Tissue Clearing and Deep Imaging of the Kidney Using Confocal and Two-Photon Microscopy

Microscopic and macroscopic evaluation of biological tissues in three dimensions is becoming increasingly popular. This trend is coincident with the emergence of numerous tissue clearing strategies, and advancements in confocal and two-photon microscopy, enabling the study of intact organs and systems down to cellular and sub-cellular resolution. In this chapter, we describe a wholemount immunofluorescence technique for labeling structures in renal tissue. This technique combined with solvent-based tissue clearing and confocal imaging, with or without two-photon excitation, provides greater structural information than traditional sectioning and staining alone. Given the addition of paraffin embedding to our method, this hybrid protocol offers a powerful approach to combine confocal or two-photon findings with histological and further immunofluorescent analysis within the same tissue

Smad4 promotes diabetic nephropathy by modulating glycolysis and OXPHOS

Diabetic nephropathy (DN) is the leading cause of end‐stage kidney disease. TGF‐β1/Smad3 signalling plays a major pathological role in DN; however, the contribution of Smad4 has not been examined. Smad4 depletion in the kidney using anti‐Smad4 locked nucleic acid halted progressive podocyte damage and glomerulosclerosis in mouse type 2 DN, suggesting a pathogenic role of Smad4 in podocytes. Smad4 is upregulated in human and mouse podocytes during DN. Conditional Smad4 deletion in podocytes protects mice from type 2 DN, independent of obesity. Mechanistically, hyperglycaemia induces Smad4 localization to mitochondria in podocytes, resulting in reduced glycolysis and oxidative phosphorylation and increased production of reactive oxygen species. This operates, in part, via direct binding of Smad4 to the glycolytic enzyme PKM2 and reducing the active tetrameric form of PKM2. In addition, Smad4 interacts with ATPIF1, causing a reduction in ATPIF1 degradation. In conclusion, we have discovered a mitochondrial mechanism by which Smad4 causes diabetic podocyte injury

Mammal-Like Organization of the Avian Midbrain Central Gray and a Reappraisal of the Intercollicular Nucleus

In mammals, rostrocaudal columns of the midbrain periaqueductal gray (PAG) regulate diverse behavioral and physiological functions, including sexual and fight-or-flight behavior, but homologous columns have not been identified in non-mammalian species. In contrast to mammals, in which the PAG lies ventral to the superior colliculus and surrounds the cerebral aqueduct, birds exhibit a hypertrophied tectum that is displaced laterally, and thus the midbrain central gray (CG) extends mediolaterally rather than dorsoventrally as in mammals. We therefore hypothesized that the avian CG is organized much like a folded open PAG. To address this hypothesis, we conducted immunohistochemical comparisons of the midbrains of mice and finches, as well as Fos studies of aggressive dominance, subordinance, non-social defense and sexual behavior in territorial and gregarious finch species. We obtained excellent support for our predictions based on the folded open model of the PAG and further showed that birds possess functional and anatomical zones that form longitudinal columns similar to those in mammals. However, distinguishing characteristics of the dorsal/dorsolateral PAG, such as a dense peptidergic innervation, a longitudinal column of neuronal nitric oxide synthase neurons, and aggression-induced Fos responses, do not lie within the classical avian CG, but in the laterally adjacent intercollicular nucleus (ICo), suggesting that much of the ICo is homologous to the dorsal PAG

The tetraspanin CD9 controls migration and proliferation of parietal epithelial cells and glomerular disease progression

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