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An intact kidney filter is vital to retention of essential proteins in the blood and removal of waste from the body. Damage to the filtration barrier results in albumin loss in the urine, a hallmark of cardiovascular disease and kidney failure. Here we found that the ion channel TRPC5 mediates filtration barrier injury. Using Trpc5-KO mice, a small-molecule inhibitor of TRPC5, Ca2+ imaging in isolated kidney glomeruli, and live imagining of podocyte actin dynamics, we determined that loss of TRPC5 or its inhibition abrogates podocyte cytoskeletal remodeling. Inhibition or loss of TRPC5 prevented activation of the small GTP-binding protein Rac1 and stabilized synaptopodin. Importantly, genetic deletion or pharmacologic inhibition of TRPC5 protected mice from albuminuria. These data reveal that the Ca2+-permeable channel TRPC5 is an important determinant of albuminuria and identify TRPC5 inhibition as a therapeutic strategy for the prevention or treatment of proteinuric kidney disease.

The cream or fat fraction of milk consists of fat droplets composed primarily of triacylglycerols that are surrounded by cellular membranes. In this review we discuss what is known about how these droplets are formed in and secreted by mammary epithelial cells during lactation. This secretion mechanism, which appears to be unique, is unlike the exocytotic mechanism used by other cell types to secrete lipids. Milk fat globules originate as small, triacylglycerol-rich, droplets that are formed on or in endoplasmic reticulum membranes. These droplets are released from endoplasmic reticulum into the cytosol as microlipid droplets coated by proteins and polar lipids. Microlipid droplets can fuse with each other to form larger cytoplasmic lipid droplets. Droplets of all sizes appear to be unidirectionally transported to apical cell regions by as yet unknown mechanisms that may involve cytoskeletal elements. These lipid droplets appear to be secreted from the cell in which they were formed by being progressively enveloped in differentiated regions of apical plasma membrane. While plasma membrane envelopment appears to be the primary mechanism by which lipid droplets are released from the cell, a mechanism involving exocytosis of lipid droplets from cytoplasmic vacuoles also has been described. As discussed herein, while we have a general overview of the steps leading to the fat globules of milk, virtually nothing is known about the molecular mechanisms involved in milk fat globule formation, intracellular transit, and secretion.

We report on the heterogeneity and diversity of lipid droplets (LDs) in early stages of adipogenesis by elucidating the cell and molecular biology of amphiphilic and cytoskeletal proteins regulating and stabilizing the generation of LDs in human adipose cells. A plethora of distinct and differently sized LDs was detected by a brief application of adipocyte differentiation medium and additional short treatment with oleic acid. Using these cells and highly specific antibodies for LD-binding proteins of the perilipin (PLIN) family, we could distinguish between endogenously derived LDs (endogenous LDs) positive for perilipin from exogenously induced LDs (exogenous LDs) positive for adipophilin, TIP47 and S3-12. Having optimized these stimulation conditions, we used early adipogenic differentiation stages to investigate small-sized LDs and concentrated on LD-protein associations with the intermediate-sized filament (IF) vimentin. This IF protein was described earlier to surround lipid globules, showing spherical, cage-like structures. Consequently - by biochemical methods, by immunofluorescence microscopy and by electron- and immunoelectron microscopy - various stages of emerging lipid globules were revealed with perilipin as linking protein between LDs and vimentin. For this LD-PLIN-Vimentin connection, a model is now proposed, suggesting an interaction of proteins via opposed charged amino acid domains respectively. In addition, multiple sheaths of smooth endoplasmic reticulum cisternae surrounding concentrically nascent LDs are shown. Based on our comprehensive localization studies we present and discuss a novel pathway for the LD formation.

Purpose Liposarcomas are the most common soft tissue sarcomas of adults. The identification of lipoblastic cells in soft tissue sarcomas is mandatory for the diagnosis of most subtypes of liposarcomas but may be difficult in conventional histology. The present study focuses on the expression and possible diagnostic impact of two PAT family proteins, perilipin 1/perilipin and perilipin 2/adipophilin in human liposarcomas. Methods Eighty-seven cases of liposarcomas and 30 cases of non-lipomatous sarcomas were investigated immunohistochemically for perilipin 1 and 2 using entire tissue sections. Statistical analyses were performed using appropriate tests. Results Most liposarcomas and non-lipomatous sarcomas displayed positivity for perilipin 2. In contrast, while more than two-thirds of liposarcomas presented perilipin 1 positivity, all non-lipomatous sarcomas studied were negative for this marker, with statistical significance ( p  < 0.001). Perilipin 1 expression increased with adipocytic differentiation of liposarcoma subtypes showing statistical significance ( p  < 0.001). Non-lipomatous sarcomas demonstrated variable expression levels of perilipin 2. The expression level of perilipin 2 appeared to be correlated with tumor cell degeneration, e.g., through hypoxia. Conclusions Perilipin 2 is not well suitable for distinction between liposarcomas and non-lipomatous sarcomas. However, perilipin 1 appeared to be a highly specific marker for liposarcoma and adipocytic differentiation in sarcomas with intermediate sensitivity.

