This work presents a new in vivo model of segmental kidney injury using kidney GFP transgenic zebrafish. The model allows for the induction of the targeted ablation of kidney epithelial cells to show the cellular mechanisms of nephron injury and repair.
Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore adequate kidney function after supportive treatment. However, a better understanding of how nephron cell death and repair occur on the cellular level is required to minimize cell death and to enhance the regenerative process. The zebrafish pronephros is a good model system to accomplish this goal because it contains anatomical segments that are similar to the mammalian nephron. Previously, the most common model used to study kidney injury in fish was the pharmacological gentamicin model. However, this model does not allow for precise spatiotemporal control of injury, and hence it is difficult to study cellular and molecular processes involved in kidney repair. To overcome this limitation, this work presents a method through which, in contrast to the gentamicin approach, a specific Green Fuorescent Protein (GFP)-expressing nephron segment can be photoablated using a violet laser light (405 nm). This novel model of AKI provides many advantages that other methods of epithelial injury lack. Its main advantages are the ability to "dial" the level of injury and the precise spatiotemporal control in the robust in vivo animal model. This new method has the potential to significantly advance the level of understanding of kidney injury and repair mechanisms.
Acute Kidney Injury (AKI)1,2, which also can be referred to as acute renal failure, is broadly defined as a sudden impairment in kidney function3. While the level of understanding of this condition has been enhanced remarkably over the years, morbidity and mortality rates have remained high1,2. The current treatment for this condition is mostly supportive, as results from multiple clinical trials of drug therapy have been negative4,5. The kidney is unique in that it has the ability to repair itself. Therefore, supportive therapy after an early diagnosis of AKI is the best way to limit morbidity6. However, it is difficult to detect AKI early, and the mortality rate is a staggering 50-80% for those who require dialysis5. With the ability of the kidneys to repair themselves and the lack of treatment options for this condition, it is important to develop methods to enhance this nephron regeneration process.
There have been many different models used for AKI research that includes different agents of injury and animal models. In terms of agents of kidney damage, the aminoglycoside antibiotic gentamicin has been used as a nephrotoxic agent that leads to AKI7,8. However, several groups have found that gentamicin treatment is lethal to the zebrafish embryo9. It causes tubular damage that is too serious for embryo recovery, making the study of regeneration difficult without some type of intervention. Mammalian models, like the mouse and rat, are also considered valuable, but they face many limitations during the study of AKI. Perhaps the main disadvantage of rodent models is the difficulty in visualizing the rodent kidney and thus determining the precise spatiotemporal processes leading to epithelial death and repair.
Johnson et al. have reported a laser ablation-based technique to induce acute kidney injury in embryonic and larval zebrafish9. They used pulsed laser ablation to damage the kidney after an intramuscular injection with dextran conjugates. The fluorescence from dextran conjugates allows for the visualization of damage and regeneration in the tubule epithelium9. This model overcomes the two limitations mentioned above, but it does not allow for graded levels of injury and is difficult to carry out on large, arbitrary cell groups.
The new laser ablation-based zebrafish model of AKI described here addresses all of the above limitations. The pronephric kidney in larval zebrafish is a mature, functioning organ that contains segments similar to the mammalian nephron, including a glomerulus, proximal and distal tubules, and a collecting duct10. Zebrafish larvae are also optically transparent, making it feasible to observe the kidney through fluorescence techniques. Thus, zebrafish are a valuable in vivo model of AKI, and the larval pronephric kidney (5-12 days-post-fertilization (dpf)) can be used to study the cellular and molecular processes involved in kidney injury and repair.
This paper presents a method through which specific Green Fluorescent Protein (GFP)-expressing nephron segments can be photoablated using a low-energy (compared to a pulsed-laser system) violet laser light (405 nm). The GFP fluorescence allows for the targeting of a group of cells, making the changes that occur visible through the observation of GFP photobleaching. In addition, GFP (by absorbing violet light) serves as an energy sink to potentiate the injury in GFP-expressing kidney cells. Time-lapse microscopy can then be used to study the repair process. Studies have found cell proliferation, cell migration, and cell metaplasia11,12,13 to all be potential processes that may play an important role in kidney repair. However, the relative importance of these processes and the details of their interplay have been difficult to uncover due to the limitations of existing models of AKI. Using this novel approach, it was possible to show that cell migration plays a central role in kidney repair after acute injury14.
