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>

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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 reagents and consumables required to perform the acid maceration method are readily available in most basic laboratories, the only major requirement being access to an inverted microscope for glomerular counting. In terms of expense, it is estimated that the acid maceration method costs just a couple of hundred dollars per animal, which is not a lot when compared to costs associated with other methods of whole kidney glomerular counting, such as magnetic resonance imaging, which can range into thousands of dollars per animal of scanning time and technical expertise.
Second, the acid maceration method involves significantly less tissue-processing time compared to other methods of whole kidney nephron number such as the dissector/fractionator method24. From start to finish, the acid maceration method requires less than 24 hours of total time, of which less than 1 - 2 hours is spent processing kidneys and then counting the individual glomeruli per experimental animal. In contrast, the dissector/fractionator method is labor-intensive, requiring an estimated 15 hours of labor (4 - 6 hours of sectioning, 2 - 3 hours of staining, and 4 - 5 hours of counting time) per kidney, not including the 48 - 72 hours needed to process the kidneys in glycol methacrylate24. Because of the advantages of cost and time, the acid maceration method is especially useful in studies where estimates of whole kidney nephron number need to be made in large numbers of animals with or without genetic or pharmacological interventions. Both the cost and time involved with the dissector/fractionator method and magnetic resonance imaging have been considered a major limiting factor in their adoption in most research laboratories.
Finally, the acid maceration method provides estimates of whole kidney nephron number that are comparable to measures using more sophisticated methods such as magnetic resonance imaging26. For example, using cationic ferritin-labeling of glomeruli and three-dimensional image processing to count every nephron in a single kidney from the Sprague-Dawley rat, magnetic resonance imaging yielded on average counts of 34,000 individual glomeruli per kidney26. In non-cationic ferritin-labeled kidneys, magnetic resonance imaging yielded counts of 2,000 nephron-like structures due to the counting of non-glomerular regions of kidney with magnetic signals of similar magnitude and shape as ferritin-labeled glomeruli. When these artifacts are subtracted from the counts determined by cationic ferritin-labeled kidneys, the effective number of glomeruli determined, using magnetic resonance imaging, is closer to 32,000 nephrons per kidney26. In this same study, validation experiments yielded average glomerular counts of 31,000 per kidney using the acid maceration method26. Thus, the acid maceration method produces estimates of whole kidney nephron number that are within &#60; 5% of those determined using state-of-the-art techniques such as magnetic resonance imaging.
Although there are several key advantages associated with the acid maceration method, investigators should also be aware of the limitations associated with this method. One limitation relates to the use of whole kidney. As the whole kidney is dissolved and homogenized, information regarding the spatial distribution of glomeruli within the kidney cortex cannot be determined. If knowing the intrarenal distribution of glomerular volume is important, then the use of magnetic resonance imaging would be a better-suited method.
Compared to other methods, the use of the acid maceration method appears to slightly underestimate total nephron number. The smaller estimates are likely due, in part, to the dissolution of a small percentage of glomeruli in the acid or during the maceration of the kidney. In addition, with aging or disease, there is a loss of functional nephrons, which, though not contributing to urine formation, may be more sensitive to acid maceration and may be destroyed during processing, thus contributing to lower estimates of total nephron number. Thus, care should be used when collecting and processing kidneys so as not to leave any kidney tissue in vivo when chopping the kidney, or in the syringes when extruding tissue samples. Overall, however, the underestimation of total nephron number due to tissue processing appears to be relatively small.
As the counting of glomeruli using the acid maceration method is subjective, the underestimation of nephron number may also be reflective of observer bias, which can be easily overcome by measurements being performed by two separate investigators blinded to information regarding experimental animal or condition. If there is a significant concern as to what constitutes a glomerulus, an alternative approach would involve infusing experimental animals with iron oxide before euthanasia. When infused intravenously, iron oxide will enter and become trapped in the glomerulus; thus, processed glomeruli should stain dark black, allowing for a greater identification of glomeruli when counting30.
The acid maceration method estimates whole kidney nephron number based on the measurement of small samples (3 x 0.5 mL or 1.5 mL) as opposed to measurements of the entire solution of kidney (50 mL). The acid maceration method is highly reproducible in nature, and glomeruli counts within samples of a single kidney and within experimental groups are found to be highly consistent. Moreover, representative results reported in the present study from wild-type C57Bl/6 mice and rats born with a single kidney (Figure&#160;2 and 3) are highly consistent with previous findings, as well as with ranges previously reported (Table 1)5,31. If desired, additional measurements could be easily made to better estimate the total nephron number per kidney.
While the acid maceration technique provides reliable and repeatable estimates of nephron number, this method is not recommended for obtaining measurements of glomerular volume, as exposure to acid most likely produces alterations in glomeruli volume. As the determination of glomerular volume is important in determining the whole kidney glomerular surface area, it is recommended that such measurements be made using histological and stereological methods24,25.&#160;Particularly, stereological methods that make use of glycol methacrylate embed kidneys in order to limit tissue swelling and expansion and, thus, maintain glomerular geometry as close as possible to that in vivo.
Finally, another limitation of the acid maceration technique, and one that is common to other methods of assessing nephron number, is that no information regarding glomerular function (such as glomerular filtration rate) can be ascertained from glomeruli once processed. Thus, while individual glomeruli can be counted, its functional status (urine-forming or non-urine-forming) cannot be determined using the acid maceration method. Similarly, no longitudinal information can be ascertained using the acid maceration method in individual mice. Instead, separate groups of animals at various time points are required to gain insight into changes in nephron number over an animal&#39;s lifespan or pre- or postgenetic or pharmacological intervention.
In summary, the acid maceration method is a low-cost, high-throughput, and efficient method to estimate whole kidney nephron number. The acid maceration method provides a high degree of reproducibility as evidenced by the two examples presented here, as well as by those previously reported using this method18,19,20,21,22,28. While the use of the acid maceration method was described for use in the mouse (and rat), the acid maceration method can also be scaled for use in larger species, such as the dog and pig27,32.

