Vps34 deficiency reveals the importance of endocytosis for podocyte homeostasis.

The molecular mechanisms that maintain podocytes and consequently, the integrity of the glomerular filtration barrier are incompletely understood. Here, we show that the class III phosphoinositide 3-kinase vacuolar protein sorting 34 (Vps34) plays a central role in modulating endocytic pathways, maintaining podocyte homeostasis. In mice, podocyte-specific conditional knockout of Vps34 led to early proteinuria, glomerular scarring, and death within 3-9 weeks of age. Vps34-deficient podocytes exhibited substantial vacuolization and foot process effacement. Although the formation of autophagosomes and autophagic flux were impaired, comparisons between podocyte-specific Vps34-deficient mice, autophagy-deficient mice, and doubly deficient mice suggested that defective autophagy was not primarily responsible for the severe phenotype caused by the loss of Vps34. In fact, Rab5-positive endosomal compartments, endocytosis, and fluid-phase uptake were severely disrupted in Vps34-deficient podocytes. Vps34 deficiency in nephrocytes, the podocyte-like cells of Drosophila melanogaster, resulted in a block between Rab5- and Rab7-positive endosomal compartments. In summary, these data identify Vps34 as a major regulator of endolysosomal pathways in podocytes and underline the fundamental roles of endocytosis and fluid-phase uptake for the maintenance of the glomerular filtration barrier.

Molecular fingerprinting of the podocyte reveals novel gene and protein regulatory networks.

A thorough characterization of the transcriptome and proteome of endogenous podocytes has been hampered by low cell yields during isolation. Here we describe a double fluorescent reporter mouse model combined with an optimized bead perfusion protocol and efficient single cell dissociation to yield more than 500,000 podocytes per mouse allowing for global, unbiased downstream applications. Combining mRNA and miRNA transcriptional profiling with quantitative proteomic analyses revealed programs of highly specific gene regulation tightly controlling cytoskeleton, cell differentiation, endosomal transport, and peroxisome function in podocytes. Strikingly, the analyses further predict that these podocyte-specific gene regulatory networks are accompanied by alternative splicing of respective genes. Thus, our 'omics' approach will facilitate the discovery and integration of novel gene, protein, and organelle regulatory networks that deepen our systematic understanding of podocyte biology.

Twist: a new link from hypoxia to fibrosis.

Sun et al. demonstrate that hypoxia causes epithelial-to-mesenchymal transition by activating Twist, a transcription factor known to mediate both acquisition of a mesenchymal phenotype and resistance to apoptosis in cancer cells. This study provides clues as to how hypoxia and transforming growth factor-beta can collaborate to drive renal fibrogenesis.

The class III phosphatidylinositol 3-kinase PIK3C3/VPS34 regulates endocytosis and autophagosome-autolysosome formation in podocytes.

Phosphatidylinositol phosphates are key regulators of vesicle identity, formation and trafficking. In mammalian cells, the evolutionarily conserved class III PtdIns 3-kinase PIK3C3/VPS34 is part of a large multiprotein complex that catalyzes the localized phosphorylation of phosphatidylinositol to phosphatidylinositol-3-phosphate (PtdIns3P). We demonstrate that PIK3C3 has a key function in vesicular trafficking, endocytosis and autophagosome-autolysosome formation in the highly specialized glomerular podocytes.

Activin-like kinase 3 is important for kidney regeneration and reversal of fibrosis.

Molecules associated with the transforming growth factor ß (TGF-ß) superfamily, such as bone morphogenic proteins (BMPs) and TGF-ß, are key regulators of inflammation, apoptosis and cellular transitions. Here we show that the BMP receptor activin-like kinase 3 (Alk3) is elevated early in diseased kidneys after injury. We also found that its deletion in the tubular epithelium leads to enhanced TGF-ß1-Smad family member 3 (Smad3) signaling, epithelial damage and fibrosis, suggesting a protective role for Alk3-mediated signaling in the kidney. A structure-function analysis of the BMP-Alk3-BMP receptor, type 2 (BMPR2) ligand-receptor complex, along with synthetic organic chemistry, led us to construct a library of small peptide agonists of BMP signaling that function through the Alk3 receptor. One such peptide agonist, THR-123, suppressed inflammation, apoptosis and the epithelial-to-mesenchymal transition program and reversed established fibrosis in five mouse models of acute and chronic renal injury. THR-123 acts specifically through Alk3 signaling, as mice with a targeted deletion for Alk3 in their tubular epithelium did not respond to therapy with THR-123. Combining THR-123 and the angiotensin-converting enzyme inhibitor captopril had an additive therapeutic benefit in controlling renal fibrosis. Our studies show that BMP signaling agonists constitute a new line of therapeutic agents with potential utility in the clinic to induce regeneration, repair and reverse established fibrosis.

Methylation determines fibroblast activation and fibrogenesis in the kidney.

