Quantifying Podocyte Number in a Small Sample Size of Glomeruli with CUBIC to Evaluate Podocyte Depletion of db/db Mice

Abstract

The loss of podocyte is crucial for diagnosis and prognosis of diabetic kidney disease, whereas commonly two-dimensional methods for quantifying podocyte number existed with issues of low fidelity and accuracy. In this study, clear, unobstructed brain imaging cocktails and computational analysis (CUBIC), one of three-dimensional optical clearing approaches, was used which combines tissue clearing, immunolabeling, and a light-sheet microscope to image and evaluate podocytes in C57BL/6 (C57) and db/db mice. We discovered that 77 podocytes per glomerulus were in C57 mice. On the subject of db/db mice, there were 74 podocytes by the age of 8 w, 72 podocytes by the age of 12 w, and 66 podocytes by the age of 16 w, compared with 76 podocytes in the control group, suggesting that there was a significant decrease in podocyte number in db/db mice with the age of 16 w, showing a trend which positively correlated to the deterioration of kidney function. Sample size estimation using the PASS software revealed that taking 5%, 7.5%, and 10% of the mean podocyte number per glomerulus as the statistical allowable error and 95% as total confidence interval, 33, 15, and 9 glomeruli were independently needed to be sampled in C57 mice to represent the overall glomeruli to calculate podocyte number. Furthermore, in the control group of db/db mice, 36, 18, and 11 glomeruli were needed, compared with 46, 24, and 14 glomeruli in db/db mice by the age of 8 w, 43, 21, and 12 glomeruli by the age of 12 w, and 52, 27, and 16 by the age of 16 w. These findings indicated that precise quantification of podocyte number could judge the progression of diabetic kidney disease. In addition, a small number of glomeruli could be actually representative of the whole sample size, which indicated apparent practicability of CUBIC for clinical use.

Pericyte Loss in Diseases

“…The loss of podocytes serves as an early pathological marker and contributes to proteinuric glomerulopathies in DN [70,71,73]. The number of podocytes is decreased in both type 1 and type 2 diabetic patients [74,75], and podocyte loss has also been observed in diabetic mice [76,77] and rats [78,79]. Podocyte apoptosis is the most common mechanism of podocyte loss and has been extensively documented [71,[80][81][82].…”

Therapeutic potential of Ganoderma lucidum polysaccharide peptide in Doxorubicin-induced nephropathy: modulation of renin-angiotensin system and proteinuria

“…Proteinuria, characterized by abnormal excretion of protein in the urine, is a prominent characteristic of DOX nephropathy and a significant global healthcare concern, affecting several hundred million people worldwide (Yamashita et al, 2017). Proteinuria is frequently accompanied by increased permeability of the glomerular filtration barrier, which is primarily brought on by podocyte loss (Shi et al, 2023). This barrier acts as a highly specific filtration system within the kidneys, facilitating the elimination of waste products while retaining essential proteins in circulation.…”