Albumin-associated free fatty acids induce macropinocytosis in podocytes

Podocytes are specialized epithelial cells in the kidney glomerulus that play important structural and functional roles in maintaining the filtration barrier. Nephrotic syndrome results from a breakdown of the kidney filtration barrier and is associated with proteinuria, hyperlipidemia, and edema. Additionally, podocytes undergo changes in morphology and internalize plasma proteins in response to this disorder. Here, we used fluid-phase tracers in murine models and determined that podocytes actively internalize fluid from the plasma and that the rate of internalization is increased when the filtration barrier is disrupted. In cultured podocytes, the presence of free fatty acids (FFAs) associated with serum albumin stimulated macropinocytosis through a pathway that involves FFA receptors, the Gβ/Gγ complex, and RAC1. Moreover, mice with elevated levels of plasma FFAs as the result of a high-fat diet were more susceptible to Adriamycin-induced proteinuria than were animals on standard chow. Together, these results support a model in which podocytes sense the disruption of the filtration barrier via FFAs bound to albumin and respond by enhancing fluid-phase uptake. The response to FFAs may function in the development of nephrotic syndrome by amplifying the effects of proteinuria

Diverse molecular causes of unsolved autosomal dominant tubulointerstitial kidney diseases

Autosomal Dominant Tubulointerstitial Kidney Disease (ADTKD) is caused by mutations in one of at least five genes and leads to kidney failure usually in mid adulthood. Throughout the literature, variable numbers of families have been reported, where no mutation can be found and therefore termed ADTKD-not otherwise specified. Here, we aim to clarify the genetic cause of their diseases in our ADTKD registry. Sequencing for all known ADTKD genes was performed, followed by SNaPshot minisequencing for the dupC (an additional cytosine within a stretch of seven cytosines) mutation of MUC1. A virtual panel containing 560 genes reported in the context of kidney disease (nephrome) and exome sequencing were then analyzed sequentially. Variants were validated and tested for segregation. In 29 of the 45 registry families, mutations in known ADTKD genes were found, mostly in MUC1. Sixteen families could then be termed ADTKD-not otherwise specified, of which nine showed diagnostic variants in the nephrome (four in COL4A5, two in INF2 and one each in COL4A4, PAX2, SALL1 and PKD2). In the other seven families, exome sequencing analysis yielded potential disease associated variants in novel candidate genes for ADTKD; evaluated by database analyses and genome-wide association studies. For the great majority of our ADTKD registry we were able to reach a molecular genetic diagnosis. However, a small number of families are indeed affected by diseases classically described as a glomerular entity. Thus, incomplete clinical phenotyping and atypical clinical presentation may have led to the classification of ADTKD. The identified novel candidate genes by exome sequencing will require further functional validation

A novel domain regulating degradation of the glomerular slit diaphragm protein podocin in cell culture systems.

Mutations in the gene NPHS2 are the most common cause of hereditary steroid-resistant nephrotic syndrome. Its gene product, the stomatin family member protein podocin represents a core component of the slit diaphragm, a unique structure that bridges the space between adjacent podocyte foot processes in the kidney glomerulus. Dislocation and misexpression of slit diaphragm components have been described in the pathogenesis of acquired and hereditary nephrotic syndrome. However, little is known about mechanisms regulating cellular trafficking and turnover of podocin. Here, we discover a three amino acids-comprising motif regulating intracellular localization of podocin in cell culture systems. Mutations of this motif led to markedly reduced degradation of podocin. These findings give novel insight into the molecular biology of the slit diaphragm protein podocin, enabling future research to establish the biological relevance of podocin turnover and localization

The domain podocin^340–350 regulates the subcellular localization of podocin.

<p>A–D. Various truncations of Flag-tagged podocin were coexpressed with eGfp-tagged CD63 in HeLa-cells. Immunofluorescence using anti-Flag antibody revealed a primarily membranous staining pattern for podocin^1–285 and podocin^1–340 (a and b). In contrast, podocin^1–350 and podocin wild type were shown to also localize to intracellular vesicles costaining with CD63 hinting at a crucial role of podocin^340–350 in determining the subcellular localization of podocin (c and d).</p

Podocin^{TVV339,340,341} regulates the turnover of podocin through a lipid raft-independent mechanism.