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><tbody><tr><td>Isoflurane anesthesia</td><td>Abbott Laboratories</td><td>05260-05</td><td></td></tr><tr><td>Isoflurane vaporizor system &amp;amp; flow gauge</td><td>Braintree Scientific</td><td>VP I</td><td>Include medical grade oxygen supply</td></tr><tr><td>Leica Inverted Microscope DMIL LED</td><td>Leica Microsystems</td><td>DMIL LED</td><td>Any make also suitable</td></tr><tr><td>Digital water bath</td><td>Fisher Scientific</td><td>2239</td><td>Any make also suitable</td></tr><tr><td>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><td>Micro dissecting forceps 4 1/4 in.</td><td>Biomed Res Instruments, Inc</td><td>10-1760</td><td>Curved tip</td></tr><tr><td>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><td>Hexagonal polystyrene weighing dish</td><td>Fisher Scientific</td><td>02-2002-100</td><td>Any make also suitable</td></tr><tr><td>Razor blades</td><td>Fisher Scientific</td><td>12-640</td><td>Single edge carbon steel 0.009</td></tr><tr><td>Gauze sponges 4 x 4 in. 8 ply</td><td>Fisher Scientific</td><td>MSD-1400250</td><td></td></tr><tr><td>10x concentrate phosphate buffered saline (PBS)</td><td>Sigma Aldrich</td><td>P5493-4L</td><td>Dilute to 1x&amp;nbsp;</td></tr><tr><td>6 N Hydrocholric acid solution</td><td>Sigma Aldrich</td><td>3750-32</td><td></td></tr><tr><td>15 mL conical centrifuge tube</td><td>Fisher Scientific</td><td>14-959-70C</td><td>Any make also suitable</td></tr><tr><td>50 mL conical centrifuge tube</td><td>Fisher Scientific</td><td>14-959-49A</td><td>Any make also suitable</td></tr><tr><td>Disposable 5 mL syringe</td><td>Cole Palmer</td><td>EW-07944-06</td><td>Any make also suitable</td></tr><tr><td>18G1.5 disposable needle</td><td>Fisher Scientific</td><td>14-826-5D</td><td>Any make also suitable</td></tr><tr><td>21G1.5 disposable needle</td><td>Fisher Scientific</td><td>14-826-5B</td><td>Any make also suitable</td></tr><tr><td>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><td>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 the first experimental example, total nephron numbers were determined in mice (male C57BL/6, 6 weeks of age) infused with vehicle (saline) or angiotensin II for 14 days. In vehicle-infused mice, nephron number was 12,411 &#177; 248 nephrons per kidney as determined using the acid maceration method (Figure 2). These estimates are consistent with ranges of whole kidney nephron number reported previously in mice (Table 1). Angiotensin increased atrial pressure by approximately 40 mmHg following 14 days of infusion and was associated with a significant reduction in nephron number (9,122 &#177; 193 nephrons per kidney) of nearly 26% (Figure 2). These data suggest that angiotensin II infusion is not only associated with hypertension but, also, a significant reduction in nephron number is associated with nephron loss due to sclerosis or glomerular injury.
In the second experimental example, previous findings of nephron number in a genetic rat model of renal agenesis, the heterogeneous stock-derived model of unilateral renal agenesis (HSRA) rat, were confirmed. The HSRA model exhibits incomplete penetrance phenotypes for defects of the kidney and urinary tract, with some animals being born normal (with two kidneys) and others born with one kidney. In rats born with two kidneys (male hA-Control; 12 weeks of age), estimates of nephron number using the acid maceration method were 27,288 &#177; 336 nephrons per kidney (Figure 3). In contrast, in rats born with a solitary kidney (male hA-Solitary; 4 weeks of age) estimates of nephron number were found to be significantly lower at 24,594 &#177; 883 nephrons per kidney (Figure 3). Estimates of nephron number attained in these samples are also consistent with ranges previously reported in the rat (Table 1). Note that these data are consistent with previous findings in which hA-Solitary rats were found to exhibit decreased nephron endowment or numbers (compared to one control kidney) most likely reflective of the underlying genetic factors associated with being born with a single kidney31.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58599/58599fig1.jpg" /> 
