Published: November 1st, 2018
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 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 glomerulus is a highly specialized tuft of capillaries responsible for the filtration of circulating plasma. It forms the beginning of the nephron, which is the basic functional unit of the kidney. Glomerular function is defined by a uniquely fenestrated capillary endothelium, the slit diaphragm of podocytes, and an intervening basement membrane. These layers form a semipermeable barrier to allow for the selective excretion of substances into the filtrate. Water, ions, and other small molecules generally pass through, while larger molecules are retained in the plasma. Podocytes are specialized epithelial cells that spread over the basement membrane, surrounding the capillaries with cytoplasmic projections known as foot processes. The foot processes of adjacent podocytes interdigitate and are crossed by slit diaphragms comprised of proteins such as nephrin, podocin, P-cadherin, and ZO-11. In cross section, these foot processes are evenly arranged over the basement membrane. In diseased glomeruli, the foot processes become grossly abnormal or "effaced," leading to abnormal leak of plasma contents into the filtrate. As such, glomerular damage is generally characterized by the presence of abnormally large amounts of protein (e.g., proteinuria) and/or red blood cells (e.g., hematuria) in the urine. In addition, injured podocytes lose expression of nephrin as well as its regulator Wilms Tumor 1 (WT1), a key protein responsible for maintenance of differentiation2,3. The glomeruli are a primary target of damage in diabetic nephropathy and other glomerulonephritides such as minimal change disease, membranous nephropathy, and focal segmental glomerulosclerosis. These diseases are major causes of progressive kidney failure and the development of end-stage renal disease, a condition in which survival relies upon dialysis or renal transplantation. Therefore, it is important to study glomeruli to better understand chronic kidney disease (CKD) pathology.
A cell culture system is critical to studying glomerular biology. Due to its central role in generating the slit diaphragm, as well as the existence of specific proteinuric diseases due to slit diaphragm protein mutations, much research has understandably utilized the podocyte in isolation. This has led to the generation of primary podocyte cell lines to utilize in vitro. These cells can be cultured under a variety of conditions and can even be grown on permeable supports to assess permeability4. However, the isolation of proliferating cells often selects dedifferentiated cells that have lost some of their podocyte markers. This has led to the generation of conditionally immortalized podocytes derived from a transgenic mouse strain carrying a temperature-sensitive mutant of the SV40 large T gene (e.g. immortomouse), which could be grown in culture but also be differentiated to express a full array of podocyte markers5. These methods of primary culture have been pivotal in understanding podocyte biology4,6,7.
Nevertheless, cultures containing single cell types lack the intercellular relationships that occur in vivo as well as the support structure and matrices, and monolayers of these cells do not necessarily recapitulate the three-dimensional architecture of glomeruli. The immortalized podocytes can also be cumbersome and challenging to culture8, and require possession of either the immortomouse or a starting aliquot of cells from established investigators to get started. Further, the glomerulus is comprised of not only podocytes, but also capillary endothelial cells and the basement membrane, as well as mesangial cells which provide support for the structure. It is therefore useful to develop an ex vivo approach available to all investigators for the study of intact glomeruli that retain their native architecture as well as all the cells constituting the normal glomerulus.
In 1958, Cook and Pickering described the first isolation of glomeruli from the rabbit kidney. After observations that fat emboli became lodged in glomeruli, they postulated that particles of the same size could be used to specifically isolate these structures. Indeed, the infusion of iron oxide particles into the kidney led to the trapping of these particles in glomeruli. After mechanical dissociation and sieving of the kidney, the glomeruli could be isolated intact and with purity through the use of magnetic separation9. In 1971, Misra showed that the iron oxide infusions could be omitted, and glomerular isolation achieved with sieving of minced human, dog, rabbit or rat kidney tissue10. This technique has been modified since then depending on the goal of the investigators but has essentially resulted in purified preparations that could be further studied or from which primary cell cultures could be established11,12,13,14,15,16,17.
Here we describe a protocol for the isolation of intact viable glomeruli from the rat kidney. The entire protocol takes just a few hours. Although they do not proliferate, experimental plans of any size can be supported by simply increasing the number of kidneys as starting material. While there are published protocols for the magnetic bead separation of glomeruli, they require an intravenous injection of beads, are more expensive, and may alter biology since the beads are either retained by the glomeruli in culture or require glomerular "lysing" and removal by centrifugation19. Compared to mouse glomeruli, the larger size of rat glomeruli (nearly 100 µm in two month old rats18) makes it much easier to separate them from other kidney structures using a simple sieving technique.
As evidence of their usefulness, we have characterized the glomeruli to demonstrate the different cell types. They can also be exposed to agents known to injure glomeruli in vivo, and we demonstrate the adverse effects of protamine sulfate (PS) on these cultures. PS is a polycation that neutralizes the anionic sites along the glomerular capillary wall20. This neutralization has a dramatic effect on the glomerular filtration barrier and therefore increases proteinuria and foot process effacement. These glomeruli can be assessed with immunoblots for key proteins such as nephrin and WT1 to assess overall health. Furthermore, their structure can be evaluated with light, immunofluorescence, and electron microscopy.
