Name: estimation of nephron number in whole kidney using the acid maceration method
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Description: Watch this Scientific Journal Video about Estimation of Nephron Number in Whole Kidney using the Acid Maceration Method at JoVE.com

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

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</p...

<ol>
	<li>Hall, J. E., Guyton, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Guyton and Hall Textbook of Medical Physiology. , (2016).</li><li>Wang, X., Garrett, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nephron+number,+hypertension,+and+CKD:+physiological+and+genetic+insight+from+humans+and+animal+models.">Nephron number, hypertension, and CKD: physiological and genetic insight from humans and animal models.</a> Physiological Genomics. 49 (3), 180-192 (2017).</li><li>Didion, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+genetic+model+to+explore+the+Brenner+hypothesis:+Linking+nephron+endowment+and+number+with+hypertension.">A novel genetic model to explore the Brenner hypothesis: Linking nephron endowment and number with hypertension.</a> Medical Hypotheses. 106, 6-9 (2017).</li><li>Brenner, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nephron+adaptation+to+renal+injury+or+ablation.">Nephron adaptation to renal injury or ablation.</a> American Journal of Physiology. 249 (3 Pt 2), F324-F337 (1985).</li><li>Didion, S. P., Wang, X., Garrett, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+correlation+between+blood+pressure+and+nephron+endowment+in+a+genetic+model+of+hypertension.">Direct correlation between blood pressure and nephron endowment in a genetic model of hypertension.</a> Hypertension. 68, A052 (2016).</li><li>Luyckx, V. A., Brenner, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+clinical+importance+of+nephron+mass.">The clinical importance of nephron mass.</a> Journal of the American Society of Nephrology. 21 (6), 898-910 (2010).</li><li>Mackenzie, H. S., Brenner, B. 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D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypertension,+glomerular+hypertrophy+and+nephrosclerosis:+the+effect+of+race.">Hypertension, glomerular hypertrophy and nephrosclerosis: the effect of race.</a> Nephrology Dialysis Transplantation. 29 (7), 1399-1409 (2014).</li><li>Puelles, V. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glomerular+number+and+size+variability+and+risk+for+kidney+disease.">Glomerular number and size variability and risk for kidney disease.</a> Current Opinion in Nephrology and Hypertension. 20 (1), 7-15 (2010).</li><li>Brenner, B. M., Garcia, D. L., Anderson, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glomeruli+and+blood+pressure.+Less+of+one,+more+of+the+other?.">Glomeruli and blood pressure. Less of one, more of the other?.</a> American Journal of Hypertension. 1 (4 Pt 1), 335-347 (1988).</li><li>Clark, A. T., Bertram, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+regulation+of+nephron+endowment.">Molecular regulation of nephron endowment.</a> American Journal of Physiology. 276 (4 Pt 2), F485-F497 (1999).</li><li>Benz, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+glomerular+alterations+in+genetically+determined+low+nephron+number.">Early glomerular alterations in genetically determined low nephron number.</a> American Journal of Physiology Renal Physiology. 300 (2), F521-F530 (2011).</li><li>Zhao, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+fibroblast+growth+factor+receptors+1+and+2+in+the+ureteric+bud.">Role of fibroblast growth factor receptors 1 and 2 in the ureteric bud.</a> Developmental Biology. 276 (2), 403-415 (2004).</li><li>Sims-Lucas, S., Caruana, G., Dowling, J., Kett, M. M., Bertram, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Augmented+and+accelerated+nephrogenesis+in+TGF-beta2+heterozygous+mutant+mice.">Augmented and accelerated nephrogenesis in TGF-beta2 heterozygous mutant mice.</a> Pediatric Research. 63 (6), 607-612 (2008).</li><li>Cullen-McEwen, L. A., Kett, M. M., Dowling, J., Anderson, W. P., Bertram, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nephron+number,+renal+function,+and+arterial+pressure+in+aged+GDNF+heterozygous+mice.">Nephron number, renal function, and arterial pressure in aged GDNF heterozygous mice.</a> Hypertension. 41 (2), 335-340 (2003).</li><li>Stelloh, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prematurity+in+mice+leads+to+reduction+in+nephron+number,+hypertension+and+proteinuria.">Prematurity in mice leads to reduction in nephron number, hypertension and proteinuria.</a> Translational Research. 159 (2), 80-89 (2012).</li><li>Galinsky, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+intra-amniotic+lipopolysaccharide+on+nephron+number+in+preterm+fetal+sheep.">Effect of intra-amniotic lipopolysaccharide on nephron number in preterm fetal sheep.</a> American Journal of Physiology Renal Physiology. 301 (2), F280-F285 (2011).</li><li>Bertam, J. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+and+how+we+determine+nephron+number.">Why and how we determine nephron number.</a> Pediatric Nephrology. 29 (4), 575-580 (2014).</li><li>Nyengaard, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stereologic+methods+and+their+application+in+kidney+research.">Stereologic methods and their application in kidney research.</a> Journal of the American Society of Nephrology. 10 (5), 1100-1123 (1999).</li><li>Beeman, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+glomerular+number+and+size+in+perfused+kidneys+using+MRI.">Measuring glomerular number and size in perfused kidneys using MRI.</a> American Journal of Physiology Renal Physiology. 300 (6), F1454-F1457 (2011).</li><li>Damadian, R. V., Shawayri, E., Bricker, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+existence+of+non-urine+forming+nephrons+in+the+diseased+kidney+of+the+dog.">On the existence of non-urine forming nephrons in the diseased kidney of the dog.</a> Journal of Laboratory and Clinical Medicine. 65, 26-39 (1965).</li><li>Bonvalet, J. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+method+for+the+isolation+of+mouse+glomeruli.">Improved method for the isolation of mouse glomeruli.</a> Journal of the American Society of Nephrology. 2 (4), 944-946 (1991).</li><li>Wang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nephron+deficiency+and+predisposition+to+renal+injury+in+a+novel+one-kidney+genetic+model.">Nephron deficiency and predisposition to renal injury in a novel one-kidney genetic model.</a> Journal of the American Society of Nephrology. 26 (7), 1634-1646 (2015).</li><li>Lodrup, A. B., Karstoft, K., Dissing, T. H., Pedersen, M., Nyengaard, J. 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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 ...