Figure 1:&#160;Key identifiers of glomeruli for counting and assessing whole kidney nephron number. Glomeruli are readily identifiable by their spherical structure, as indicated by the arrows. Additional identifiers include a reddish hue due to blood-filled capillaries, as well as pre- or post-arterioles (or tubules) that may remain attached to the body of individual glomeruli following processing (like the one identified at the bottom of this micrograph). Also present in this image are remnants of tubules and blood vessels. Magnification = 10X; scale bar = 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58599/58599fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58599/58599fig2.jpg" /> 
Figure 2: Nephron counting in mice. (A) Representative image of glomeruli from a single mouse kidney plated in a 22.1-mm flat-bottomed well of a 12-well culture plate, as seen using an inverted microscope. Glomeruli and renal tubules are seen at moderate density when diluted by a factor of two (0.5 mL of kidney solution plus 0.5 mL of 1x PBS). (B) Effect of a 14-day vehicle or angiotensin II infusion on nephron number in male C57Bl/6 mice as assessed by glomerular counting. The angiotensin II infusion at 1,000 ng/kg/min (via osmotic minipump) was associated with hypertension and a marked a reduction in nephron number as compared to the nephron number in mice infused with vehicle. Nephron numbers in vehicle-infused mice are consistent with previously reported estimates of nephron number in mice (Table 1). Vehicle: n = 3, angiotensin II: n = 3, *p &#60; 0.05. Error bars = SE. Magnification = 4X; scale bar = 250 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58599/58599fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58599/58599fig3.jpg" /> 
Figure 3: Nephron counting in rats. (A) Representative image of glomeruli from a single rat kidney plated in a 22.1-mm flat-bottomed well of a 12-well culture plate, as seen using an inverted microscope. Glomeruli and renal tubules are seen at a significantly higher density as compared to that in the mouse. (B) Quantification of the nephron number in HSRA-Control and HSRA-S rats reveals that nephron number is less in HSRA-S as compared to HSRA-C rats and consistent with previous findings in this model31. HSRA-C, rats born with two kidneys: n = 3; HSRA-S, rats born with a solitary kidney: n = 3,*p &#60;0.05. Error bars = SE. Magnification = 4X; scale bar = 250 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58599/58599fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Species</td>
			<td>Nephron Number per Kidney</td>
			<td>Reference</td>
		</tr>
		<tr>
			<td>Mouse</td>
			<td>9,000-21,000&#160;</td>
			<td>18-22,28</td>
		</tr>
		<tr>
			<td>Rat</td>
			<td>13,000-27,000</td>
			<td>31,33</td>
		</tr>
		<tr>
			<td>Sheep</td>
			<td>200,000-800,000</td>
			<td>23,29,34</td>
		</tr>
		<tr>
			<td>Pig</td>
			<td>1,600,000-4,600,000</td>
			<td>32,35</td>
		</tr>
		<tr>
			<td>Human</td>
			<td>500,000-2,000,000</td>
			<td>8-15</td>
		</tr>
	</tbody>
</table>
Table 1: Reported ranges of nephron number in several species.