In many human cancers, lipogenic pathways are activated; in some tumors, such as hepatocellular carcinoma, this is reflected by the presence of visible lipid droplets. Yet, the biology of steatogenesis in malignant tumors is largely unknown. We have recently shown that lipid droplet-associated proteins of the PAT-family, named after their constituents perilipin (perilipin 1), adipophilin (perilipin 2), and TIP47 (perilipin 3) are differentially expressed in hepatic steatogenesis. We have comprehensively investigated PAT-expression in neoplastic steatogenesis as well as in respective normal tissues with immunohistology and electron microscopy as well as protein biochemical and molecular biological methods. By staining for PAT-proteins, we found lipid droplet accumulation to be a frequent phenomenon of carcinoma cells. Although adipophilin and TIP47 stained almost ubiquitously the rim of lipid droplets in various tumor types, especially those with clear cell phenotype, perilipin was restricted to lipid droplets of hepatocellular adenoma and carcinoma, sebaceous adenoma and carcinoma, and lipomatous tumors. In hepatocellular carcinoma, perilipin, adipophilin, and TIP47 were coexpressed, and showed regional heterogeneity with a predominantly mutually exclusive localization pattern. In step-wise carcinogenesis, adipophilin expression correlated with the proliferation rate and was upregulated during early tumorigenesis, whereas perilipin was often lost during hepatocarcinogenesis. In conclusion, expression analysis of PAT-proteins showed that by far more carcinomas contain (PAT-positive) lipid droplets than expected by conventional light microscopy. PAT-proteins, such as perilipin, are differentially expressed in different tumor types and thus may support diagnostic considerations. Because inhibition of lipogenesis has been shown to exert antineoplastic effects, PAT-proteins may represent targets for interventive strategies.

Exposure of cells to stress, particularly oxidative stress, leads to misfolding of proteins and, if they are not refolded or degraded, to cytoplasmic protein aggregates. Protein aggregates are characteristic features of a variety of chronic toxic and degenerative diseases, such as Mallory bodies (MBs) in hepatocytes in alcoholic and non-alcoholic steatohepatitis, neurofibrillary tangles in neurons in Alzheimer's, and Lewy bodies in Parkinson's disease. Using 2D gel electrophoresis and mass spectrometry, we identified p62 as a novel MB component. p62 and cytokeratins (CKs) are major MB constituents; HSP 70, HSP 25, and ubiquitinated CKs are also present. These proteins characterize MBs as a prototype of disease-associated cytoplasmic inclusions generated by stress-induced protein misfolding. As revealed by transfection of tissue culture cells overexpressed p62 did not induce aggregation of regular CK filaments but selectively bound to misfolded and ubiquitinated CKs. The general role of p62 in the cellular response to misfolded proteins was substantiated by detection of p62 in other cytoplasmic inclusions, such as neurofibrillary tangles, Lewy bodies, Rosenthal fibers, intracytoplasmic hyaline bodies in hepatocellular carcinoma, and α1-antitrypsin aggregates. The presence of p62 along with other stress proteins and ubiquitin in cytoplasmic inclusions indicates deposition as aggregates as a third line of defense against misfolded proteins in addition to refolding and degradation.