<p>A. Differentiated podocytes stably expressing Flag-tagged podocin, podocin^1–285 or podocin^{TVV339,340,341AAA} were exposed to the translation inhibitor cycloheximide for the times as indicated and analyzed per western blot using anti-Flag antibody (a). Actin levels detected by anti-actin antibody served as loading control. Podocin^1–285 and podocin^{TVV339,340,341AAA} were shown to be more stable than podocin wild type, consistent with a regulatory role of podocin^{TVV339,340,341} in its degradation. (b) Summarizes the results of three experiments. Podocin levels were normalized to actin levels. B. (a) shows a schematic comparison between the PHB-domain proteins podocin and stomatin. Umlauf et al. proved a motif partially overlapping with podocin^{TVV339,340,341} to play a crucial role in lipid raft binding. (b) HEK293T cells were transfected with the plasmids as indicated, lysed in 1% TX-100 on ice and subjected to flotation gradient centrifugation to prepare detergent-resistant membranes (DRM). In contrast to the control protein transferrin receptor, both podocin wild type and podocin^{TVV339,340,341AAA} were detected in DRM. C. Graphical representation of the structure prediction analysis of podocin using the I-Tasser algorithm revealed exposed position of podocin^{TVV339, 340,341}.</p

Podocin localizes to endosomal vesicles. Immunofluorescence for transiently expressed, V5- (a–c, e) or Flag-tagged (d) podocin using anti-V5 or anti-Flag antibody in HeLa cells.

<p>Costainings of endogenous calnexin (a), endogenous golgin-97 (b), endogenous EEA1 (c), Lysotracker Red (d) and eGfp-tagged and transiently expressed CD63 (e) as markers for the endoplasmatic reticulum, Golgi apparatus, early endosomes, acidic organelles and late endosomes respectively, displayed that podocin localizes to the endosomal compartment. Analogously to HeLa cells, transiently expressed V5-tagged podocin colocalizes with eGFP tagged CD63/LAMP3 in cultured human podocytes (f).</p

Podocin^{TVV339, 340,341} regulates the surface expression of podocin.

<p>A. Amino acid-triplets within podocin^335–350 were mutated using a Quickchange approach. Mutated plasmids were transiently expressed in HeLa cells and localization of mutants was assessed by immunofluorescence using anti-Flag antibody. In contrast to the other mutants, podocin^{TVV339,340,341AAA} was shown to localize in a predominantly membranous pattern similar to podocin^1–285 (a–d). B. Immunofluorescence of Flag-tagged podocin wild type, podocin^1–285 and podocin^{TVV339, 340,341AAA} confirmed localization in transgenic differentiated podocytes to be analogous to HeLa cells (a–c). C. 293T-cells were transfected with plasmids expressing either CD16-7-*, CD16-7-podocin^286–385 or CD16-7-podocin^{286–385 TVV339,340,341AAA} and cell surface expression was analyzed by FACS. As transfected cells also expressed Gfp driven from an internal ribosomal site from the same vector, gates were set to include Gfp-positive cells only. Cell surface expression of CD16-7-* and CD16-7-podocin^{286–385TVV339,340,341AAA,} was comparable (b), while less CD16-7-podocin^286–385 could be detected at the plasma membrane, consistent with a role of podocin^{TVV339,340,341} in regulating internalization of podocin (a).</p

A C-terminal domain regulates plasma membrane localization of podocin.

<p>A. Immunofluorescence for a transiently expressed, Flag-tagged truncation of podocin (podocin^1–285) reveals increased plasma membrane localization in comparison with podocin wild type in HeLa-cells (b and a respectively). B. Schematic representation of constructs consisting of the extracellular domain of CD16 and the transmembrane domain of CD7 fused to different parts of podocin. C. Immunofluorescence using anti-CD16 antibody showed primarily membranous staining patterns for CD-16-7-* and CD16-7-podocin^1–285 (a, b). In contrast, immunofluorescence for CD16-7-podocin^286–385 revealed multiple vesicular structures (c), thereby proving analogous localization of CD16-fusion constructs and V5/Flag-tagged constructs.</p

Ablation of the mTORC2 component rictor in brain or Purkinje cells affects size and neuron morphology

The mammalian target of rapamycin (mTOR) assembles into two distinct multi-protein complexes called mTORC1 and mTORC2. Whereas mTORC1 is known to regulate cell and organismal growth, the role of mTORC2 is less understood. We describe two mouse lines that are devoid of the mTORC2 component rictor in the entire central nervous system or in Purkinje cells. In both lines neurons were smaller and their morphology and function were strongly affected. The phenotypes were accompanied by loss of activation of Akt, PKC, and SGK1 without effects on mTORC1 activity. The striking decrease in the activation and expression of several PKC isoforms, the subsequent loss of activation of GAP-43 and MARCKS, and the established role of PKCs in spinocerebellar ataxia and in shaping the actin cytoskeleton strongly suggest that the morphological deficits observed in rictor-deficient neurons are mediated by PKCs. Together our experiments show that mTORC2 has a particularly important role in the brain and that it affects size, morphology, and function of neurons