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
<ol>
	<li>Weigh the mouse (or other species) and euthanize it with an isoflurane (5% - 8%) overdose or pentobarbital (150 mg/kg intraperitoneal injection).</li>
	<li>Once the mouse is euthanized, open its abdominal cavity using fine surgical scissors along the midline.</li>
	<li>Carefully lift the intestines and reproductive adipose to the right side of the abdominal cavity. By gross dissection, isolate the left kidney. Using fine surgical scissors, cut the left renal artery and vein and carefully remove the left kidney, placing the kidney into an appropriately labeled (mouse number/identifier) weigh boat containing phosphor-buffered saline (PBS).</li>
	<li>Repeat the procedure for the right kidney.</li>
	<li>Remove each kidney from its respective weigh boat and place it onto a surgical gauze pre-moistened with PBS.</li>
	<li>Leaving the kidney on the surgical gauze, quickly remove any adherent non-renal tissue (such as perirenal adipose or adrenal gland) followed by the removal of the renal capsule. Weigh each kidney individually, recording the weight of the left and right kidney separately in a laboratory notebook.</li>
</ol>
2. Homogenization, Incubation, and Straining Procedures
<ol>
	<li>Once each kidney is weighed, drain each weigh boat of PBS and place the kidneys back into the appropriately labeled weigh boat. Using a clean razor blade, cut the kidney in half, lengthwise. Place each kidney half facing down and cut each half into 2-mm or smaller pieces.</li>
	<li>Using the same razorblade, carefully collect and place the chopped kidney pieces into a labeled 15-mL conical tube (mouse number/identifier; left versus right kidney).</li>
	<li>Repeat the procedure for the opposite kidney, using a new razor blade. Place the chopped kidney into a separately labeled 15-mL conical tube.</li>
	<li>In a well-ventilated fume hood, add 5 mL of 6 M hydrochloric (HCl) acid to each 15-mL conical tube.</li>
	<li>Replace the cap to the conical tube, gently agitate the kidney/HCl mixture, and place the 15-mL conical tube into a preheated water bath set at 37 &#176;C for 90 min (*120 min for rat kidneys).</li>
	<li>Briefly agitate each 15-mL tube every 15 min during the incubation in order to ensure that all tissue is exposed to HCl acid.</li>
	<li>Insert an 18-G needle into a 5-mL syringe (*10-mL syringe for rat) and carefully remove the syringe plunger. Place the syringe in a 50-mL conical tube (tube #1) in a fume hood.</li>
	<li>Remove the kidney/HCl solution from the water bath and pour the tissue solution into the open end of the syringe and set the 15-mL conical tube aside in a test tube rack. Carefully replace the plunger and slowly push the plunger so as to extrude the solution through the needle and into tube #1.</li>
	<li>Wash the 15-mL conical tube with 5 mL of PBS solution. Swirl the PBS in the 15-mL conical tube so as to solubilize any remaining kidney tissue.</li>
	<li>Again, carefully remove the plunger from the 5-mL syringe containing the 18-G needle and pour the contents from the 15-mL conical tube into the open end of the syringe. Carefully replace the plunger and flush the syringe by gently pushing down on the plunger, into tube #1. Repeat this process 2x (performed 3x in total).</li>
	<li>Insert a 21-G needle into a new 5-mL syringe (*10-mL syringe for rat) and carefully remove the syringe plunger. Place the syringe with the 21-G needle attached into a new 50-mL conical tube (tube #2).</li>
	<li>Pour the contents from tube #1 into the open end of the syringe containing the 21-G needle. Carefully insert the plunger and flush the syringe by gently pushing down on the syringe plunger and placing the extruded solution into tube #2.</li>
	<li>Wash tube #1 with 5 mL of PBS solution. Swirl the PBS in tube #1 so as to solubilize any remaining kidney tissue.</li>
	<li>Again, carefully remove the plunger from the 5-mL syringe containing the 18-G needle and pour the contents from tube #1 into the open end of the syringe. Carefully replace the plunger and flush the syringe by gently pushing down on the plunger, extruding solution into tube #2. Repeat this process 2x (performed 3x in total).</li>
	<li>Bring the total volume of tube #2 up to 50 mL by adding additional PBS, up to the 50-mL line on tube #2.</li>
	<li>Incubate tube #2 containing the kidney tissue solution in a tube rack on a rocker plate in a refrigerator set at 4 &#176;C overnight (minimum 8 - 10 h).</li>
</ol>
3. Counting of Glomeruli and Extrapolation of the Nephron Number
<ol>
	<li>Remove tube #2 from the refrigerator and resuspend the pelleted tissue by gently inverting the tube several times in order to create a homogenous solution. We recommend counting glomeruli within 5 d after processing.</li>
	<li>Carefully aliquot 500 &#181;L of the kidney solution into a single well of a 12-well plate. Repeat this 2x, placing each aliquot into a separate well so that there are three wells of kidney solution per kidney, for analysis in triplicate.</li>
	<li>Add 500 &#181;L of PBS to each of the three wells containing the kidney solution, for a 1:1 dilution.</li>
	<li>Using an inverted microscope, count the number of glomeruli per well. Counting is aided by using a grid of 16 separate sections placed on the bottom of each well. Count the number of glomeruli per each gridded section and then sum the count per grid to get the total number of glomeruli per well. Glomeruli are readily identifiable by their spherical structure. Additional identifiers included a reddish hue due to blood-filled capillaries, as well as pre- or post-arterioles that remain attached to the body of individual glomeruli (Figure 1).</li>
	<li>Add the total number of glomeruli counted per each of the three wells and then divide them by three for the average number of glomeruli per 500 &#181;L of kidney solution. If the variance in the average number of glomeruli per well is greater than 10%, repeat the nephron-counting procedure, paying close attention to the homogeneous nature of the kidney solution. Multiply the number of glomeruli counted per well times 100 for the average number of glomeruli per kidney. Total nephron number can be expressed per kidney or, using kidney weight, per mg or g of tissue.</li>
</ol>