This study was carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the NYIT College of Osteopathic Medicine Institutional Animal Care and Use Committee (NYITCOM IACUC). All surgery and in vivo experimentation was performed under tricaine anesthesia, and all efforts were made to minimize suffering.
1. Obtaining and Maintaining Embryos
2. Mounting the Zebrafish for Live-imaging
3. Laser Ablation
4. Time-lapse Microscopy with Propidium Iodide Staining
Please note that this protocol was successfully used with a number of kidney GFP transgenic lines, including ET(krt8:EGFP)sqet11-9, ET(krt8:EGFP)sqet33-d10, and Tg(atp1a1a.4:GFP). The example results shown here were obtained using the ET(krt8:EGFP)sqet11-9 line.
Figure 1 shows the example photoablation protocol. The average GFP intensity is monitored inside the region of interest (Figure 1A, and Movie 1). An example average intensity trace is shown in Figure 1B.
As shown in Figure 2, after exposure to 405 nm laser light, GFP fluorescence continues to disappear one cell at a time until, at 220 min, the entire ablated segment loses 100% of its GFP positivity. This loss of GFP fluorescence is indeed due to cell death and not, for example, the downregulation of GFP expression. This is evidenced by the appearance of red fluorescent propidium iodide-positive nuclei in the cells that lose GFP positivity.
The extent of cell death can be assessed by measuring average GFP fluorescence in the ablated segment and comparing it to the mean GFP intensity immediately anterior and posterior to the ablated segment. Figure 3 shows the relationship between the extent of laser exposure (measured by the initial amount of photobleaching) and the extent of cell death in the exposed segment. It shows that, by varying the initial exposure, it is possible to "dial" the injury and the response of the injured epithelium. With 10-20% of initial GFP photobleaching, virtually no reduction of GFP positivity is observed at 5 h post-injury. 50 and 60% photobleaching leads to a complete disappearance of GFP at 5 h, while 30 and 40% bleaching leads to intermediate results, with 50% estimated death observed at about 35% photobleaching. These results are supported by PI staining (Figure 3B). 20% photobleaching results in virtually no PI staining at 100 min post-ablation. 50% photobleaching leads to continuous PI positivity in the ablated epithelium after 60 min, while 30 and 40% photobleaching leads to intermediate PI incorporation. As can be seen from this data, this methodology allows for the induction of graded amounts of epithelial injury and for the study of the epithelial response to lethal and sub-lethal epithelial damage.
Figure 1: Photoablation Procedure. (A) Schematic showing embryo/larva orientation, laser exposure, and the region-of-interest (ellipse) measurement of the average fluorescence to monitor the amount of GFP photobleaching; see Movie 1. The rectangular box indicates the scan window. (B) An example of an actual region-of-interest average GFP intensity trace before, during, and after 405 nm laser exposure. Average GFP intensities on four consecutive measurements (shown inside green boxes) are shown before (Y) and after (X) the photoablation. The ratio X/Y is used as a measure of the total light exposure and is plotted on the X axis in Figure 3. Please click here to view a larger version of this figure.
Figure 2: Epithelial Cell Death after 405 nm Laser Exposure. Example sequential frames showing the disappearance of GFP and the appearance of propidium iodide staining after photoablation (40% initial photobleaching). The frames are at 0, 130, 170, and 220 min after violet-light (405 nm) exposure. The disappearance of GFP coincides with the appearance of red fluorescent PI nuclear positivity. Please note that PrI fluorescence is detected by using the red channel. The red fluorescence seen outside the kidney is due to chromophores and the presence of PI in the gut lumen. Scale bar = 100 µm. Please click here to view a larger version of this figure.
