Epithelial RAC1-dependent cytoskeleton dynamics controls cell mechanics, cell shedding and barrier integrity in intestinal inflammation

Objective: Increased apoptotic shedding has been linked to intestinal barrier dysfunction and development of inflammatory bowel diseases (IBD). In contrast, physiological cell shedding allows the renewal of the epithelial monolayer without compromising the barrier function. Here, we investigated the role of live cell extrusion in epithelial barrier alterations in IBD. Design: Taking advantage of conditional GGTase and RAC1 knockout mice in intestinal epithelial cells (Pggt1biΔIEC and Rac1iΔIEC mice), intravital microscopy, immunostaining, mechanobiology, organoid techniques and RNA sequencing, we analysed cell shedding alterations within the intestinal epithelium. Moreover, we examined human gut tissue and intestinal organoids from patients with IBD for cell shedding alterations and RAC1 function. Results: Epithelial Pggt1b deletion led to cytoskeleton rearrangement and tight junction redistribution, causing cell overcrowding due to arresting of cell shedding that finally resulted in epithelial leakage and spontaneous mucosal inflammation in the small and to a lesser extent in the large intestine. Both in vivo and in vitro studies (knockout mice, organoids) identified RAC1 as a GGTase target critically involved in prenylation-dependent cytoskeleton dynamics, cell mechanics and epithelial cell shedding. Moreover, inflamed areas of gut tissue from patients with IBD exhibited funnel-like structures, signs of arrested cell shedding and impaired RAC1 function. RAC1 inhibition in human intestinal organoids caused actin alterations compatible with arresting of cell shedding. Conclusion: Impaired epithelial RAC1 function causes cell overcrowding and epithelial leakage thus inducing chronic intestinal inflammation. Epithelial RAC1 emerges as key regulator of cytoskeletal dynamics, cell mechanics and intestinal cell shedding. Modulation of RAC1 might be exploited for restoration of epithelial integrity in the gut of patients with IBD

Early ultrastructural changes in biopsies from patients with symptomatic CKDu (Sym-CKDu)

Introduction Although the investigation of chronic kidney disease of uncertain etiology (CKDu) has identified many possible influencing factors in recent years, the exact pathomechanism of this disease remains unclear. Methods In this study, we collected 13 renal biopsies from patients with symptomatic CKDu (Sym-CKDu) from Sri Lanka with well-documented clinical and socioeconomic factors. We performed light microscopy and electron microscopic evaluation for ultrastructural analysis which was compared to 100 biopsies from German patients with 20 different kidney diseases. Results Of the Sri Lankan patients, most were men (12/13), frequently employed in agriculture (50%), and showed symptoms such as feverish feeling (83.3%), dysuria (83.3%), and arthralgia (66.6%). Light microscopic evaluation using activity and chronicity score revealed that cases represented early stages of CKDu except for two biopsies which showed additional signs of diabetes. Most glomeruli showed only mild changes, such as podocyte foot process effacement on EM. We found a spectrum of early tubulointerstitial changes including partial loss of brush border in proximal tubules, detachment of tubular cells, enlarged vacuoles, and mitochondrial swelling associated with loss of cristae and dysmorphic lysosomes with electron-dense aggregates. None of these changes occurred exclusively in Sym-CKDu, however they were significantly more frequent in these cases than in the control cohort. Conclusion In conclusion, our findings confirm the predominant and early alterations of tubular structure in CKDu that can occur without significant glomerular changes. The ultrastructural changes do not provide concrete evidence of the cause of CKDu but were significantly more frequent in Sym-CKDu compared to the controls