<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. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fewer+nephrons+at+birth:+a+missing+link+in+the+etiology+of+essential+hypertension?.">Fewer nephrons at birth: a missing link in the etiology of essential hypertension?.</a> American Journal of Kidney Diseases. 26 (1), 91-98 (1995).</li><li>Nyengaard, J. R., Bendtsen, T. F. <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+in+relation+to+age,+kidney+weight,+and+body+surface+in+normal+man.">Glomerular number and size in relation to age, kidney weight, and body surface in normal man.</a> The Anatomical Record. 232 (2), 194-201 (1992).</li><li>Denic, A., Glassock, R. J., Rule, A. 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E., 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+number,+glomerular+volume,+renal+disease+and+hypertension.">Nephron number, glomerular volume, renal disease and hypertension.</a> Current Opinion in Nephrology and Hypertension. 17 (3), 258-265 (2008).</li><li>Hoy, W. E., 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=Distribution+of+volumes+of+individual+glomeruli+in+kidneys+at+autopsy:+association+with+age,+nephron+number,+birth+weight+and+body+mass+index.">Distribution of volumes of individual glomeruli in kidneys at autopsy: association with age, nephron number, birth weight and body mass index.</a> Clinical Nephrology. 74 (Suppl 1), S105-S122 (2010).</li><li>Hughson, M. 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. 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P., 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=Number+of+glomeruli+in+normal+and+hypertrophied+kidneys+of+mice+and+guinea-pigs.">Number of glomeruli in normal and hypertrophied kidneys of mice and guinea-pigs.</a> The Journal of Physiology. 269 (3), 627-641 (1977).</li><li>Bains, R. K., Sibbons, P. D., Murray, R. D., Howard, C. V., Van Velzen, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stereological+estimation+of+the+absolute+number+of+glomeruli+in+the+kidneys+of+lambs.">Stereological estimation of the absolute number of glomeruli in the kidneys of lambs.</a> Research in Veterinary Science. 60 (2), 122-125 (1996).</li><li>Assmann, K. J., van Son, J. P., Koene, R. 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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The authors have nothing to disclose.

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.

<table border="0" cellpadding="0" cellspacing="0" style="width:873px;" width="872">
	<tbody>
		<tr height="21">
			<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>
</table>

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).
In th...

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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 ...

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9.9K 조회수.  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.