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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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10.0K Views.  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.

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

Quantifying Glomerular Permeability of Fluorescent Macromolecules Using 2-Photon Microscopy in Munich Wistar Rats

Kidney diseases involving urinary loss of large essential macromolecules, such as serum albumin, have long been thought to be caused by alterations in the permeability barrier comprised of podocytes, vascular endothelial cells, and a basement membrane working in unison. Data from our laboratory using intravital 2-photon microscopy revealed a more permeable glomerular filtration barrier (GFB) than previously thought under physiologic conditions, with retrieval of filtered albumin occurring in an early subset of cells called proximal tubule cells (PTC)1,2,3.
Previous techniques used to study renal filtration and establishing the characteristic of the filtration barrier involved micropuncture of the lumen of these early tubular segments with sampling of the fluid content and analysis4. These studies determined albumin concentration in the luminal fluid to be virtually non-existent; corresponding closely to what is normally detected in the urine. However, characterization of dextran polymers with defined sizes by this technique revealed those of a size similar to serum albumin had higher levels in the tubular lumen and urine; suggesting increased permeability5.
Herein is a detailed outline of the technique used to directly visualize and quantify glomerular fluorescent albumin permeability in vivo. This method allows for detection of filtered albumin across the filtration barrier into Bowman's space (the initial chamber of urinary filtration); and also allows quantification of albumin reabsorption by proximal tubules and visualization of subsequent albumin transcytosis6. The absence of fluorescent albumin along later tubular segments en route to the bladder highlights the efficiency of the retrieval pathway in the earlier proximal tubule segments. Moreover, when this technique was applied to determine permeability of dextrans having a similar size to albumin virtually identical permeability values were reported2. These observations directly support the need to expand the focus of many proteinuric renal diseases to included alterations in proximal tubule cell reclamation.

A technique utilizing high resolution intavital 2-photon microscopy to directly visualize and quantify gloemrular filtration in surface glomeruli. This method allows for direct determination of permeability characteristics of macromolecules in both normal and diseased states.

Kidney  diseases involving urinary loss of large essential macromolecules, such as  serum albumin, have long been thought to be caused by alterations in ...

Reliable and High Efficiency Extraction of Kidney Immune Cells

Immune system activation occurs in multiple kidney diseases and pathophysiological processes. The immune system consists of both adaptive and innate components and multiple cell types. Sometimes, the cell type of interest is present in very low numbers among the large numbers of total cells isolated from the kidney. Hence, reliable and efficient isolation of kidney mononuclear cell populations is important in order to study the immunological problems associated with kidney diseases. Traditionally, tissue isolation of kidney mononuclear cells have been performed via enzymatic digestions using different varieties and strengths of collagenases/DNAses yielding varying numbers of viable immune cells. Recently, with the development of the mechanical tissue disruptors for single cell isolation, the collagenase digestion step is avoided and replaced by a simple mechanical disruption of the kidneys after extraction from the mouse. Herein, we demonstrate a simple yet efficient method for the isolation of kidney mononuclear cells for every day immune cell extractions. We further demonstrate an example of subset analysis of immune cells in the kidney. Importantly, this technique can be adapted to other soft and non-fibrous tissues such as the liver and brain.

Techniques that are reliable and efficient for the isolation of kidney immune cells are needed for downstream applications. This requires surface antibody labeling of a small number of kidney immune cells. Herein, we describe a concise method for isolation of kidney immune cells that seemingly achieves this goal.

Immune system activation occurs in multiple kidney diseases and pathophysiological processes.  The immune system consists of both adaptive and innate ...