Overall, this protocol is accessible to most investigators (one only needs access to the animals and some simple equipment). With morphological features left undamaged, the researcher is able to analyze the glomeruli and see how other important cell types and matrix preservation in the glomeruli affect function and disease progression, a shortcoming of podocyte cultures.
All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of University of Pittsburgh.
1. Isolation of Rat Glomeruli
2. Injury of Glomeruli
3. Preparing Glomeruli for Analysis
The protocol from the time of euthanasia to isolation of the glomeruli can be completed in as little as 2 h and has a high throughput and efficiency. With proper utilization of the technique, the yield of glomeruli per rat kidney ranges from 6,000 - 10,000 glomeruli when starting with 8 kidneys. The final suspension is densely packed with glomeruli and has an overall purity > 95%, showing minimal contamination from tubular segments or other cell types (Figure 1A, B). In addition, the OCT-embedded glomeruli can be stained with Hematoxylin and Eosin (H & E) stains to see the morphology (Figure 1C). These glomeruli maintain their structure throughout the whole protocol, even after processing. We demonstrate that isolated glomeruli possess intact and viable podocytes (Figure 2A), mesangial cells (Figure 2B), and endothelial cells (Figure 2C).
Once isolated, the glomeruli can be exposed to well-known chemical injuries to simulate in vivo pathology. In this case, protamine sulfate (PS) was chosen for its ability to disrupt the charge of the glomerular filtration barrier, which eventually leads to foot process effacement. PS-treated glomeruli have a striking reduction in nephrin (red) and a number of nuclei positive for WT1 (green) via immunofluorescence (Figure 3A, B). The glomeruli can also be prepared for transmission electron microscopy (Figure 3C). Control samples have normal podocyte morphology and foot processes whereas the PS-treated glomeruli have foot process effacement (Figure 3D), which is a sign of podocyte dysfunction and is seen in in vivo models using PS21. This also corresponded to a decrease in nephrin detected by immunoblotting (Figure 3E, F).
To determine viability, cleaved caspase-3 was assessed as a marker of apoptosis. Using immunofluorescence, cleaved caspase-3 first appears in a few cells starting 2 h after isolation (Figure 4). The number of cells expressing this protease became more abundant over time, with the highest levels seen at 24 and 48 h. This suggests that apoptosis does occur relatively early in culture and that downstream applications should be performed soon after isolation for best results.
Figure 1. Typical appearance of rat glomerular cultures after isolation. (A) Brightfield image of glomerular culture. Although we chose a field in which there is a single renal tubule in this micrograph (arrowhead), the cultures obtained are generally > 95% pure. Scale bar equals 100 µm. (B) Enlarged image of a single glomerulus. (C) Hematoxylin and eosin stain of a single glomerulus. Scale bars in B and C equal 10 µm. Please click here to view a larger version of this figure.
Figure 2. Constituent cell types are retained in isolated glomeruli. Confocal immunofluorescence microscopy was performed for nephrin (red, podocytes) and cell-specific markers to identify podocytes, mesangial cells, and the endothelium. (A) Costain for nephrin and WT1 (green, podocytes). (B) Costain for nephrin and PDGFR-β (green, mesangial cells, arrow). (C) Costain for nephrin and CD31 (green, endothelial cells, vessels in cross section marked by arrowheads). Scale bar = 25 µm on all panels. Please click here to view a larger version of this figure.
Figure 3. Podocyte injury can be induced in vitro using isolated rat glomeruli. (A) Confocal immunofluorescence microscopy for nephrin and WT1 in an untreated glomerular culture. Note the linear nephrin staining (red) and presence of WT1-positive nuclei (green) which are typically seen when there are healthy podocytes. (B) After protamine sulfate treatment, nephrin expression is decreased and non-linear, and WT1 is absent, indicating podocyte injury. (C) Transmission electron microscpy (TEM) image showing foot processes, basement membrane, and a fenestrated endothelium typical of normal glomerular structure. Bar equals 500 nm. (D) After protamine sulfate, foot processes are elongated or effaced (arrowheads), which indicates podocyte injury. (E) Western blot for nephrin showing decreased nephrin after protamine sulfate treatment. (F) Actin loading control for western blot is shown. Please click here to view a larger version of this figure.
Figure 4. Assessment of cell viability in isolated glomeruli. Confocal immunofluorescence microscopy for cleaved caspase-3 (green) was performed. A nephrin co-stain was performed to easily locate the glomeruli. While there was no cleaved caspase-3 positivity at 0 and 1 h after isolation of glomeruli, fluorescence signal in a few cells was noted at 2 h. Progressively more cells turned positive the longer after isolation they were examined. The greatest number of caspase-3 positive cells were noted at 24 and 48 h. Please click here to view a larger version of this figure.