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

A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize their blood glucose levels, islet transplantation has been proposed to be a potential treatment for type 1 diabetes 1,2. More recently, advances in human islet transplantation have further strengthened this view 1,3. However, two major limitations prevent islet transplantation from being a widespread clinical reality: (a) the requirement for large numbers of islets per patient, which severely reduces the number of potential recipients, and (b) the need for heavy immunosuppression, which significantly affects the pediatric population of patients due to their vulnerability to long-term immunosuppression. Strategies that can overcome these limitations have the potential to enhance the therapeutic utility of islet transplantation.
Islet transplantation under the mouse kidney capsule is a widely accepted model to investigate various strategies to improve islet transplantation. This experiment requires the isolation of high quality islets and implantation of islets to the diabetic recipients. Both procedures require surgical steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol. We also briefly discuss different transplantation models: syngeneic, allogeneic, syngeneic autoimmune, and allogeneic autoimmune.

Transplantation of isolated islets has been proposed to be a potential treatment for type 1 diabetes. Here we describe a method to isolate islets from mouse pancreata and transplant them to the subcapsular space of the kidney.

Murine 아일릿 절연 및 Subcapsular 신장 이식을위한 방법

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize ...

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The &alpha;-test: Rapid Cell-free CD4 Enumeration Using Whole Saliva

There is an urgent need for affordable CD4 enumeration to monitor HIV disease. CD4 enumeration is out of reach in resource-limited regions due to the time and temperature restrictions, technical sophistication, and cost of reagents, in particular monoclonal antibodies to measure CD4 on blood cells, the only currently acceptable method. A commonly used cost-saving and time-saving laboratory strategy is to calculate, rather than measure certain blood values. For example, LDL levels are calculated using the measured levels of total cholesterol, HDL, and triglycerides1. Thus, identification of cell-free correlates that directly regulate the number of CD4+ T cells could provide an accurate method for calculating CD4 counts due to the physiological relevance of the correlates.
The number of stem cells that enter blood and are destined to become circulating CD4+ T cells is determined by the chemokine CXCL12 and its receptor CXCR4 due to their influence on locomotion2. The process of stem cell locomotion into blood is additionally regulated by cell surface human leukocyte elastase (HLECS) and the HLECS-reactive active &alpha;1proteinase inhibitor (&alpha;1PI, &alpha;1antitrypsin, SerpinA1)3. In HIV-1 disease, &alpha;1PI is inactivated due to disease processes 4. In the early asymptomatic categories of HIV-1 disease, active &alpha;1PI was found to be below normal in 100% of untreated HIV-1 patients (median=12 &mu;M, and to achieve normal levels during the symptomatic categories4, 5. This pattern has been attributed to immune inactivation, not to insufficient synthesis, proteolytic inactivation, or oxygenation. We observed that in HIV-1 subjects with &gt;220 CD4 cells/&mu;l, CD4 counts were correlated with serum levels of active &alpha;1PI (r2=0.93, p&lt;0.0001, n=26) and inactive &alpha;1PI (r2=0.91, p&lt;0.0001, n=26) 5. Administration of &alpha;1PI to HIV-1 infected and uninfected subjects resulted in dramatic increases in CD4 counts suggesting &alpha;1PI participates in regulating the number of CD4+ T cells in blood 3.
With stimulation, whole saliva contains sufficient serous exudate (plasma containing proteinaceous material that passes through blood vessel walls into saliva) to allow measurement of active &alpha;1PI and the correlation of this measurement is evidence that it is an accurate method for calculating CD4 counts. Briefly, sialogogues such as chewing gum or citric acid stimulate the exudation of serum into whole mouth saliva. After stimulating serum exudation, the activity of serum &alpha;1PI in saliva is measured by its capacity to inhibit elastase activity. Porcine pancreatic elastase (PPE) is a readily available inexpensive source of elastase. PPE binds to &alpha;1PI forming a one-to-one complex that prevents PPE from cleaving its specific substrates, one of which is the colorimetric peptide, succinyl-L-Ala-L-Ala-L-Ala-p-nitroanilide (SA3NA). Incubating saliva with a saturating concentration of PPE for 10 min at room temperature allows the binding of PPE to all the active &alpha;1PI in saliva. The resulting inhibition of PPE by active &alpha;1PI can be measured by adding the PPE substrate SA3NA. (Figure 1). Although CD4 counts are measured in terms of blood volume (CD4 cells/&mu;l), the concentration of &alpha;1PI in saliva is related to the concentration of serum in saliva, not to volume of saliva since volume can vary considerably during the day and person to person6. However, virtually all the protein in saliva is due to serum content, and the protein content of saliva is measurable7. Thus, active &alpha;1PI in saliva is calculated as a ratio to saliva protein content and is termed the &alpha;1PI Index. Results presented herein demonstrate that the &alpha;1PI Index provides an accurate and precise physiologic method for calculating CD4 counts.