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&#39;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 endowment refers to the total number of nephrons which an individual is born with, whereas nephron number is the total number of nephrons measured at any time post-birth3. The term nephron number and glomerular number are often used interchangeably. Because there is only one glomerulus per nephron, the assessment of glomeruli number is an important surrogate for estimating nephron number.
The assessment of nephron endowment and nephron number is of clinical interest as studies have demonstrated an association between nephron endowment and reduced nephron numbers with an increased incidence of cardiovascular disease4,5,6,7,8,9,10,11,12,13,14,15. Based on findings in kidneys at autopsy, Brenner observed that hypertensive individuals presented with a lower total number of nephrons than normotensive individuals16. Thus, Brenner hypothesized that there is an inverse relationship between nephron number and the risk of developing hypertension later in life. Brenner also hypothesized that a reduction in nephron number was compensated for by the nephrons that remained. In order to maintain the normal filtration rate in the kidney, residual nephrons compensate by increasing their glomerular surface area (glomerular hypertrophy), thereby working to mitigate any adverse effect of nephron loss on renal function4,16.
While protective in the short-term, glomerular hypertrophy, in the long-term, leads to increased sodium and fluid retention, increased extracellular fluid volume, and increases in arterial blood pressure, leading to a vicious cycle of further increases in glomerular capillary pressure, glomerular hyperfiltration, and nephron scarring (sclerosis) and injury4,16.
Obtaining estimates or counts of nephron number offer a couple of experimental advantages: 1) it provides information regarding the process of nephrogenesis, which can then be linked to specific genes or factors in the embryo or maternal-fetal environment, and 2) there is an association of nephron number with cardiovascular disease and, thus, there is the potential that estimates of nephron number could be used to predict future cardiovascular risk2,17,18,19,20,21,22. In addition to the maternal-fetal environment, several diseases directly impact nephron number and renal function, including atherosclerosis, diabetes, hypertension, and even normal aging2,9,10,11,12,22,23. Thus, assessment of whole kidney nephron number is important to understand both the genetic and environmental factors that affect nephrogenesis (i.e., nephron endowment) and nephron number over the course of a person&#39;s life and the resulting effects on renal function and cardiovascular health.
Currently, there are several methods available for the determination and quantification of nephron number, each with its own advantages and limitations24,25,26,27,28,29,30. Sophisticated methods for determining whole kidney nephron number include stereological methods, such as the dissector/fractionator method, and magnetic resonance imaging25,26. Often considered the gold-standard for determining whole kidney nephron number, the dissector/fractionator method is both expensive and time-consuming. Recent advances and improvement in magnetic resonance imaging and processing have provided the tools to count each and every nephron individually. However, magnetic resonance imaging is not only time-consuming but also extremely expensive. In addition, both the dissector/fractionator method and magnetic resonance imaging requires advanced technical expertise, thus limiting the use of such methods in the majority of research laboratories.
Most methods of determining nephron number make counts or estimates based on the identification of glomeruli, as they are readily identifiable structurally. In this paper, the acid maceration method for estimating nephron number in whole kidney is described and demonstrated27. The acid maceration method is fast, reliable, and significantly less expensive than other methods, such as the dissector/fractionator method and magnetic resonance imaging. Moreover, the acid maceration method provides highly repeatable estimates of nephron number that have been reported to be within the range of those determined using magnetic resonance imaging26.

