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

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.

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This method can help to answer key questions in the field of kidney disease. Particularly in the understanding of the glomerulus in normal and diseased states. Glomerular biology can be difficult to study due to the variety of cell types present and their unique structure.

The main advantage of this technique is to provide a readily available source of intact glomeruli using simple methods and equipment. This technique has implications for our understanding of the filtration barrier in the normal and diseased kidney. In particular, it can shed light on our understanding of proteinuric chronic kidney disease in which glomerular injury leads to the abnormally high secretion of serum proteins into the urine.

Generally, individuals new to this technique will have difficulty obtaining adequate yield and purity in the final sample. Demonstrating the procedure will be Brittney Rush, a technician from my laboratory. To begin, use hair clippers to remove hair from the anterior abdomen of a euthanized rat.

Use 70%ethanol to clean the exposed skin. Then, using surgical scissors, make a midline incision in the skin. To expose internal organs, make a midline incision through the muscle layer.

Locate and isolate the kidneys and place into a steril 50 milliliter plastic conical tube with 30 milliliters of Hank's buffered salt solution or HBSS placed on ice. Transfer the tube on ice to a sterile cell culture hood. Transfer the kidneys to a sterile Petri dish containing five milliliters of HBSS placed on ice.

Use scissors and sharp forceps to remove and discard perirenal fat. To remove and discard the capsules surrounding the kidney, make a small superficial incision and then use sharp forceps to gently pull it away from the kidney. Place the kidneys on a sterile gauze in a second Petri dish with five milliliters of HBSS and divide each kidney in half through a mid-sagittal section.

Use a scalpel to remove and discard the medulla which can be identified by its darker color and central location. Transfer the remaining pieces containing predominantly the kidney cortex, into a third Petri dish with five milliliters of HBSS. Use a sterile razor blade to mince them until the pieces are less than one millimeter in size or until a paste is formed.

Use 1%BSA PBS to wet the top and bottom of a 180 micrometer sieve over a 500 milliliter waste beaker. This step is critical as it coats the sieve with protein and reduces the adherence of glomeruli which will then improve the yield. Place the mixed cortex on a small edge of the sieve.

Use the textured plunger flange of a 10 milliliter disposable syringe to mush the tissue through the sieve into a bottom pan placed on ice. Rinse periodically with HBSS using as little as possible to avoid sample dilution and reuse the fluid collecting in the bottom pan to rinse the sieve. After the completed sieving, carefully wash the bottom of the sieve with HBSS from the bottom of the pan to capture any loosely adhered glomeruli.

Divide all of the fluid from the bottom pan into 10 milliliter syringes equipped with 20 gauge needles. Pass the fluid through the needle at least three times into the bottom pan. For the last collection, keep the glomeruli containing fluid in the syringe until ready to pass it through the 90 micrometer sieve.

Meanwhile, wash the bottom pan by flushing it with 1%BSA PBS and into a waste beaker. Use 1%BSA PBS to wet the top and bottom of a 90 micrometer sieve and then place it on the top of the bottom pan. Apply the sample in the syringes to one edge of the sieve.

Use a textured plunger flange to mush the tissue through the sieve as done previously. Use the solution from the bottom pan to wash the tissue and collect everything from one edge of the sieve. Once sieving is complete, carefully wash the bottom of the sieve with HBSS buffer from the bottom of the pan to capture any glomeruli that may be loosely adhered.

Collect all the fluid from the bottom pan into a 50 milliliter plastic conical tube. Use 1%BSA PBS to wash the bottom pan into a waste beaker. Then use 1%BSA PBS to wet the top and bottom of a 75 micrometer sieve.

Place the sieve on top of the bottom pan placed on ice. Apply the sample to one edge of the sieve with liquid flowing through easily. Rinse the top of the sieve with a minimum of 20 milliliters of 1%BSA PBS into a waste beaker and carefully wash the bottom of the sieve.

To collect the glomeruli that remained on the top of the 75 micrometer sieve, rinse through the sieve upside-down with as much HBSS as needed to collect glomeruli in a Petri dish. Collect the sieved glomeruli into a 50 milliliter plastic conical tube on ice. After centrifuging at 1800 times G for five minutes at four degrees Celsius, remove the supernatant carefully using a pipette.

Re-suspend the pellet in 10 milliliters of cold HBSS. Combine the samples and repeat the centrifugation. Re-suspend the glomeruli in five milliliters of HBSS.

To count the total glomeruli, place a 10 microliter drop of the sample on a glass slide, count under a microscope and multiple by 500 to get the total yield. Isolated structures should consist of nearly all glomeruli without tubular structures. If tubules are present and would affect your downstream application, they can be removed by applying the entire sample to the 90 micrometer sieve and repeating the protocol from that point onward.

The protocol described here is efficient in isolating glomeruli and the final suspension is densely packed with glomeruli with a purity greater than 95%and minimal contamination from tubular segments or other cell types. These glomeruli can be stained with hematoxylin and eosin stains to view their morphology. Furthermore, they maintain their structure throughout the whole protocol even after processing.

They retain intact and viable podocytes, mesangial cells and endothelial cells. The isolated glomeruli have been exposed to chemical injuries to simulate in vivo pathology. Specifically protamine sulfate which disrupts the charge of the glomerular filtration barrier.

Contrary to healthy glomeruli, protamine sulfate treated glomeruli, have a prominent reduction in nephron and a number of nuclei positive for WT1. When viewed with transmission electron microscopy, healthy glomeruli show typical foot processes. After protamine sulfate treatment, foot processes are elongated or effaced indicating podocyte injury.

To determine cell viability in isolated glomeruli, cleaved caspase three was observed as an apoptosis marker. There was no cleaved caspase three at zero and one hours after isolation of glomeruli. A few cells showed signs of apoptosis at two and four hours with a progressive increase over time.

The greatest number of apoptotic cells were noted at 24 and 48 hours. Based on these results, we recommend that isolated glomeruli be used immediately after isolation for most experiments. When performing this procedure, it is important to work quickly and efficiently.

From the moment the kidney is isolated, glomeruli begin to deteriorate. The faster you isolate the glomeruli, the more time you will have to pose, isolation, manipulation. After isolation, isolated glomeruli can be exposed to chemical or biological agents to stimulate physiological or pathologic conditions.

And specimens can be process for protein or RNA isolation, histology or immunofluorescence and electron microscopy. Since its development in the 1950s, this technique has aided researchers in studying glomerular biology. Overall, this protocol provides a method to evaluate the morphologic and cellular characteristics of intact glomeruli in normal and diseased states.

This method can help our understanding of proteinuric chronic kidney disease and aid in the development of future therapies.

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

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