The &amp;#945;-test: Rapid Cell-free CD4 Enumeration Using Whole Saliva

A CD4 enumeration method, the &#x03B1;-test, is described which uses whole saliva to provide rapid and accurate CD4 counts. The &alpha;-test costs pennies and eliminates the need for technical training, costly reagents such as monoclonal antibodies, instrumentation, refrigeration, transport of samples, as well as collection and handling of blood.

α-테스트 : 전체 타액을 사용하여 빠른 셀 프리 CD4의 열거

There is an urgent need for affordable CD4 enumeration to monitor HIV disease. CD4 enumeration is out of reach in resource-limited regions due to the time ...

Mouse Kidney Transplantation: Models of Allograft Rejection

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely replicates both the technical and pathological processes that occur in human renal transplantation. Although mouse models of allogeneic rejection in organs other than the kidney exist, and are more technically feasible, there is evidence that different organs elicit disparate rejection modes and dynamics, for instance the time course of rejection in cardiac and renal allograft differs significantly in certain strain combinations. This model is an attractive tool for many reasons despite its technical challenges. As inbred mouse strain haplotypes are well characterized it is possible to choose donor and recipient combinations to model acute allograft rejection by transplanting across MHC class I and II loci. Conversely by transplanting between strains with similar haplotypes a chronic process can be elicited were the allograft kidney develops interstitial fibrosis and tubular atrophy. We have modified the surgical technique to reduce operating time and improve ease of surgery, however a learning curve still needs to be overcome in order to faithfully replicate the model. This study will provide key points in the surgical procedure and aid the process of establishing this technique.

Here, we present a protocol to study the immunology of rejection. The surgical model presented reports a short operating time and a concise technique. Depending on the donor-recipient strain combination, the transplanted kidney may develop acute cellular rejection or chronic allograft damage, defined by interstitial fibrosis and tubular atrophy.

마우스 신장 이식 : 동종 이식 거부의 모델

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely ...

Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation

Kidney transplantation has become a well-established treatment option for patients with end-stage renal failure. The persisting organ shortage remains a serious problem. Therefore, the acceptance criteria for organ donors have been extended leading to the usage of marginal kidney grafts. These marginal organs tolerate cold storage poorly resulting in increased preservation injury and higher rates of delayed graft function. To overcome the limitations of cold storage, extensive research is focused on alternative normothermic preservation methods.
Ex vivo normothermic organ perfusion is an innovative preservation technique. The first experimental and clinical trials for ex vivo lung, liver, and kidney perfusions demonstrated favorable outcomes.
In addition to the reduction of cold ischemic injury, the method of normothermic kidney storage offers the opportunity for organ assessment and repair. This manuscript provides information about kidney retrieval, organ preservation techniques, and isolated ex vivo normothermic kidney perfusion (NEVKP) in a porcine model. Surgical techniques, set up for the perfusion solution and the circuit, potential assessment options, and representative results are demonstrated.

Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation

The severe organ shortage has resulted in increased use of marginal kidney grafts for transplantation. This has triggered interest in alternative storage methods, since marginal grafts especially tolerate cold storage poorly. The technique of normothermic ex vivo kidney perfusion (NEVKP) represents a novel preservation method for kidney grafts prior to &#160;transplantation.

정상 체온<em&gt; 전의 VIVO</em&gt; 신장의 보존을위한 신장 관류는 이식 전에 이식

Kidney transplantation has become a well-established treatment option for patients with end-stage renal failure. The persisting organ shortage remains a ...

Intravascular Delivery of Biologics to the Rat Kidney

The renal microvascular compartment plays an important role in the progression of kidney disease and hypertension, leading to the development of End Stage Renal Disease with high risk of death for cardiovascular events. Moreover, recent clinical studies have shown that renovascular structure and function may have a great impact on functional renal recovery after surgery. Here, we describe a protocol for the delivery of drugs into the renal artery of rats. This procedure offers significant advantages over the frequently used systemic administration as it may allow a more localized therapeutic effect. In addition, the use of rodents in pharmacodynamic analysis of preclinical studies may be cost effective, paving the way for the design of translational experiments in larger animal models. Using this technique, infusion of rat recombinant Vascular Endothelial Growth Factor (VEGF) protein in rats has induced activation of VEGF signaling as shown by increased expression of FLK1, pAKT/AKT, pERK/ERK. In summary, we established a protocol for the intrarenal delivery of drugs in rats, which is simple and highly reproducible.

