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

The acid maceration method can help answer key questions about the kidney and how specific genes contribute to or influence nephron development as well as how specific conditions, such as diabetes or hypertension, affect nephron number. This is important as a total number of nephrons an individuals born with is determined during fetal development and loss of nephrons has been associated with renal pathology. The main advantages of the acid maceration method are that it is faster, more reproducible, inexpensive, and less time consuming than other nephron counting methods, like magnetic resonance imaging.

This technique facilitates the correlation of the impact in nephron number on renal function, which is important to further our understanding of how specific genes influence nephron function and development. The study of genes or factors that influence overall nephron number will be instrumental in the development of pharmacological or genetic approaches designed to limit disease associated nephron loss. With good experimental technique and practice, individuals new to this method should readily gain mastery of the acid maceration method allowing reliable and reproducible estimations of whole kidney nephron numbers.

Begin by using fine surgical scissors to open the mouse abdominal cavity along the midline. Carefully shift the intestines and reproductive adipose to the right side of the animal. Using gross dissection, isolate the left kidney and cut the left renal artery and vein to carefully remove the kidney placing the organ into an appropriately labeled weigh boat of PBS.

Collect the right kidney in the same manner and place both kidneys onto a surgical gauze pre-moistened with PBS. Quickly remove any adherent non-renal tissue and the renal capsules. Then weight each kidney individually recording the weight of the left and right organ separately.

After weighing, place each kidney back into an appropriately labeled weigh boat without PBS and use a clean razor blade to cut each organ in half lengthwise. Place each kidney half cut side down and cut each half into two millimeter or smaller pieces. Using the same razor blade, carefully transfer the chopped kidney pieces into an appropriately labeled 15 milliliter conical tube.

In a well ventilated fume hood, add five milliliters of six molar hydrochloric acid to each tube. Next, gently agitate the kidney acid mixtures before placing the tubes into a 37 degree Celsius water bath for 90 minutes. At the end of the acid maceration, attach one 18 gauge needle to one five milliliter syringe per kidney and carefully remove the syringe plungers.

Place the syringes in individual 50 milliliter conical tubes in the fume hood and pour the kidney solutions into the open end of each syringe setting the empty tubes aside in a test tube rack. Then carefully replace the plungers for the slow extrusion of each solution through the needles into the 50 milliliter tubes. Wash the 15 milliliter conical tubes with five milliliters of PBS solution per tube swirling the PBS to solubilize any remaining kidney tissue.

Extrude the wash contents into the appropriate 50 milliliter tube as just demonstrated. After extruding the third wash, equip each syringe with a 21 gauge needle. Extrude the contents of the 50 milliliter tubes into a second set of 50 milliliter tubes through the smaller bore needle washing and extruding the first 50 milliliter tube contents three times with fresh PBS as demonstrated.

After the third wash and extrusion, bring the total volume in each tube up to 50 milliliters with additional PBS and place the tubes in a rack on a rocker plate at four degrees Celsius overnight. Within five days of processing, place a grid containing 16 separate sections onto the bottom of six wells of a 12 well plate. Gently invert the tubes of kidney solution several times to resuspend the pelleted tissues.

Carefully add three 500 microliter aliquots of each homogenized solution per kidney to each of three wells of the 12 well plate. Dilute the contents of each well with an equal volume of PBS. Place the plate onto the stage of an inverted microscope.

Identify the glomeruli by their spherical structure, reddish hue, and the pre or post arterials attached to the bodies of individual glomeruli. Count the number of glomeruli per each gridded section to sum the count per grid for calculation of the total number of glomeruli per well. Then multiply the number of glomeruli per well times 100 to obtain the average number of glomeruli per kidney.

In this representative experiment, the total nephron number in mice infused with vehicle for 14 days was about 12, 500 nephrons per kidney. Angiotensin two infusion however, increased the atrial pressure by approximately 40 millimeters of mercury and was associated with a significant nearly 26%reduction in the total nephron number. In this genetic model of rat renal agenesis, animals born with two kidneys were calculated to contain about 27, 500 nephrons per kidney after acid maceration as demonstrated, while rats born with a solitary kidney were found to have significantly lower total nephron numbers.

Estimates from both experiments were consistent with the ranges previously reported in rats. The acid maceration method is ideal for estimating nephron numbers in whole kidneys, especially in laboratories not equipped with more labor intensive and cost prohibitive techniques for counting nephrons, such as the disector fractionator method or magnetic resonance imaging. This method allows a high throughput, efficient, and reproducible estimation of whole kidney nephron number that are within 5%of the numbers determined by magnetic resonance imaging.

The assessment of nephron number is of clinical relevance as reduced nephron numbers have been associated with an increased risk of cardiovascular disease, such as chronic kidney disease. Don't forget that working with hydrochloric acid can be extremely hazardous and that precautions should be taken, such as wearing gloves, protective eyewear, and a laboratory coat.