Fibrogenesis is a pathological wound repair process that fails to cease, even when the initial insult has been removed. Fibroblasts are principal mediators of fibrosis, and fibroblasts from fibrotic tissues fail to return to their quiescent stage, including when cultured in vitro. In a search for underlying molecular mechanisms, we hypothesized that this perpetuation of fibrogenesis is caused by epigenetic modifications. We demonstrate here that hypermethylation of RASAL1, encoding an inhibitor of the Ras oncoprotein, is associated with the perpetuation of fibroblast activation and fibrogenesis in the kidney. RASAL1 hypermethylation is mediated by the methyltransferase Dnmt1 in renal fibrogenesis, and kidney fibrosis is ameliorated in Dnmt1(+/-) heterozygous mice. These studies demonstrate that epigenetic modifications may provide a molecular basis for perpetuated fibroblast activation and fibrogenesis in the kidney.

Catalase protects tumor cells from apoptosis induction by intercellular ROS signaling.

Transformed cells are subject to intercellular induction of apoptosis by neighbouring nontransformed cells and to autocrine apoptotic self-destruction. Both processes depend on extracellular superoxide anion generation by the transformed cells and on the release of peroxidase from both nontransformed and transformed cells. This concerted action results in HOCl synthesis, HOCl-superoxide anion interaction and generation of apoptosis-inducing hydroxyl radicals. In contrast to transformed cells, ex vivo tumor cells are resistant against intercellular induction of apoptosis and autocrine apoptotic self-destruction. Resistance of tumor cells against intercellular ROS signaling depends on interference through catalase expression on the membrane. Intercellular ROS signaling of tumor cells can be restored when i) exogenous HOCl is added; ii) exogenous hydrogen peroxide is supplied, or iii) catalase is inhibited. These findings define the biochemical basis for specific apoptosis induction in tumor cells through re-establishment of intercellular ROS signaling, a potential novel approach in tumor prevention and therapy.

Modulation of intercellular ROS signaling of human tumor cells.

Tumor cells are resistant against apoptosis-inducing intercellular reactive oxygen species (ROS) signaling but can be resensitized by the inhibition of catalase. Hydrogen peroxide exhibits a dual role in the modulation of intercellular ROS signaling. When suboptimal concentrations of the catalase inhibitior 3-aminotriazole (3-AT) are applied, additional exogenous hydrogen peroxide shifts apoptosis induction to its optimum. When hydrogen peroxide is added at optimal concentrations of 3-AT, or when higher concentrations of 3-AT are applied, the subsequent consumption between HOCl and hydrogen peroxide blunts overall apoptosis induction. These supraoptimal conditions can be brought back to the optimum through excess myeloperoxidase (MPO), partial removal of hydrogen peroxide through the catalase mimetic EUK-134 or partial inhibition of NADPH oxidase. Exogenous nitric oxide (NO) interferes with HOCl signaling through consumption of hydrogen peroxide. Site-specific generation of hydroxyl radicals at the cell membrane of tumor cells induces apoptosis, whereas random HOCl-superoxide anion interaction, and ferrous iron-induced Fenton chemistry of HOCl inhibit intercellular ROS signaling.

Nitric oxide mediates apoptosis induction selectively in transformed fibroblasts compared to nontransformed fibroblasts.

Nitric oxide (NO) mediates apoptosis induction in fibroblasts with constitutive src or induced ras oncogene expression, whereas nontransformed parental cells and revertants are not affected. This direct link between the transformed phenotype and sensitivity to NO-mediated apoptosis induction seems to be based on the recently described extracellular superoxide anion generation by transformed cells, as NO-mediated apoptosis induction in transformed cells is inhibited by extracellular superoxide dismutase (SOD), by SOD mimetics and by apocynin, an inhibitor of NADPH oxidase. Furthermore, nonresponsive nontransformed cells can be rendered sensitive for NO-mediated apoptosis induction when they are supplemented with xanthine oxidase/xanthine as an extracellular source for superoxide anions. As superoxide anions and NO readily interact in a diffusion-controlled reaction to generate peroxynitrite, peroxynitrite seems to be the responsible apoptosis inducer in NO-mediated apoptosis induction. In line with this conclusion, NO-mediated apoptosis induction in superoxide anion-generating transformed cells is inhibited by the peroxynitrite scavengers ebselen and FeTPPS. Moreover, direct application of peroxynitrite induces apoptosis both in transformed and nontransformed cells, indicating that peroxynitrite is no selective apoptosis inducer per se, but that selective apoptosis induction in transformed cells by NO is achieved through selective peroxynitrite generation. The interaction of NO with target cell derived superoxide anions represents a novel concept for selective apoptosis induction in transformed cells. This mechanism may be the basis for selective apoptosis induction by natural antitumor systems (like macrophages, natural killer cells, granulocytes) that utilize NO for antitumor action. Apoptosis induction mediated by NO involves mitochondrial depolarization and is blocked by Bcl-2 overexpression.