Figure 3: Dose Response of Epithelial Injury to the Amount of Photoablation. (A) Photoablation is measured by the percent of initial reduction in GFP fluorescence in the exposed segment. This is compared to the decline in average GFP fluorescence in the injured segment at 5 h post-injury. This measurement is normalized to the average amount of fluorescence upstream and downstream of the injured segment. Target doses were 10, 20, 30, 40, 50, and 60% of the initial photobleaching. The actual amounts are slightly larger, amounting to 14.8, 24.4, 33.8, 45.4, 56.5, and 62.1% photobleaching, which result in 85.2, 75.6, 66.2, 54.6, 43.5, and 37.9% remaining fluorescence, n = 2 – 6/target group. The error bars represent the standard deviation along the X (percent of remaining fluorescence immediately after photoablation) and Y (percent of remaining fluorescence 5 h after photoablation) axes. (B-E) PI staining parallels the overall disappearance of GFP positivity. Virtually no PI staining is observed at 20% (80% remaining fluorescence) photobleaching at 100 min post-laser exposure (B, right panel). (C and D) 30 and 40% photobleaching (70 and 60% remaining fluorescence) show increasing numbers of PrI-positive cells (right panels), and (E) at 50% photobleaching, virtually 100% PI positivity is observed at 60 min post-injury (right panel). The left panels in (B-E) show the initial amount of photobleaching. Scale bar = 100 µm. Please click here to view a larger version of this figure.
Transgenic | Expression Pattern | Referencias |
Tg(wt1b:GFP) | Glomerulus, some PT | [11] |
Tg(atp1a1a.4:GFP) | Distal to glomerulus | [12] |
Tg(cdh17:GFP) | Distal to glomerulus | [9,10] |
Tg(ret1:GFP) | Late DT, PD | [13] |
Tg(enpep:GFP) | Distal to glomerulus | [14] |
Tg(cd41:GFP) | Multiciliated cells | [15] |
ET(krt8:EGFP)sqet11-9 | Straight PT, early DT | [16,17] |
ET(krt8:EGFP)sqet33-d10 | Convoluted PT | [16,17] |
PT = Proximal Tubule | ||
DT = Distal Tubule | ||
PD – Pronephric Duct |
Table 1: Examples of Kidney GFP Zebrafish Lines. Some representative zebrafish lines are listed here, indicating which segment of the pronephric kidney is labeled in a particular transgenic line.
Movie 1: Animation Summary of the Photoablation Procedure. In the first part of the movie, proper embryo/larva orientation is demonstrated. The two kidney branches are shown in green. A glass probe is used to orient the fish in agarose. This is done if a control, non-injured branch is desired. Angling the fish allows for laser exposure on only one branch of the pronephric kidney. In the second part of the movie, the laser ablation procedure is outlined. The ellipse indicates the region of interest within the segment undergoing ablation used to monitor the amount of initial photobleaching. The rectangular window allows for the precise adjustment of the size and position of the ablated segment. Please click here to view this video. (Right-click to download.)
It should be noted that the total laser power varies between systems. However, using percent GFP photobleaching allows for a readout of total energy delivered to the fluorescent kidney, independent of the variation in laser power and compensated for by the length of exposure. Keep in mind, however, that the response of different tissues to this method of photoablation varies. Even during kidney maturation, it is significantly more difficult to obtain a 50% reduction in GFP fluorescence in younger embryos than in mature larvae. These differences in tissue responsiveness to photoablation should be taken into account when modifying and adapting this protocol to different applications. Thus, it is critical to monitor the percent reduction in GFP fluorescence to properly gauge the amount of injury and the predicted response of the injured kidney epithelium.