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			<td height="21" style="height:21px;width:353px;">Isoflurane anesthesia</td>
			<td style="width:239px;">Abbott Laboratories</td>
			<td style="width:191px;">05260-05</td>
			<td style="width:91px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isoflurane vaporizor system &#38; flow gauge</td>
			<td>Braintree Scientific</td>
			<td>VP I</td>
			<td>Include medical grade oxygen supply</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leica Inverted Microscope DMIL LED</td>
			<td>Leica Microsystems</td>
			<td>DMIL LED</td>
			<td>Any make also suitable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Digital water bath</td>
			<td>Fisher Scientific</td>
			<td align="right">2239</td>
			<td>Any make also suitable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ToughCut Fine surgical scissors</td>
			<td>Fine Science Tools</td>
			<td>14058-11</td>
			<td>25 mm cutting edge, 11.5 cm length; Tips: sharp-sharp; Tip shape: straight</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro dissecting forceps 4 1/4 in.</td>
			<td>Biomed Res Instruments, Inc</td>
			<td>10-1760</td>
			<td>Curved tip</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plexiglass board 5 in. x 7 in.</td>
			<td>any source suitable</td>
			<td>n/a</td>
			<td>Any make also suitable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hexagonal polystyrene weighing dish</td>
			<td>Fisher Scientific</td>
			<td>02-2002-100</td>
			<td>Any make also suitable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Razor blades</td>
			<td>Fisher Scientific</td>
			<td>12-640</td>
			<td>Single edge carbon steel 0.009</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gauze sponges 4 x 4 in. 8 ply</td>
			<td>Fisher Scientific</td>
			<td>MSD-1400250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10x concentrate phosphate buffered saline (PBS)</td>
			<td>Sigma Aldrich</td>
			<td>P5493-4L</td>
			<td>Dilute to 1x&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6 N Hydrocholric acid solution</td>
			<td>Sigma Aldrich</td>
			<td>3750-32</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15 mL conical centrifuge tube</td>
			<td>Fisher Scientific</td>
			<td>14-959-70C</td>
			<td>Any make also suitable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50 mL conical centrifuge tube</td>
			<td>Fisher Scientific</td>
			<td>14-959-49A</td>
			<td>Any make also suitable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable 5 mL syringe</td>
			<td>Cole Palmer</td>
			<td>EW-07944-06</td>
			<td>Any make also suitable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">18G1.5 disposable needle</td>
			<td>Fisher Scientific</td>
			<td>14-826-5D</td>
			<td>Any make also suitable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">21G1.5 disposable needle</td>
			<td>Fisher Scientific</td>
			<td>14-826-5B</td>
			<td>Any make also suitable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">12-well multiple-well cell culture plates with lid</td>
			<td>Cole Palmer</td>
			<td>#FW-01959-06</td>
			<td>Any make also suitable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polypropylene modular test tube rack</td>
			<td>Cole Palmer</td>
			<td>#EW-06733-00</td>
			<td>Capable of accommodating 15 and 50 mL conical tubes; any make also suitable</td>
		</tr>
	</tbody>
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estimation of nephron number in whole kidney using the acid maceration method

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.

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

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 ...

