Lectin-mediated, time-efficient, and high-yield sorting of different morphologically intact nephron segments.

Area of Science:

• Nephrology
• Molecular Biology
• Biotechnology

Background:

• The kidney's nephrons are complex structures with distinct segments, crucial for filtration and reabsorption.
• Conventional methods for isolating nephron segments are laborious, time-consuming, and yield limited material.
• There is a need for efficient and high-yield techniques to obtain pure nephron segments for research.

Purpose of the Study:

• To develop a novel, rapid, and cost-effective method for isolating multiple kidney nephron segments simultaneously.
• To preserve the 3D morphology of isolated nephron segments for downstream analysis.
• To enable high-purity isolation of nephron segments from mouse and human kidney samples.

Main Methods:

• Combined low-cost fluorophore-conjugated lectins or agglutinins (Flaggs) with flow sorting.
• Utilized specific Flaggs (SNA-Cy3, LTL-FITC, SBA-PB) for differential labeling of nephron segments.
• Employed a 200-µm nozzle and 5 psi for sorting glomeruli, proximal convoluted tubules, and distal convoluted tubules.
• Identified connecting tubules/collecting ducts via dual labeling and thick ascending limbs by lack of labeling.

Main Results:

• Simultaneous separation of different nephron segments with preserved 3D morphology in under 3 hours.
• Achieved high yields of protein (37-521 ng/s) and RNA (0.71-16.71 ng/s) per sorted segment.
• Demonstrated high purity of sorted segments with a median enrichment of 96.1-fold for mRNA expression levels.

Conclusions:

• The novel Flaggs and flow sorting method is simple, cost-effective, and widely applicable.
• This technique provides high yields of pure, morphologically intact renal tubule materials.
• The method has the potential to significantly advance nephron segment-specific research.