Renal Parenchymal Area and Kidney Collagen Content

The extent of renal scarring in chronic kidney disease (CKD) can only be ascertained by highly invasive, painful and sometimes risky tissue biopsy. Interestingly, CKD-related abnormalities in kidney size can often be visualized using ultrasound. Nevertheless, not only does the ellipsoid formula used today underestimate true renal size but also the relation governing renal size and collagen content remains unclear. We used coronal kidney sections from healthy mice and mice with renal disease to develop a new technique for estimating the renal parenchymal area. While treating the kidney as an ellipse with the major axis the polar distance, this technique involves extending the minor axis into the renal pelvis. The calculated renal parenchymal area is remarkably similar to the measured area. Biochemically determined kidney collagen content revealed a strong and positive correlation with the calculated renal parenchymal area. The extent of renal scarring, i.e. kidney collagen content, can now be computed by making just two renal axial measurements which can easily be accomplished via noninvasive imaging of this organ. Kidney Collagen Calculator Introduction Given the prevalence of diabetes, hypertension and Metabolic Syndrome, chronic kidney disease (CKD) is reaching epidemic proportions across the world [1,2]. Characterized by scarring or accumulation of fibrillar collagen within the renal interstitium, CKD is associated with a progressive decline in glomerular filtration rate (GFR) reflected by rising serum creatinine (SCr). However, existing disease is often diagnosed late because clinically meaningful changes in SCr occur long after substantial and irreversible scar formation [3,4]. Further compounding both disease diagnosis and prognosis is the fact that highly invasive renal biopsy remains the mainstay for determining the extent of renal scarring [5]. Interestingly, fibrosis-related abnormalities in kidney dimension can be visualized by noninvasive sonography [6-8]. Renal length or major axis, renal width or minor axis and renal thickness measurements can be incorporated into an ellipsoid formula to yield kidney size [911]. Unfortunately, there is insufficient information relating renal dimension with the amount of tissue interstitial collagen. Second, the standard ellipsoid formula underestimates true kidney size confounding any inferences of tissue collagen content [9-11]. In the present study, we used a mouse model of CKD to develop a modified elliptical formula that better represents true renal parenchymal area. We then formulated a relationship between calculated renal parenchymal area and total tissue collagen. Kidney Collagen Calculator Methods Animal Model: The study protocol, designed to induce renal fibrosis in mice, was submitted to and approved by the Angion Biomedica Corp. Institutional Animal Care and Use and Committee. Animals were allowed to acclimatize for a minimum of 5 days prior to use and had free access to water and standard rodent chow. Adult male CD-1 mice (~30-35 g) were anesthetized with ketamine (25 mg/kg, intraperitoneal) and xylazine (5 mg/kg, ip) and placed on a heating pad table to maintain ~37.5°C core body temperature. A midline laparotomy was made and the right kidney removed was removed. Extended release buprenorphine (0.65 mg/kg, subcutaneous) was administered prior to returning animals to their cages. One week later, animals were placed on 1% NaCl (drinking water) and deoxycorticosterone acetate (DOCA, 1 mg/kg, subcutaneous) injected twice weekly for the first 3 weeks [12,13]. Eight weeks after nephrectomy, animals were sacrificed and the left kidney retrieved. Age-matched, surgery-and DOCA-naive animals on regular drinking water were used as the baseline control. Left kidneys from these animals were retrieved at sacrifice. Kidneys were weighed and sliced coronally under a dissecting microscope (4X). One half of the kidney was placed in 10% formalin for subsequent sectioning (coronal sections 5 μm apart) and staining with hematoxylin & eosin (H&E). The other half of the kidney was weighed and submitted to hydroxyproline analysis using a previously described method [ 14]. Total kidney hydroxyproline values were converted to total kidney collagen (μg/kidney) content [14 ]. Renal Parenchymal Area: H&E-stained renal sections were photographed (Nikon) and analyzed using NISElements D 3.1 software by an observer blinded to the collagen content of that kidney. Images were superimposed on a precalibrated grid and the major (a), minor (b) and the extended minor (be) axes (mm) measured (see Figs 1 and 3). Renal parenchymal area (mm) was measured using the “area measurement” tool available in the software and also calculated from equations 1 and 2. Data Analysis: Both collagen and the corresponding renal parenchymal area measurement were obtained from a total of 30 kidneys, 10 from the healthy cohort and 20 from the diseased cohort. Microsoft Excel 2010 curve fitting software was used to generate all scatterplots. Since a linear relation was observed between the 2 variables in each of the scatterplots, both Pearson product moment (r) and Spearman’s rho (rs) were calculated Kidney Collagen Calculator from the trend line. To determine whether the relationship between the 2 variables was significant, r or rs and the sample size (n=30) were entered into an online calculator [15]. A p <0.05 was considered to be statistically significant. Kidney Collagen Calculator Results Shown in Fig 1 is an H&E-stained renal coronal section from a uninephrectomized mouse administered DOCA and 1% NaCl (drinking water) with the major (a) and minor (b) axes delineated. Fig 1: Renal Parenchyma Area. An H&E-stained coronal section (4X) from the left kidney of a uninephrectomized mouse administered DOCA and NaCl. The section has been superimposed on a 1 mm grid. The white bar represents renal length or the major axis (a) whereas the yellow bar represents renal width or the minor axis (b). The renal parenchymal area can be measured using a precalibrated measuring tool or calculated from a and b. a