Immunology and Infection

Assessment of Kidney Function in Mouse Models of Glomerular Disease

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function in diabetic nephropathy and podocyte-specific VEGF-A knock-out mice; therefore, this protocol describes the full kidney work-up used in our lab to assess these mouse models of glomerular disease, enabling a vast amount of information regarding kidney and glomerular function to be obtained from a single mouse. In comparison to alternative methods presented in the literature to assess glomerular function, the use of the method outlined in this paper enables the glomerular phenotype to be fully evaluated from multiple aspects. By using this method, the researcher can determine the kidney phenotype of the model and assess the mechanism as to why the phenotype develops. This vital information on the mechanism of disease is required when examining potential therapeutic avenues in these models. The methods allow for detailed functional assessment of the glomerular filtration barrier through measurement of the urinary albumin creatinine ratio and individual glomerular water permeability, as well as both structural and ultra-structural examination using the Periodic Acid Schiff stain and electron microscopy. Furthermore, analysis of the genes dysregulated at the mRNA and protein level enables mechanistic analysis of glomerular function. This protocol outlines the generic but adaptable methods that can be applied to all mouse models of glomerular disease.

This protocol describes a full kidney work-up that should be carried out in mouse models of glomerular disease. The methods allow for detailed functional, structural, and mechanistic analysis of glomerular function, which can be applied to all mouse models of glomerular disease.

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function ...

An Efficient Sieving Method to Isolate Intact Glomeruli from Adult Rat Kidney

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by proteinuria which can eventually lead to chronic and end-stage renal disease. The glomerulus is a complex apparatus responsible for the filtration of plasma from the body. In disease, structural integrity is lost and allows for the abnormal leakage of plasma contents into the urine. A method to isolate and examine glomeruli in culture is critical for the study of these diseases. In this protocol, an efficient method of retrieving intact glomeruli from adult rat kidneys while conserving structural and morphological characteristics is described. This process is capable of generating high yields of glomeruli per kidney with minimal contamination from other nephron segments. With these glomeruli, injury conditions can be mimicked by incubating them with a variety of chemical toxins, including protamine sulfate, which causes foot process effacement and proteinuria in animal models. Degree of injury can be assessed using transmission electron microscopy, immunofluorescence staining, and western blotting. Nephrin and Wilms Tumor 1 (WT1) levels can also be assessed from these cultures. Due to the ease and flexibility of this protocol, the isolated glomeruli can be utilized as described or in a way that best suits the needs of the researcher to help better study glomerular health and structure in diseased states.

The main focus of this protocol is to efficiently isolate viable primary glomeruli cultures with minimal contaminants for use in a variety of downstream applications. The isolated glomeruli retain structural relationships between component cell types and can be cultured ex vivo for a short time.

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by ...

Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys are composed of functional units known as nephrons, which are highly conserved with other vertebrates, including mammals. Research in zebrafish has recently demonstrated that two distinctive phenomena transpire after adult nephrons incur damage: first, there is robust regeneration within existing nephrons that replaces the destroyed tubule epithelial cells; second, entirely new nephrons are produced from renal progenitors in a process known as neonephrogenesis. In contrast, humans and other mammals seem to have only a limited ability for nephron epithelial regeneration. To date, the mechanisms responsible for these kidney regeneration phenomena remain poorly understood. Since adult zebrafish kidneys undergo both nephron epithelial regeneration and neonephrogenesis, they provide an outstanding experimental paradigm to study these events. Further, there is a wide range of genetic and pharmacological tools available in the zebrafish model that can be used to delineate the cellular and molecular mechanisms that regulate renal regeneration. One essential aspect of such research is the evaluation of nephron structure and function. This protocol describes a set of labeling techniques that can be used to gauge renal composition and test nephron functionality in the adult zebrafish kidney. Thus, these methods are widely applicable to the future phenotypic characterization of adult zebrafish kidney injury paradigms, which include but are not limited to, nephrotoxicant exposure regimes or genetic methods of targeted cell death such as the nitroreductase mediated cell ablation technique. Further, these methods could be used to study genetic perturbations in adult kidney formation and could also be applied to assess renal status during chronic disease modeling.

The zebrafish adult kidney is an excellent system for renal regeneration and disease studies. An essential aspect of such research is the assessment of nephron structure and function. This protocol describes several methodologies that can be implemented to assess nephron tubule composition and to evaluate renal reabsorption.

