Patient-derived podocyte spheroids reveal new insights into the etiopathogenesis of Alport syndrome.

Alport syndrome (AS) is a rare disease characterized by defective glomerular basement membranes, caused by mutations in COL4A3, COL4A4, and COL4A5, which synthesize collagen type IV. Patients present with progressive proteinuria, hematuria and podocyte loss. There is currently no cure for Alport syndrome, and this is mainly due to its complex and variable pathogenesis, as well as the lack of models that can faithfully mimic the human phenotype. Here we have developed a novel human culture model of Alport syndrome and used it to study the effects of different mutations on podocyte development and biology. First, we established a differentiation protocol that allowed us to generate podocyte spheroids from patient-derived human induced pluripotent stem cells (hiPSCs). We have then carried out discovery proteomics and demonstrated that a total of 178 proteins were differentially expressed between Alport (AS1 and AS3) and control (LT) podocytes. GO analysis indicated alterations in several metabolic pathways, such as oxidative phosphorylation, RNA maturation, chromatin condensation, and proliferation. Although functional assays showed no changes in lactate production and mitochondrial potential compared to healthy controls, immunofluorescence and electron microscopy analysis showed key morphological changes related to the phenotypical maturation of Alport podocytes. Moreover, the studied mutations led to persistent proliferation, increased reactive oxygen species (ROS) production and the concomitant expression of peroxisome proliferator-activated receptors α and γ (PPARα and PPARγ) in podocytes. These data on patient-derived podocytes provide evidence that collagen mutations, in addition to playing a central role in the defective development of the glomerular filtration barrier, cause significant alterations in podocyte development and metabolism very early in development, even before the formation of the filtering apparatus. In conclusion, our study provides a new methodological platform for the differentiation of podocytes and to study human podocytopathies in a personalized manner, and reveals new insights into the etiopathogenesis and pathobiology of Alport syndrome.

Effects of ozone on stratum corneum lipid integrity and assembly.

The stratum corneum (SC) acts as the main barrier of the skin against exogenous substances (e.g. air pollutants) and against the loss of endogenous substances such as water. The SC consists of keratin-rich dead cells surrounded by crystalline lamellar lipid regions. The main lipid classes are ceramides (CERs), free fatty acids (FFAs), and cholesterol (CHOL). Tropospheric ozone (O3) is a potent oxidant compound that reacts instantly with biological molecules such as lipids and proteins. Although it has been reported that O3 induces biological responses at the cellular level, to the best of our knowledge, there is no information related to the damages O3 can cause at the level of the SC extracellular lipid matrix. The aim of our work was to investigate which SC lipid subclasses are prone to oxidation when exposed to O3 and how the changes in chemical structures affect the lipid organization in a stratum corneum substitute (SCS) membrane. Ultimately, the barrier properties of the SCS were examined. Our studies revealed that O3 induces chemical modifications of the unsaturated bonds in CERs and CHOL. The appearance of carbonyl groups at the headgroup level and the removal of the linoleate moiety of omega­O­acylceramides (CER EOS) impact the lamellar organization of the lipid assembly and to a lesser extent the lateral packing of the lipids. Unexpectedly, these changes improved the barrier function of the SCS.

Cultivating a Three-dimensional Reconstructed Human Epidermis at a Large Scale.

A three-dimensional human epidermis model reconstructed from neonatal primary keratinocytes is presented. Herein, a protocol for the cultivation process and the characterization of the model is described. Neonatal primary keratinocytes are grown submerged on permeable polycarbonate inserts and lifted to the air-liquid interface three days after seeding. After fourteen days of stimulation with defined growth factors and ascorbic acid in high calcium culture medium, the model is fully differentiated. Histological analysis revealed a completely stratified epidermis, mimicking the morphology of native human skin. To characterize the model and its barrier functions, protein levels and localization specific for early-stage keratinocyte differentiation (i.e., keratin 10), late-stage differentiation (i.e., involucrin, loricrin, and filaggrin) and tissue adhesion (i.e., desmoglein 1), were assessed by immunofluorescence. The tissue barrier integrity was further evaluated by measuring transepithelial electrical resistance. Reconstructed human epidermis was responsive to proinflammatory stimuli (i.e., lipopolysaccharide and tumor necrosis factor alpha), leading to increased cytokine release (i.e., interleukin 1 alpha and interleukin 8). This protocol represents a straightforward and reproducible in vitro method to cultivate reconstructed human epidermis as a tool to assess environmental effects and a broad range of skin-related studies.

Bench approaches to study the detrimental cutaneous impact of tropospheric ozone.

Being exposed to ground-level ozone (O3), as it is often the case in polluted cities, is known to have a detrimental impact on skin. O3 induces antioxidant depletion and lipid peroxidation in the upper skin layers and this effect has repercussions on deeper cellular layers, triggering a cascade of cellular stress and inflammatory responses. Repetitive exposure to high levels of O3 may lead to chronic damages of the cutaneous tissue, cause premature skin aging and aggravate skin diseases such as contact dermatitis and urticaria. This review paper debates about the most relevant experimental approaches that must be considered to gather deeper insights about the complex biological processes that are activated when the skin is exposed to O3. Having a better understanding of O3 effects on skin barrier properties and stress responses could help the whole dermato-cosmetic industry to design innovative protective solutions and develop specific cosmetic regime to protect the skin of every citizen, especially those living in areas where exposure to high levels of O3 is of concern to human health.