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Recent clinical trials of drugs enhancing cyclic guanosine monophosphate (cGMP) signaling for cardiovascular diseases have renewed interest in cGMP biology within the kidney. However, the role of cGMP signaling in glomerular endothelial cells (GECs) and podocytes remains largely unexplored. Using acute kidney slices from mice expressing the FRET-based cGMP biosensor cGi500 in endothelial cells or podocytes enabled real-time visualization of cGMP. Stimulation with atrial natriuretic peptide (ANP) or SNAP (NO donor) and various phosphodiesterase (PDE) inhibitors elevated intracellular cGMP in both cell types. GECs showed a transient cGMP response upon particulate or soluble guanylyl cyclase activation, while the cGMP response in podocytes reached a plateau following ANP administration. Co-stimulation (ANP + SNAP) led to an additive response in GECs. The administration of PDE inhibitors revealed a broader basal PDE activity in GECs dominated by PDE2a. In podocytes, basal PDE activity was mainly restricted to PDE3 and PDE5 activity. Our data demonstrate the existence of both guanylyl cyclase pathways in GECs and podocytes with cell-specific differences in cGMP synthesis and degradation, potentially suggesting new therapeutic options for kidney diseases., (© 2024. The Author(s).)

In recent years functional multiphoton (MP) imaging of vital mouse tissues and stimulation emission depletion (STED) imaging of optically cleared tissues allowed new insights into kidney biology. Here, we present a novel workflow where MP imaging of calcium signals can be combined with super-resolved STED imaging for morphological analysis of the slit diaphragm (SD) within the same glomerulus. Mice expressing the calcium indicator GCaMP3 in podocytes served as healthy controls or were challenged with two different doses of nephrotoxic serum (NTS). NTS induced glomerular damage in a dose dependent manner measured by shortening of SD length. In acute kidney slices (AKS) intracellular calcium levels increased upon disease but showed a high variation between glomeruli. We could not find a clear correlation between intracellular calcium levels and SD length in the same glomerulus. Remarkably, analysis of the SD morphology of glomeruli selected during MP calcium imaging revealed a higher percentage of completely disrupted SD architecture than estimated by STED imaging alone. Our novel co-imaging protocol is applicable to a broad range of research questions. It can be used with different tissues and is compatible with diverse reporters and target proteins., (© 2024. The Author(s).)

Mycophenolate mofetil (MMF) is applied in proteinuric kidney diseases, but the exact mechanism of its effect on podocytes is still unknown. Our previous in vitro experiments suggested that MMF can ameliorate podocyte damage via restoration of the Ca 2+ -actin cytoskeleton axis. The goal of this study was to characterize podocyte biology during MMF treatment in nephrotoxic serum (NTS) nephritis (NTN). NTN was induced in three-week old wild-type mice. On day 3, half of the mice were treated with MMF (100 mg/kgBW/d p.o.) for one week. On day 10, we performed proteomic analysis of glomeruli as well as super-resolution imaging of the slit diaphragm. For multiphoton imaging of Ca 2+ concentration ([Ca 2+ ] i ), the experimental design was repeated in mice expressing podocyte-specific Ca 2+ sensor. MMF ameliorated the proteinuria and crescent formation induced by NTS. We identified significant changes in the abundance of proteins involved in Ca 2+ signaling and actin cytoskeleton regulation, which was further confirmed by direct [Ca 2+ ] i imaging in podocytes showing decreased Ca 2+ levels after MMF treatment. This was associated with a tendency to restoration of podocyte foot process structure. Here, we provide evidence that MPA has a substantial direct effect on podocytes. MMF contributes to improvement of [Ca 2+ ] i and amelioration of the disorganized actin cytoskeleton in podocytes. These data extend the knowledge of direct effects of immunosuppressants on podocytes that may contribute to a more effective treatment of proteinuric glomerulopathies with the least possible side effects., (© 2023. Springer Nature Limited.)

Intracellular calcium ([Ca²⁺]i) signaling mediates physiological and pathological processes in multiple organs, including the renal podocyte; however, in vivo podocyte [Ca²⁺]i dynamics are not fully understood. Here we developed an imaging approach that uses multiphoton microscopy (MPM) to directly visualize podocyte [Ca²⁺]i dynamics within the intact kidneys of live mice expressing a fluorescent calcium indicator only in these cells. [Ca²⁺]i was at a low steady-state level in control podocytes, while Ang II infusion caused a minor elevation. Experimental focal podocyte injury triggered a robust and sustained elevation of podocyte [Ca²⁺]i around the injury site and promoted cell-to-cell propagating podocyte [Ca²⁺]i waves along capillary loops. [Ca²⁺]i wave propagation was ameliorated by inhibitors of purinergic [Ca²⁺]i signaling as well as in animals lacking the P2Y2 purinergic receptor. Increased podocyte [Ca²⁺]i resulted in contraction of the glomerular tuft and increased capillary albumin permeability. In preclinical models of renal fibrosis and glomerulosclerosis, high podocyte [Ca²⁺]i correlated with increased cell motility. Our findings provide a visual demonstration of the in vivo importance of podocyte [Ca²⁺]i in glomerular pathology and suggest that purinergic [Ca²⁺]i signaling is a robust and key pathogenic mechanism in podocyte injury. This in vivo imaging approach will allow future detailed investigation of the molecular and cellular mechanisms of glomerular disease in the intact living kidney.