Un enfoque basado en microscopía electrónica de barrido para explorar la remodelación del espacio subpodocítico en...

Ultrastructural analysis of the subpodocyte space (SPS) is essential to understanding podocyte injury in diabetic nephropathy, where SPS expansion represents an early marker of glomerular dysfunction. While traditionally assessed by transmission electron microscopy (TEM), we previously established a scanning electron microscopy (SEM) protocol enabling high-resolution SPS visualization in perfused kidney tissue. However, perfusion-based fixation, though effective in animal models, is not feasible in patients during the biopsy retrieval. To overcome these limitations, we developed a rapid SEM protocol based on secondary electron detection, specifically optimized for non-perfused samples. Using a paired design in healthy and diabetic (BTBR ob/ob) mice, we compared perfused and contralateral non-perfused kidneys, showing that SPS morphology and quantification remained accurate even in the absence of perfusion. The protocol was successfully applied to human kidney biopsies from controls and diabetic patients, allowing reliable SPS assessment and ultrastructural analysis across entire glomerular cross-sections. This optimized SEM protocol provides a reliable and versatile approach for SPS analysis in both non-perfused preclinical models and human kidney biopsies, thus broadening its applicability to clinically relevant settings.