The acid maceration method can help answer key questions about the kidney and how specific genes contribute to or influence nephron development as well as how specific conditions, such as diabetes or hypertension, affect nephron number. This is important as a total number of nephrons an individuals born with is determined during fetal development and loss of nephrons has been associated with renal pathology. The main advantages of the acid maceration method are that it is faster, more reproducible, inexpensive, and less time consuming than other nephron counting methods, like magnetic resonance imaging.This technique facilitates the correlation of the impact in nephron number on renal function, which is important to further our understanding of how specific genes influence nephron function and development. The study of genes or factors that influence overall nephron number will be instrumental in the development of pharmacological or genetic approaches designed to limit disease associated nephron loss. With good experimental technique and practice, individuals new to this method should readily gain mastery of the acid maceration method allowing reliable and reproducible estimations of whole kidney nephron numbers.Begin by using fine surgical scissors to open the mouse abdominal cavity along the midline. Carefully shift the intestines and reproductive adipose to the right side of the animal. Using gross dissection, isolate the left kidney and cut the left renal artery and vein to carefully remove the kidney placing the organ into an appropriately labeled weigh boat of PBS.Collect the right kidney in the same manner and place both kidneys onto a surgical gauze pre-moistened with PBS. Quickly remove any adherent non-renal tissue and the renal capsules. Then weight each kidney individually recording the weight of the left and right organ separately.After weighing, place each kidney back into an appropriately labeled weigh boat without PBS and use a clean razor blade to cut each organ in half lengthwise. Place each kidney half cut side down and cut each half into two millimeter or smaller pieces. Using the same razor blade, carefully transfer the chopped kidney pieces into an appropriately labeled 15 milliliter conical tube.In a well ventilated fume hood, add five milliliters of six molar hydrochloric acid to each tube. Next, gently agitate the kidney acid mixtures before placing the tubes into a 37 degree Celsius water bath for 90 minutes. At the end of the acid maceration, attach one 18 gauge needle to one five milliliter syringe per kidney and carefully remove the syringe plungers.Place the syringes in individual 50 milliliter conical tubes in the fume hood and pour the kidney solutions into the open end of each syringe setting the empty tubes aside in a test tube rack. Then carefully replace the plungers for the slow extrusion of each solution through the needles into the 50 milliliter tubes. Wash the 15 milliliter conical tubes with five milliliters of PBS solution per tube swirling the PBS to solubilize any remaining kidney tissue.Extrude the wash contents into the appropriate 50 milliliter tube as just demonstrated. After extruding the third wash, equip each syringe with a 21 gauge needle. Extrude the contents of the 50 milliliter tubes into a second set of 50 milliliter tubes through the smaller bore needle washing and extruding the first 50 milliliter tube contents three times with fresh PBS as demonstrated.After the third wash and extrusion, bring the total volume in each tube up to 50 milliliters with additional PBS and place the tubes in a rack on a rocker plate at four degrees Celsius overnight. Within five days of processing, place a grid containing 16 separate sections onto the bottom of six wells of a 12 well plate. Gently invert the tubes of kidney solution several times to resuspend the pelleted tissues.Carefully add three 500 microliter aliquots of each homogenized solution per kidney to each of three wells of the 12 well plate. Dilute the contents of each well with an equal volume of PBS. Place the plate onto the stage of an inverted microscope.Identify the glomeruli by their spherical structure, reddish hue, and the pre or post arterials attached to the bodies of individual glomeruli. Count the number of glomeruli per each gridded section to sum the count per grid for calculation of the total number of glomeruli per well. Then multiply the number of glomeruli per well times 100 to obtain the average number of glomeruli per kidney.In this representative experiment, the total nephron number in mice infused with vehicle for 14 days was about 12, 500 nephrons per kidney. Angiotensin two infusion however, increased the atrial pressure by approximately 40 millimeters of mercury and was associated with a significant nearly 26%reduction in the total nephron number. In this genetic model of rat renal agenesis, animals born with two kidneys were calculated to contain about 27, 500 nephrons per kidney after acid maceration as demonstrated, while rats born with a solitary kidney were found to have significantly lower total nephron numbers.Estimates from both experiments were consistent with the ranges previously reported in rats. The acid maceration method is ideal for estimating nephron numbers in whole kidneys, especially in laboratories not equipped with more labor intensive and cost prohibitive techniques for counting nephrons, such as the disector fractionator method or magnetic resonance imaging. This method allows a high throughput, efficient, and reproducible estimation of whole kidney nephron number that are within 5%of the numbers determined by magnetic resonance imaging.The assessment of nephron number is of clinical relevance as reduced nephron numbers have been associated with an increased risk of cardiovascular disease, such as chronic kidney disease. Don't forget that working with hydrochloric acid can be extremely hazardous and that precautions should be taken, such as wearing gloves, protective eyewear, and a laboratory coat.

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