The administration of drugs for recovery of kidney function requires control of the localization and distribution of the therapeutic compound. Here, we describe in detail a simple technique for intrarenal delivery of drugs in rats. This procedure may be easily performed with no mortality and high reproducibility.

쥐의 신장에 생물의 혈관 배달

The renal microvascular compartment plays an important role in the progression of kidney disease and hypertension, leading to the development of End Stage ...

A Method for Quantifying Upper Limb Performance in Daily Life Using Accelerometers

A key reason for referral to rehabilitation services after stroke and other neurological conditions is to improve one's ability to function in daily life. It has become important to measure a person's activities in daily life, and not just measure their capacity for activity in the structured environment of a clinic or laboratory. A wearable sensor that is now enabling measurement of daily movement is the accelerometer. Accelerometers are commercially-available devices resembling large wrist watches that can be worn throughout the day. Data from accelerometers can quantify how the limbs are engaged to perform activities in peoples' homes and communities. This report describes a methodology to collect accelerometry data and turn it into clinically-relevant information. First, data are collected by having the participant wear two accelerometers (one on each wrist) for 24 h or longer. The accelerometry data are then downloaded and processed to produce four different variables that describe key aspects of upper limb activity in daily life: hours of use, use ratio, magnitude ratio, and the bilateral magnitude. Density plots can be constructed that visually represent the data from the 24 h wearing period. The variables and their resultant density plots are highly consistent in neurologically-intact, community-dwelling adults. This striking consistency makes them a useful tool for determining if upper limb daily performance is different from normal. This methodology is appropriate for research studies investigating upper limb dysfunction and interventions designed to improve upper limb performance in daily life in people with stroke and other patient populations. Because of its relative simplicity, it may not be long before it is also incorporated in clinical neurorehabilitation practice.

This protocol describes a method to quantify upper limb performance in daily life using wrist-worn accelerometers.

가속도계를 사용하여 일상 생활에서 상지 성능을 정량화하는 방법

A key reason for referral to rehabilitation services after stroke and other neurological conditions is to improve one's ability to function in daily life. ...

A Novel Clinical Grade Isolation Method for Human Kidney Perivascular Stromal Cells

Mesenchymal Stromal Cells (MSCs) are tissue homeostatic and immune modulatory cells that have shown beneficial effects in kidney diseases and transplantation. Perivascular Stromal Cells (PSCs) share characteristics with bone marrow MSCs (bmMSCs). However, they also possess, most likely due to local imprinting, tissue-specific properties and play a role in local tissue homeostasis. This tissue specificity may result in tissue specific repair, also within the human kidney. We previously showed that human kidney PSCs (kPSCs) have enhanced kidney epithelial wound healing whereas bmMSCs did not have this potential. Moreover, kPSCs can ameliorate kidney injury in vivo. Therefore, kPSCs constitute an interesting source for cell therapy, particularly for kidney diseases and renal transplantation. Here we show the detailed isolation and culture method for kPSCs from transplant-grade human kidneys based on whole-organ perfusion of digestive enzymes via the renal artery and enrichment for the perivascular marker NG2. In this way, large cell quantities can be obtained that are suitable for cellular therapy.

Here we present a novel clinical grade isolation and culture method for kidney Perivascular Stromal Cells (kPSCs) based on whole organ perfusion with digestive enzymes and NG2-cell enrichment. With this method, it is possible to acquire sufficient cell numbers for cellular therapy.

인간의 신장 혈관 주위 Stromal 세포에 대 한 새로운 임상 등급 격리 방법

Mesenchymal Stromal Cells (MSCs) are tissue homeostatic and immune modulatory cells that have shown beneficial effects in kidney diseases and ...

