Automated image analyses of glomerular hypertrophy in a mouse model of diabetic nephropathy

Background: Glomerular hypertrophy is a hallmark of kidney injury in metabolically induced renal disease such as obesity-associated glomerulopathies and diabetic nephropathy (DN). Methods: Using light sheet fluorescent microscopy (LSFM) and 3D image analysis, we tested algorithms for automated and unbiased quantification of total glomerular numbers and individual glomerular volume in the uninephrectomized (UNx) db/db mouse model of DN. Results: Six weeks after surgery, db/db and UNx db/db mice showed increased urine albumin-to-creatinine ratio (ACR) compared to db/+ control mice. Prior to sacrifice glomeruli were labeled in vivo by injecting tomato lectin. Whole-kidney LSFM 3D image analysis revealed that mean glomerular volume was significantly increased in UNx db/db mice compared to db/+ mice. Moreover, analysis of individual glomerular volume showed a shift in volume distribution towards larger glomerular and thereby demonstrating additive effects of diabetes and UNx on induction of glomerular hypertrophy. The automized quantification showed no significant differences in glomerular numbers among db/+, db/db and UNx db/db mice. These data correlated with glomerular numbers as quantified by subsequent stereological quantification. Conclusions: Overall, LSFM coupled with automated 3D histomorphometric analysis was demonstrated to be advantageous for unbiased assessment of glomerular volume and numbers in mouse whole-kidney samples. Furthermore, we show that injection of fluorescently labelled lectin and albumin can be used as markers of nephron segments in the mouse kidneys, thus enabling functional assessment of kidney physiology, pathology and pharmacology in preclinical rodent models of kidney disease.