Novel 3D analysis using optical tissue clearing documents the evolution of murine rapidly progressive glomerulonephritis.

Recent developments in optical tissue clearing have been difficult to apply for the morphometric analysis of organs with high cellular content and small functional structures, such as the kidney. Here, we establish combinations of genetic and immuno-labelling for single cell identification, tissue clearing and subsequent de-clarification for histoimmunopathology and transmission electron microscopy. Using advanced light microscopy and computational analyses, we investigated a murine model of crescentic nephritis, an inflammatory kidney disease typified by immune-mediated damage to glomeruli leading to the formation of hypercellular lesions and the rapid loss of kidney function induced by nephrotoxic serum. Results show a graded susceptibility of the glomeruli, significant podocyte loss and capillary injury. These effects are associated with activation of parietal epithelial cells and formation of glomerular lesions that may evolve and obstruct the kidney tubule, thereby explaining the loss of kidney function. Thus, our work provides new high-throughput endpoints for the analysis of complex tissues with single-cell resolution.

The role of TRPC6 calcium channels and P2 purinergic receptors in podocyte mechanical and metabolic sensing.

Podocyte calcium (Ca2+) signaling plays important roles in the (patho)physiology of the glomerular filtration barrier. Overactivation of podocyte transient receptor potential canonical (TRPC) channels including TRPC6 and purinergic signaling via P2 receptors that are known mechanosensors can increase podocyte intracellular Ca2+ levels ([Ca2+]i) and cause cell injury, proteinuria and glomerular disease including in diabetes. However, important mechanistic details of the trigger and activation of these pathways in vivo in the intact glomerular environment are lacking. Here we show direct visual evidence that podocytes can sense mechanical overload (increased glomerular capillary pressure) and metabolic alterations (increased plasma glucose) via TRPC6 and purinergic receptors including P2Y2. Multiphoton microscopy of podocyte [Ca2+]i was performed in vivo using wild-type and TRPC6 or P2Y2 knockout (KO) mice expressing the calcium reporter GCaMP3/5 only in podocytes and in vitro using freshly dissected microperfused glomeruli. Single-nephron intra-glomerular capillary pressure elevations induced by obstructing the efferent arteriole lumen with laser-induced microthrombus in vivo and by a micropipette in vitro triggered >2-fold increases in podocyte [Ca2+]i. These responses were blocked in TRPC6 and P2Y2 KO mice. Acute elevations of plasma glucose caused >4-fold increases in podocyte [Ca2+]i that were abolished by pharmacological inhibition of TRPC6 or P2 receptors using SAR7334 or suramin treatment, respectively. This study established the role of Ca2+ signaling via TRPC6 channels and P2 receptors in mechanical and metabolic sensing of podocytes in vivo, which are promising therapeutic targets in conditions with high intra-glomerular capillary pressure and plasma glucose, such as diabetic and hypertensive nephropathy.