The seminiferous tubules and the excurrent ducts of the mammalian testis are physiologically separated from the mesenchymal tissues and the blood and lymph system by a special structural barrier to paracellular translocations of molecules and particles: the “blood–testis barrier”, formed by junctions connecting Sertoli cells with each other and with spermatogonial cells. In combined biochemical as well as light and electron microscopical studies we systematically determine the molecules located in the adhering junctions of adult mammalian (human, bovine, porcine, murine, i.e., rat and mouse) testis. We show that the seminiferous epithelium does not contain desmosomes, or “desmosome-like” junctions, nor any of the desmosome-specific marker molecules and that the adhering junctions of tubules and ductules are fundamentally different. While the ductules contain classical epithelial cell layers with E-cadherin-based adherens junctions (AJs) and typical desmosomes, the Sertoli cells of the tubules lack desmosomes and “desmosome-like” junctions but are connected by morphologically different forms of AJs. These junctions are based on N-cadherin anchored in cytoplasmic plaques, which in some subforms appear thick and dense but in other subforms contain only scarce and loosely arranged plaque structures formed by α- and β-catenin, proteins p120, p0071 and plakoglobin, together with a member of the striatin family and also, in rodents, the proteins ZO-1 and myozap. These N-cadherin-based AJs also include two novel types of junctions: the “areae adhaerentes”, i.e., variously-sized, often very large cell-cell contacts and small sieve-plate-like AJs perforated by cytoplasm-to-cytoplasm channels of 5–7 nm internal diameter (“cribelliform junctions”). We emphasize the unique character of this epithelium that totally lacks major epithelial marker molecules and structures such as keratin filaments and desmosomal elements as well as EpCAM- and PERP-containing junctions. We also discuss the nature, development and possible functions of these junctions.

Lipid droplets (LDs) are spherical accumulations of apolar lipids and other hydrophobic substances and are generally surrounded by a thin cortical layer of specific amphiphilic proteins (APs). These APs segregate the LDs from the mostly polar components of the cytoplasm. We have studied LDs in epithelium-derived cell cultures and in particular characterized proteins from the perilipin (PLIN) gene family - in mammals consisting of the proteins Perilipin, Adipophilin, TIP47, S3-12 and MLDP/OXPAT (PLIN 1-5). Using a large number of newly generated and highly specific mono- and polyclonal antibodies specific for individual APs, and using improved LD isolation methods, we have enriched and characterized APs in greater detail and purity. The majority of lipid-AP complexes could be obtained in the top layer fractions of density gradient centrifugation separations of cultured cells, but APs could also be detected in other fractions within such separations. The differently sized LD complexes were analyzed using various biochemical methods and mass spectrometry as well as immunofluorescence and electron- in particular immunoelectron-microscopy. Moreover, by immunoprecipitation, protein-protein binding assays and by immunoelectron microscopy we identified a direct linkage between LD-binding proteins and the intermediate-sized filaments (IF) cytokeratins 8 and 18 (also designated as keratins K8 and K18). Specifically, in gradient fractions of higher density supposedly containing small LDs, we received as co-precipitations cytidylyl-, palmitoyl- and cholesterol transferases and other specific enzymes involved in lipid metabolism. So far, common proteomic studies have used LDs from top layer fractions only and did not report on these transferases and other enzymes. In addition to findings of short alternating hydrophobic/hydrophilic segments within the PLIN protein family, we propose and discuss a model for the interaction of LD-coating APs with IF proteins.

The molecular mechanism underlying milk fat globule secretion in mammary epithelial cells ostensibly involves the formation of complexes between plasma membrane butyrophilin and cytosolic xanthine oxidoreductase. These complexes bind adipophilin in the phospholipid monolayer of milk secretory granules, the precursors of milk fat globules, enveloping the nascent fat globules in a layer of plasma membrane and pinching them off the cell. However, using freeze-fracture immunocytochemistry, we find these proteins in locations other than those previously inferred. Significantly, butyrophilin in the residual plasma membrane of the fat globule envelope is concentrated in a network of ridges that are tightly apposed to the monolayer derived from the secretory granule, and the ridges coincide with butyrophilin labeling in the globule monolayer. Therefore, we propose that milk fat globule secretion is controlled by interactions between plasma membrane butyrophilin and butyrophilin in the secretory granule phospholipid monolayer rather than binding of butyrophilin-xanthine oxidoreductase complexes to secretory granule adipophilin.

Whole-organ engineering is an innovative field of regenerative medicine with growing translational perspectives. Recent reports suggest the feasibility of decellularization and repopulation of entire human size hearts. However, little is known about the susceptibility of epicardial adipose tissue (EAT) to decellularization. In this study, human size hearts of ovine donors were subjected to perfusion-based decellularization using detergent solutions. Upon basic histological evaluation and total DNA measurement myocardial regions prove largely decellularized while EAT demonstrated cellular remnants, further confirmed by transmission electron microscopy. Western blot analysis showed a significant reduction in lipid-associated and cardiac proteins. However, gas chromatography revealed unchanged proportional composition of fatty acids in EAT of decellularized whole hearts. Finally, cell culture medium conditioned with EAT from decellularized whole hearts had a significant deleterious effect on cardiac fibroblasts. These data suggest that perfusion decellularization of human size whole hearts provides inconsistent efficacy regarding donor material removal from myocardial regions as opposed to EAT.