Whole Body and Regional Quantification of Active Human Brown Adipose Tissue Using 18F-FDG PET/CT

In endothermic animals, brown adipose tissue (BAT) is activated to produce heat for defending body temperature in response to cold. BAT&#39;s ability to expend energy has made it a potential target for novel therapies to ameliorate obesity and associated metabolic disorders in humans. Though this tissue has been well studied in small animals, BAT&#39;s thermogenic capacity in humans remains largely unknown due to the difficulties of measuring its volume, activity, and distribution. Identifying and quantifying active human BAT is commonly performed using 18F-Fluorodeoxyglucose (18F-FDG) positron emission tomography and computed tomography (PET/CT) scans following cold-exposure or pharmacological activation. Here we describe a detailed image-analysis approach to quantify total-body human BAT from 18F-FDG PET/CT scans using an open-source software. We demonstrate the drawing of user-specified regions of interest to identify metabolically active adipose tissue while avoiding common non-BAT tissues, to measure BAT volume and activity, and to further characterize its anatomical distribution. Although this rigorous approach is time-consuming, we believe it will ultimately provide a foundation to develop future automated BAT quantification algorithms.

Whole Body and Regional Quantification of Active Human Brown Adipose Tissue Using 18F-FDG PET/CT

Using free, open-source software, we have developed an analytical approach to quantify total and regional brown adipose tissue (BAT) volume and metabolic activity of BAT using 18F-FDG PET/CT.

In endothermic animals, brown adipose tissue (BAT) is activated to produce heat for defending body temperature in response to cold. BAT&#39;s ability to ...

A High-Throughput In Situ Method for Estimation of Hepatocyte Nuclear Ploidy in Mice

When the liver is injured, hepatocyte numbers decrease, while cell size, nuclear size and ploidy increase. The expansion of non-parenchymal cells such as cholangiocytes, myofibroblasts, progenitors and inflammatory cells also indicate chronic liver damage, tissue remodeling and disease progression. In this protocol, we describe a simple high-throughput approach for calculating changes in the cellular composition of the liver that are associated with injury, chronic disease and cancer. We show how information extracted from two-dimensional (2D) tissue sections can be used to quantify and calibrate hepatocyte nuclear ploidy within a sample and enable the user to locate specific ploidy subsets within the liver in situ. Our method requires access to fixed/frozen liver material, basic immunocytochemistry reagents and any standard high-content imaging platform. It serves as a powerful alternative to standard flow cytometry techniques, which require disruption of freshly collected tissue, loss of spatial information and potential disaggregation bias.

We present a robust, cost-effective, and flexible method for measuring changes in hepatocyte number and nuclear ploidy within fixed/cryopreserved tissue samples that does not require flow cytometry. Our approach provides a powerful sample-wide signature of liver cytology ideal for tracking the progression of liver injury and disease.

마우스에서 간세포 핵 Ploidy의 추정을 위한 situ 방법에서 높은 처리량

When the liver is injured, hepatocyte numbers decrease, while cell size, nuclear size and ploidy increase. The expansion of non-parenchymal cells such as ...

Preparation of Decellularized Kidney Scaffolds in Rats

Tissue engineering is a cutting-edge discipline in biomedicine. Cell culture techniques can be applied for regeneration of functional tissues and organs to replace diseased or damaged organs. Scaffolds are needed to facilitate the generation of three-dimensional organs or tissues using differentiated stem cells in vivo. In this report, we describe a novel method for developing vascularized scaffolds using decellularized rat kidneys. Eight-week-old Sprague-Dawley rats were used in this study, and heparin was injected into the heart to facilitate flow into the renal vessels, allowing heparin to perfuse into the renal vessels. The abdominal cavity was opened, and the left kidney was collected. The collected kidneys were perfused for 9 h using detergents, such as Triton X-100 and sodium dodecyl sulfate, to decellularize the tissue. Decellularized kidney scaffolds were then gently washed with 1% penicillin/streptomycin and heparin to remove cellular debris and chemical residues. Transplantation of stem cells with the decellularized vascular scaffolds is expected to facilitate the generation of new organs. Thus, the vascularized scaffolds may provide a foundation for tissue engineering of organ grafts in the future.

This protocol introduces a method to develop a scaffold using decellularized rat kidneys. The protocol includes decellularization and recellularization processes to confirm bioavailability. Decellularization is performed using Triton X-100 and sodium dodecyl sulfate.

쥐에 있는 세포탈구형 신장 비계의 준비

Tissue engineering is a cutting-edge discipline in biomedicine. Cell culture techniques can be applied for regeneration of functional tissues and organs ...