This is an efficient method for recovering glomeruli from rat kidney using inexpensive, reusable equipment with a simple protocol. As with all procedures, there are limitations to its usefulness. First, although we obtain > 95% purity, because of the sieving nature of the protocol and the starting material it is impossible to exclude all contaminants, and a few red blood cells and the occasional tubular segment will be present in the culture. We do not anticipate these small contaminants to be a problem for the vast majority of applications. Second, the sieving protocol relies on the use of rat kidneys, in which glomeruli are much larger than in mice. If mouse glomeruli are needed, a technique using commercial magnetic beads (e.g., Dynabeads) has been published22. Third, it has been shown that specific mRNAs (plasminogen activator inhibitor-1 and collagen I) in isolated glomeruli derive almost entirely from Bowman's capsule rather than intraglomerular cells23. This could lead to inappropriate conclusions if mRNA isolation is attempted from these glomeruli. It should be noted that in our hands the majority of glomeruli are decapsulated and should therefore lack significant contributions from Bowman's capsules. Fourth, cell death begins to occur relatively quickly under culture conditions.
We found that the apoptotic program, as evidenced by appearance of cleaved caspase-3, was activated starting at 2 h of culture. This is in general agreement with previous reports showing that apoptosis, as assessed by DNA fragmentation, TUNEL, and histologic analysis, begins to occur within 1-2 h after isolation24. However, it should also be noted that early observations showed that metabolic activity could be detected from isolated sieved glomeruli when cultured for at least 3 h, suggesting considerable cellular viability at this timepoint16. Nevertheless, based on the results, we believe that it would be prudent to utilize isolated glomeruli immediately in intended experiments for the best results. We recommend that all experiments be performed before 72 h as we have observed that the entire glomerular structure begins to deteriorate beyond that timepoint.
It is much easier to obtain higher yields when starting with 4-8 kidneys as opposed to only 2 because a certain number of glomeruli are lost due to adherence to the various sieves, but once the sieves are maximally coated there is no additional loss. For the same reason, it is important to soak the sieves in the BSA/PBS buffer prior to use as glomeruli are more likely to adhere to a dry sieve, and to limit tissue exposure to one edge of the sieve. We suggest starting with no fewer than 6 kidneys (3 rats) to obtain a reasonable yield.
Some investigators have isolated primary podocyte cultures from isolated glomeruli which tend to grow out of the glomeruli during culture17. We have noted that some isolated glomeruli will adhere to the culture dish plastic and that cells will begin to migrate out of the glomerular structure at late timepoints (72 h after isolation). The study of these cells is beyond the scope of this isolation protocol, and there is some controversy as to whether these cells are podocytes, parietal epithelial cells, or both15,25. Notably, Mundel et al., have reported that cobblestone cells harvested from sieved glomeruli may be induced to differentiate into mature podocytes under specific culturing conditions26. Some of the confusion regarding cell identity may depend on whether the isolated glomeruli are encapsulated (including Bowman's capsule which is populated by parietal epithelial cells) or decapsulated in the sieving procedure. In our hands, using sequential sieve sizes of 180, 90, and 75 µm led to the majority of glomeruli being decapsulated.
Most forms of proteinuric chronic kidney disease are due to increases in glomerular permeability. Several authors have utilized isolated glomeruli in ex vivo permeability experiments. In one method, the change in glomerular volume after altering the osmotic content of the surrounding media was used to estimate permeability27. Recently, it was established that leakage of a fluorescent probe from isolated glomeruli could be quantified to measure permeability and could be performed in rodents exposed to glomerular disease experimental models28.
We have noted that in the TEM preparation process, we sometimes see the podocyte foot processes lift off the basement membrane. We do not feel this is happening in the culture and is more likely an artifact during TEM sample preparation. Notably, even when this occurs, it is clear whether the foot processes look "normal" or are effaced.
Overall, this protocol provides a method by which one can evaluate morphologic and cellular changes in response to injury. We anticipate that it may be used as a companion technique to isolated podocyte cultures, especially when interactions with extracellular matrix or other native cell types are being considered. It holds promise for increasing understanding of proteinuric CKD, which would improve the ability to develop future therapeutics for this debilitating disease.
The authors have nothing to disclose.
This work was supported by American Heart Association grant FTF 16990086, NIH P30 DK079307, American Society of Nephrology Gottschalk Award, a University of Pittsburgh Medical Center Competitive Medical Research Fund Award, and a University of Pittsburgh Physicians Academic Foundation Award. We thank Gerard Apodaca for his technical suggestions for glomerular preservation for histologic analysis, Mara Sullivan and Ming Sun for assistance with electron microscopy, and Yingjian Li and Youhua Liu for their technical input in this protocol. We also thank Cynthia St. Hilaire for the CD31 antibody.
|Solutions, Chemicals, and Animals
|Bovine serum albumin
|Hank's Balanced Salt Solution with Ca2+ and Mg2+
|1x Phosphate Buffered Saline
|Halt Protease Inhibitors
|Cleaved caspase-3 antibody
|Poly/Bed 812 (Luft) epoxy
|Carbon dioxide chamber
|Conical tubes, 15 mL
|Conical tubes, 50 mL
|Petri dishes, 100 mm
|Sterile filter apparatus
|10 mL syringes
|600 mL beakers
|Cell culture incubator
|20 G needles
|Leica Reichart Ultracut
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