The main advantages of this method when compared other methods of kidney injury in zebrafish is that is allows for the precise control of the spatiotemporal location of the injury. Also, it allows researchers to dial the level of injury up and down. This ability to control the amount of injury should allow for the examination of the responses of epithelial cells and should assist with decision making in terms of recovery versus apoptosis versus necrosis. For example, it is possible to show that cell migration is an initial response of surviving epithelium to segmental ablation and that cell proliferation is a secondary process, likely driven by mechanical forces generated by cell migration14.
Besides the ability to study the repair process at a cellular level, there are other advantages to this methodology. First, previous photoablation techniques had limited control over the amount of photodamage9. However, this model allows for a graded control over the amount of injury, making it easier to conduct future studies. In addition, in this approach, it is possible to target arbitrary groups of GFP fluorescent cells, thus permitting the easy ablation of entire segments. While this could be achieved with the pulsed laser technique, it is more laborious, limiting the potential for experimental design. The use of GFP to target and potentiate epithelial injury is a further advantage, because there are so many GFP transgenic zebrafish available (Table 1 is only a partial list). Other fluorescent proteins expressed by transgenic species have also been studied, such as the Killer red protein, but these fish transgenics are not widely available16. An additional advantage of using GFP is that, with different laser wavelengths, GFP expression can be used either for photoablation (405 nm) or imaging (488 nm). However, it should be noted that the method published by Johnson et al.9 can be applied to non-fluorescent zebrafish and thus can be used more widely than this approach.
Another limitation of this laser ablation model is that it may not take into account all the different factors that can lead to human AKI. There may be a chain of events that lead to cell necrosis in the human body, as well as cell apoptosis that is induced during kidney injury. It is unclear if the different environment produced during laser ablation can imitate all aspects of the pathophysiology of AKI in humans10. Nonetheless, this laser ablation model has many advantages over the alternative models. By allowing for the study of kidney cell death and repair at a high resolution in real time, this method should lead to a better understanding of kidney repair mechanisms and lay a foundation for new approaches to AKI treatment.
The authors have nothing to disclose.
We would like to thank Dr. Iain Drummond and Dr. Vladimir Korzh for sharing kidney GFP transgenic lines. We would also like to thank NYITCOM for providing necessary resources to conduct this work. This study was in part supported by grants: K08DK082782, R03DK097443 (NIH), and the HSCI Pilot Grant (AV).
Petri Dishes, 35 x 10mm | Genesee Scientific | 32-103 | Procedural Usage: Step 2.4,2.7 |
Petri Dishes, 100 x 15mm | Midwest Scientific | 910 | Procedural Usage: Step 1 |
De-chorination forceps- Electron Microscopy Sciences Dumont Tweezers 5 Dumostar | Fischer Scientific | 50-241-57 | Procedural Usage: Step 2.1.1 |
Plastic Transfer Pipet | Globe Scientific | 135030 | Procedural Usage: Step 2.5, 3.6 |
Tricaine | Sigma Aldrich | A5040-25G | Procedural Usage: Step 2.3, 3.4 |
Agarose | Fischer Scientific | BP165-25 | Procedural Usage: Step 2.3 |
Pulled glass probe (manufactured manually from glass capillary tubes) | Fischer Scientific | 21-1640-2C | Procedural Usage: Step 2.4 |
Stereomicroscope | Nikon | SMZ1270 | Procedural Usage: Step 1.5 |
SOLA Light Engine | Lumencor | SOLA SM-5-LCR-SB | Procedural Usage: Step 1.5 |
Eclipse C2 Plus Confocal Microscope System | Nikon | Procedural Usage: Step 3 | |
1x E3 Solution | Recipe used to generate: 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl 2 , 0.33 mM MgSO 4 Procedural Step Usage: 1.2, 1.3, 2.2, 2.3 | ||
PTU | Sigma | P7629-10G | Procedural Step Usage: 1.3, 2.2, 3.4, and 4.2 |
NIS Elements Software | Nikon | C2+ | Procedural Usage: Step 3 |
Laser Unit | Agilent | MLC 400 | Procedural Step 3.11 |
Propidium Iodide | Sigma Aldrich | P4170-100MG | Procedural Step Usage: 4.2 |