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Medicine

Estimation of Nephron Number in Whole Kidney using the Acid Maceration Method

Published: May 22nd, 2019

DOI:

10.3791/58599

1Department of Pharmacology and Toxicology, The University of Mississippi Medical Center, 2Department of Medicine, Division of Nephrology, Rush University Medical Center, 3Department of Medicine, Division of Nephrology, The University of Mississippi Medical Center, 4Department of Neurology, The University of Mississippi Medical Center

Estimates of whole kidney nephron number are important clinically and experimentally, as there is an inverse association between nephron number and an enhanced risk of renal and cardiovascular disease.Herein, the use of the acid maceration method, which provides fast and reliable estimates of whole kidney nephron number, is demonstrated.

Nephron endowment refers to the total number of nephrons an individual is born with, as nephrogenesis in humans is completed by 36 weeks of gestation and no new nephrons are formed post-birth. Nephron number refers to the total number of nephrons measured at any point in time post-birth. Both genetic and environmental factors influence both nephron endowment and number. Understanding how specific genes or factors influence the process of nephrogenesis and nephron loss or demise is important as individuals with lower nephron endowment or number are thought to be at a higher risk of developing renal or cardiovascular disease. Understanding how environmental exposures over the course of a person's lifetime affects nephron number will also be vital in determining future disease risk. Thus, the ability to assess whole kidney nephron number quickly and reliably is a basic experimental requirement to better understand mechanisms that contribute to or promote nephrogenesis or nephron loss. Here, we describe the acid maceration method for the estimation of whole kidney nephron number based on the procedure described by Damadian, Shawayri, and Bricker, with slight modifications. The acid maceration method provides fast and reliable estimates of nephron number (as assessed by counting glomeruli) that are within 5% of those determined using more advanced, albeit expensive, methods such as magnetic resonance imaging. Moreover, the acid maceration method is an excellent high-throughput method to assess nephron number in large numbers of samples or experimental conditions.

The nephron is both the basic structural and the functional unit of the kidney1. Structurally, the nephron consists of the glomerulus (capillaries and podocytes) located within the Bowman's capsule and the renal tubule, consisting of the proximal tubule, the Loop of Henle, and the distal tubule which terminates into the collecting duct. Functionally, the role of the nephron is the filtration and reabsorption of water and electrolytes and the secretion of wastes. In general, nephrogenesis is completed at 36 weeks of gestation in humans and shortly after birth in several species such as the mouse and the rat2. Nephron ....

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Supplies and reagents listed below are for the determination of the whole kidney nephron number in one mouse, that is, two kidneys. Modifications for the use of the acid maceration method for rat are identified with asterisks. All experimental protocols conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at The University of Mississippi Medical Center.

1. Kidney Isolation Procedure

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Below are representative estimates of whole kidney nephron number from an established mouse model of hypertension and a genetic rat model of age-related chronic kidney disease. Key identifying characteristics of glomeruli, such as a spherical structure with or without attached pre- or post-arteriolar or tubular structures, are highlighted for those new to the acid maceration method (Figure 1).

In th.......

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With good experimental technique, the acid maceration method is ideal for estimating nephron number in whole kidney. Although the kidney is dissolved in acid, glomeruli remain largely intact and are readily identifiable, making the counting of individual glomeruli relatively easy and straightforward. The acid maceration technique is particularly advantageous for several reasons. First, the acid maceration method is a rapid and convenient method that requires relatively little in terms of expense and physical effort. All .......

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This work was supported in part by the National Institutes of Health, National Heart, Lung, and Blood Institute (R01HL107632).

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Name Company Catalog Number Comments
Isoflurane anesthesia Abbott Laboratories 05260-05
Isoflurane vaporizor system & flow gauge Braintree Scientific VP I Include medical grade oxygen supply
Leica Inverted Microscope DMIL LED Leica Microsystems DMIL LED Any make also suitable
Digital water bath Fisher Scientific 2239 Any make also suitable
ToughCut Fine surgical scissors Fine Science Tools 14058-11 25 mm cutting edge, 11.5 cm length; Tips: sharp-sharp; Tip shape: straight
Micro dissecting forceps 4 1/4 in. Biomed Res Instruments, Inc 10-1760 Curved tip
Plexiglass board 5 in. x 7 in. any source suitable n/a Any make also suitable
Hexagonal polystyrene weighing dish Fisher Scientific 02-2002-100 Any make also suitable
Razor blades Fisher Scientific 12-640 Single edge carbon steel 0.009
Gauze sponges 4 x 4 in. 8 ply Fisher Scientific MSD-1400250
10x concentrate phosphate buffered saline (PBS) Sigma Aldrich P5493-4L Dilute to 1x 
6 N Hydrocholric acid solution Sigma Aldrich 3750-32
15 mL conical centrifuge tube Fisher Scientific 14-959-70C Any make also suitable
50 mL conical centrifuge tube Fisher Scientific 14-959-49A Any make also suitable
Disposable 5 mL syringe Cole Palmer EW-07944-06 Any make also suitable
18G1.5 disposable needle Fisher Scientific 14-826-5D Any make also suitable
21G1.5 disposable needle Fisher Scientific 14-826-5B Any make also suitable
12-well multiple-well cell culture plates with lid Cole Palmer #FW-01959-06 Any make also suitable
Polypropylene modular test tube rack Cole Palmer #EW-06733-00 Capable of accommodating 15 and 50 mL conical tubes; any make also suitable

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