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys ...

Biology

Using 2-Photon Microscopy to Quantify the Effects of Chronic Unilateral Ureteral Obstruction on Glomerular Processes

Applying novel microscopy methods to suitable animal disease models to explore the dynamic physiology of the kidney remains a challenge. Rats with surface glomeruli provide a unique opportunity to investigate physiological and pathophysiological processes using intravital 2-photon microscopy. Quantification of glomerular capillary blood flow and vasoconstriction and dilatation in response to drugs, permeability, and inflammation are just some of the processes that can be studied. In addition, transgenic rats, i.e., podocytes labeled with fluorescent dyes and other molecular biomarker approaches, provide increased resolution to directly monitor and quantify protein-protein interactions and the effects of specific molecular alterations.
In mice, which lack surface glomeruli after four weeks of age, unilateral ureteral obstruction (UUO) for several weeks has been used to induce surface glomeruli. As this induction model does not allow for baseline studies, we quantified the effects of UUO on glomerular processes in the UUO model in Munich Wistar Fr&#246;mter (MWF) rats, which have surface glomeruli under physiologic conditions. The UUO model for five weeks or more induced significant alterations to gross renal morphology, the peritubular and glomerular microvasculature, as well as the structure and function of tubular epithelia. Glomerular and peritubular red blood cell (RBC) flow decreased significantly (p &#60; 0.01), probably due to the significant increase in the adherence of white blood cells (WBCs) within glomerular and peritubular capillaries. The glomerular sieving coefficient of albumin increased from 0.015 &#177; 0.002 in untreated MWFs to 0.045 &#177; 0.05 in 5-week-old UUO MWF rats. Twelve weeks of UUO resulted in further increases in surface glomerular density and glomerular sieving coefficient (GSC) for albumin. Fluorescent albumin filtered across the glomeruli was not reabsorbed by the proximal tubules. These data suggest that using UUO to induce surface glomeruli limits the ability to study and interpret normal glomerular processes and disease alterations.

Here, we present a protocol using 2-photon microscopy in Munich Wistar Fromter rats with surface glomeruli to quantifythe effects of prolonged ureteral obstruction on glomerular dynamics and function.

Applying novel microscopy methods to suitable animal disease models to explore the dynamic physiology of the kidney remains a challenge. Rats with surface ...

A Moxibustion Platform for Herbal Cake-Based Treatment of Rats with Renal Failure

Using Herbal-Cake-Separated Moxibustion for the Treatment of Rats with Chronic Renal Failure

In the process of moxibustion in clinical practice, subjects need to be in a stable mood and comfortable posture to avoid problems such as moxa ash falling, scalding skin, and poor curative effect. Such problems also exist in the rat moxibustion experiment. To simulate clinical practice, it is necessary to introduce an experimental instrument in animal experiments, that is, a moxibustion device with fixed rats and moxibustion treatment synchronization, which can make experimental rats receive moxibustion treatment quietly and comfortably under non-anesthesia.
Our research group designed a rat moxibustion experimental platform. The device was framed by a wooden board with a supporting base plate, multiple fixed components, and partitioned components. The device can achieve the operation mode of moxibustion in rats without binding, avoiding anesthesia and scalding and simultaneously exposing multiple acupoints on the back. This operation can avoid physical and mental injury to rats and operators, which improves the research efficiency and further promotes the development and research of moxibustion animal experiments.
The device has a simple structure, is easy to operate and popularize, is comprehensively and innovatively designed, reusable, and is suitable for rat experiments mainly based on moxibustion. This article mainly introduces the structure of the experimental platform device for rat moxibustion, the basic procedure of herbal-cake-separated-moxibustion in experimental rats using the device and describes the establishment of a rat model of chronic renal failure (CRF) and representative experimental results.

Author Spotlight: Understanding the Effect of Herbal-Cake-Separated Moxibustion in Rats with Renal Faliure

The moxibustion experimental platform can fix the rats without restriction, and the rats can undergo multi-acupoint moxibustion in the awake state, ensuring the welfare of the rats, protecting the safety of the operators, ensuring therapeutic efficacy, and improving the efficiency of the experiment.

