leading to infiltration of activated immune cells and greater glomerular injury. We found that serum IFN-γ, glomerular Cxcl10 mRNA and intra- and peri-glomerular macrophage infiltration were induced strongly during the late acute phase of PAN-NS in Wistar rats, but not in nude (Foxn1(rnu/rnu) ) rats lacking functional effector T lymphocytes. Wistar rats also developed significantly greater proteinuria than nude rats, which could be abolished by macrophage depletion. Stimulation of cultured podocytes with both IFN-γ and TNF-α markedly induced the expression of Cxcl10 mRNA and CXCL10 secretion. Together, these data support our hypothesis that increased circulating IFN-γ and glomerular TNF-α induce synergistically the production and secretion of CXCL10 by podocytes, attracting activated macrophages into kidney tissue. The study also suggests that IFN-γ, secreted from Th1 lymphocytes, may prime proinflammatory macrophages that consequently aggravate renal injury.

GIV/girdin links vascular endothelial growth factor signaling to Akt survival signaling in podocytes independent of nephrin.

Podocytes are critically involved in the maintenance of the glomerular filtration barrier and are key targets of injury in many glomerular diseases. Chronic injury leads to progressive loss of podocytes, glomerulosclerosis, and renal failure. Thus, it is essential to maintain podocyte survival and avoid apoptosis after acute glomerular injury. In normal glomeruli, podocyte survival is mediated via nephrin-dependent Akt signaling. In several glomerular diseases, nephrin expression decreases and podocyte survival correlates with increased vascular endothelial growth factor (VEGF) signaling. How VEGF signaling contributes to podocyte survival and prevents apoptosis remains unknown. We show here that Gα-interacting, vesicle-associated protein (GIV)/girdin mediates VEGF receptor 2 (VEGFR2) signaling and compensates for nephrin loss. In puromycin aminonucleoside nephrosis (PAN), GIV expression increased, GIV was phosphorylated by VEGFR2, and p-GIV bound and activated Gαi3 and enhanced downstream Akt2, mammalian target of rapamycin complex 1 (mTORC1), and mammalian target of rapamycin complex-2 (mTORC2) signaling. In GIV-depleted podocytes, VEGF-induced Akt activation was abolished, apoptosis was triggered, and cell migration was impaired. These effects were reversed by introducing GIV but not a GIV mutant that cannot activate Gαi3. Our data indicate that after PAN injury, VEGF promotes podocyte survival by triggering assembly of an activated VEGFR2/GIV/Gαi3 signaling complex and enhancing downstream PI3K/Akt survival signaling. Because of its important role in promoting podocyte survival, GIV may represent a novel target for therapeutic intervention in the nephrotic syndrome and other proteinuric diseases.

A novel missense mutation of Wilms' Tumor 1 causes autosomal dominant FSGS.

FSGS is a clinical disorder characterized by focal scarring of the glomerular capillary tuft, podocyte injury, and nephrotic syndrome. Although idiopathic forms of FSGS predominate, recent insights into the molecular and genetic causes of FSGS have enhanced our understanding of disease pathogenesis. Here, we report a novel missense mutation of the transcriptional regulator Wilms' Tumor 1 (WT1) as the cause of nonsyndromic, autosomal dominant FSGS in two Northern European kindreds from the United States. We performed sequential genome-wide linkage analysis and whole-exome sequencing to evaluate participants from family DUK6524. Subsequently, whole-exome sequencing and direct sequencing were performed on proband DNA from family DUK6975. We identified multiple suggestive loci on chromosomes 6, 11, and 13 in family DUK6524 and identified a segregating missense mutation (R458Q) in WT1 isoform D as the cause of FSGS in this family. The identical mutation was found in family DUK6975. The R458Q mutation was not found in 1600 control chromosomes and was predicted as damaging by in silico simulation. We depleted wt1a in zebrafish embryos and observed glomerular injury and filtration defects, both of which were rescued with wild-type but not mutant human WT1D mRNA. Finally, we explored the subcellular mechanism of the mutation in vitro. WT1(R458Q) overexpression significantly downregulated nephrin and synaptopodin expression, promoted apoptosis in HEK293 cells and impaired focal contact formation in podocytes. Taken together, these data suggest that the WT1(R458Q) mutation alters the regulation of podocyte homeostasis and causes nonsyndromic FSGS.

Optineurin associates with the podocyte Golgi complex to maintain its structure.

Optineurin, a cytosolic protein associated with the actin cytoskeleton, microtubules, and the Golgi complex, appears to have an important function in neurons, as mutations in its gene are causative for neurodegenerative diseases such as primary open-angle glaucoma and amyotrophic lateral sclerosis. Here, we report that optineurin is localized in podocytes of the kidney and induced upon injury following treatment with puromycin aminonucleoside. In cultured human podocytes, optineurin localizes to the Golgi complex. Optineurin depletion by RNA interference causes Golgi fragmentation. Moreover, if the Golgi complex is fragmented following microtubule destabilization induced by nocodazole treatment, optineurin dissociates from Golgi vesicles. Furthermore, optineurin colocalizes with vinculin-labeled focal contacts of cultured podocytes and with lysosome-like structures. Optineurin is essential for the survival of cultured podocytes, as optineurin depletion causes cell death. Thus, optineurin appears to play an important role in the maintenance of the podocyte Golgi complex and in the trafficking of vesicles to focal contacts and lysosomes.

cAMP signaling prevents podocyte apoptosis via activation of protein kinase A and mitochondrial fusion.