In the process of moxibustion in clinical practice, subjects need to be in a stable mood and comfortable posture to avoid problems such as moxa ash ...

Traditional Medicine and PI3K/AKT Pathway Activation in Membranous Nephropathy

Mechanism of Kemeng Fang's Inhibition of Podocyte Apoptosis in Rats with Membranous Nephropathy through the PI3K/AKT Signaling Pathway

Membranous nephropathy (MN) is a common pathological type of adult nephrotic syndrome. Up to 20% of patients with MN develop end-stage renal disease (ESRD). Podocytes have an important function in maintaining the glomerular filtration barrier and play a crucial role in the occurrence and development of proteinuria and MN. PI3K/AKT signaling pathway is involved in the entire process of podocyte growth, differentiation, and apoptosis. Kemeng Fang (KMF) is a traditional Chinese medicine formula that has been used to delay kidney injury. However, the therapeutic mechanism of KMF in MN is unclear. Here, the MN rat model was established by axillary, inguinal, and tail vein injections of cationized bovine serum albumin (C-BSA), and then KMF and PI3K inhibitor (LY294002) were administered. The data of liver function, kidney function, blood lipid, renal pathology, podocyte function, expression level of PI3K/AKT signaling pathway, and transcriptomics of rats demonstrated that KMF has a protective effect on the podocytes of MN rats by activating the PI3K/AKT signaling pathway, and it can effectively prevent the progression of MN.

The present protocol describes the establishment of a membranous nephropathy (MN) animal model, and how Kemeng Fang's inhibition reduces MN rat podocyte apoptosis by activating the PI3K/AKT signaling pathway.

Membranous nephropathy (MN) is a common pathological type of adult nephrotic syndrome. Up to 20% of patients with MN develop end-stage renal disease ...

Fluorescent Labeling of Glomeruli in Cleared Mouse Kidneys for Microscopy

An Efficient and Fast Method for Labeling and Analyzing Mouse Glomeruli

The glomeruli are fundamental units in the kidney; hence, studying the glomeruli is pivotal for understanding renal function and pathology. Biological imaging provides intuitive information; thus, it is of great significance to label and observe the glomeruli. However, the glomeruli observation methods currently in use require complicated operations, and the results may lose label details or three-dimensional (3D) information. The clear, unobstructed brain imaging cocktails and computational analysis (CUBIC) tissue clearing technology has been widely used in renal research, allowing for more accurate detection and deeper detection depth. We found that mouse glomeruli can be rapidly and effectively labeled by tail vein injection of medium molecular weight FITC-Dextran followed by the CUBIC clearing method. The cleared mouse kidney could be scanned by a light-sheet microscope (or a confocal microscope when sliced) to obtain three-dimensional image stacks of all the glomeruli in the entire kidney. Processed with appropriate software, the glomeruli signals could be easily digitized and further analyzed to measure the number, volume, and frequency of the glomeruli.

Author Spotlight: Aiding Research in Kidney Biology by Labeling Glomeruli in Cleared Tissues 

This study presents an easy-to-use, complete, and simple set of methods to label and analyze glomeruli from CUBIC-cleared mouse kidneys. Data such as glomerulus number and volume can be obtained easily and reliably using fluorescein isothiocyanate (FITC)-Dextran, light sheet fluorescence microscopy (LSFM), or common confocal microscopy and software such as Imaris.

The glomeruli are fundamental units in the kidney; hence, studying the glomeruli is pivotal for understanding renal function and pathology. Biological ...

A Whole-Mount Organ Staining Technique for Three-Dimensional Imaging of the Kidney

Source: Hasegawa, S., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/63986">Whole-Kidney Three-Dimensional Staining with CUBIC.</a> J. Vis. Exp. (2022).
This video demonstrates an assay for the whole-mount staining of murine kidneys using a tissue-clearing method called CUBIC (clear, unobstructed brain/body imaging cocktails and computational analysis) and immunofluorescent staining. The kidney is subjected to tissue clearing, wherein the kidney is delipidated, decolorized, and treated with a refractive index matching solution to make the tissue transparent for optimum three-dimensional imaging. The tissue is stained with fluorescently labeled antibodies to reveal sympathetic nerves and arteries in the tissue under a light-sheet microscope.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Animal ...