Our previous in vitro studies suggested that cyclic AMP (cAMP) signaling prevents adriamycin (ADR) and puromycin aminonucleoside (PAN)-induced apoptosis in podocytes. As cAMP is an important second messenger and plays a key role in cell proliferation, differentiation and cytoskeleton formation via protein kinase A (PKA) or exchange protein directly activated by cAMP (Epac) pathways, we sought to determine the role of PKA or Epac signaling in cAMP-mediated protection of podocytes. In the ADR nephrosis model, we found that forskolin, a selective activator of adenylate cyclase, attenuated albuminuria and improved the expression of podocyte marker WT-1. When podocytes were treated with pCPT-cAMP (a selective cAMP/PKA activator), PKA activation was increased in a time-dependent manner and prevented PAN-induced podocyte loss and caspase 3 activation, as well as a reduction in mitochondrial membrane potential. We found that PAN and ADR resulted in a decrease in Mfn1 expression and mitochondrial fission in podocytes. pCPT-cAMP restored Mfn1 expression in puromycin or ADR-treated podocytes and induced Drp1 phosphorylation, as well as mitochondrial fusion. Treating podocytes with arachidonic acid resulted in mitochondrial fission, podocyte loss and cleaved caspase 3 production. Arachidonic acid abolished the protective effects of pCPT-cAMP on PAN-treated podocytes. Mdivi, a mitochondrial division inhibitor, prevented PAN-induced cleaved caspase 3 production in podocytes. We conclude that activation of cAMP alleviated murine podocyte caused by ADR. PKA signaling resulted in mitochondrial fusion in podocytes, which at least partially mediated the effects of cAMP.

Ahsa1 and Hsp90 activity confers more severe craniofacial phenotypes in a zebrafish model of hypoparathyroidism, sensorineural deafness and renal dysplasia (HDR).

The severity of most human birth defects is highly variable. Our ability to diagnose, treat and prevent defects relies on our understanding of this variability. Mutation of the transcription factor GATA3 in humans causes the highly variable hypoparathyroidism, sensorineural deafness and renal dysplasia (HDR) syndrome. Although named for a triad of defects, individuals with HDR can also exhibit craniofacial defects. Through a forward genetic screen for craniofacial mutants, we isolated a zebrafish mutant in which the first cysteine of the second zinc finger of Gata3 is mutated. Because mutation of the homologous cysteine causes HDR in humans, these zebrafish mutants could be a quick and effective animal model for understanding the role of gata3 in the HDR disease spectrum. We demonstrate that, unexpectedly, the chaperone proteins Ahsa1 and Hsp90 promote severe craniofacial phenotypes in our zebrafish model of HDR syndrome. The strengths of the zebrafish system, including rapid development, genetic tractability and live imaging, make this an important model for variability.

A novel nuclear factor κB inhibitor, dehydroxymethylepoxyquinomicin, ameliorates puromycin aminonucleoside-induced nephrosis in mice.

BACKGROUND/AIMS: Minimal-change nephrotic syndrome (MCNS) is a kidney disease defined by selective proteinuria and hypoalbuminemia occurring in the absence of cellular glomerular infiltrates or immunoglobulin deposits. Recent observations suggest that nuclear factor κB (NF-κB) of podocyte is strongly associated with the development of proteinuria in MCNS. Dehydroxymethylepoxyquinomicin (DHMEQ) is a novel NF-κB inhibitor that potently inhibits DNA-binding activity of NF-κB, resulting in several therapeutic effects in various pathological conditions. We conducted this study to ask whether DHMEQ may ameliorate the nephrosis in mice induced by puromycin aminonucleoside (PAN), which is considered to be an animal model for MCNS. METHODS/RESULTS: Pretreatment with DHMEQ alleviated the proteinuria and reversed the serum abnormalities in mice nephrosis induced by 450 mg/kg of PAN. Increased serum interleukin-6 level in PAN-induced nephrosis was also completely suppressed by DHMEQ. Electron microscopic analyses of glo-meruli indicated that DHMEQ can inhibit the podocyte foot process effacement via blocking the translocation of podocyte NF-κB from cytoplasm to nucleus. CONCLUSIONS: These results suggest that DHMEQ can be a potential therapeutic agent for MCNS.

Nephrin phosphorylation regulates podocyte adhesion through the PINCH-1-ILK-α-parvin complex.

Nephrin, a structural molecule, is also a signaling molecule after phosphorylation. Inhibition of nephrin phosphorylation is correlated with podocyte injury. The PINCH-1-ILK-α-parvin (PIP) complex plays a crucial role in cell adhesion and cytoskeleton formation. We hypothesized that nephrin phosphorylation influenced cytoskeleton and cell adhesion in podocytes by regulating the PIP complex. The nephrin phosphorylation, PIP complex formation, and F-actin in Wistar rats intraperitoneally injected with puromycin aminonucleoside were gradually decreased but increased with time, coinciding with the recovery from glomerular/podocyte injury and proteinuria. In cultured podocytes, PIP complex knockdown resulted in cytoskeleton reorganization and decreased cell adhesion and spreading. Nephrin and its phosphorylation were unaffected after PIP complex knockdown. Furthermore, inhibition of nephrin phosphorylation suppressed PIP complex expression, disorganized podocyte cytoskeleton, and decreased cell adhesion and spreading. These findings indicate that alterations in nephrin phosphorylation disorganize podocyte cytoskeleton and decrease cell adhesion through a PIP complex-dependent mechanism.