The amazing axolotl: robust kidney regeneration following acute kidney injury

ABSTRACT

The incidence of kidney disease from acute and chronic conditions continues to escalate worldwide. Interventions to replace renal function after organ failure remain limited to dialysis or transplantation, as human kidneys exhibit a limited capacity to repair damaged cells or regenerate new ones. In contrast, animals ranging from flies to fishes and even some mammals like the spiny mouse exhibit innate abilities to regenerate their kidney cells following injury. Now, a recent study has illuminated how the Mexican salamander, Ambystoma mexicanum, most commonly known as the axolotl, possesses a kidney with remarkable similarity to humans, which can robustly regenerate following acute chemical damage. These discoveries position the axolotl as a new model that can be used to advance our understanding about the fundamental mechanisms of kidney regeneration.

Perspective

The kidney is a vital organ with the principal functions of facilitating waste excretion, osmoregulation and fluid homeostasis. The basic functional unit of the kidney is the nephron, a specialized epithelial structure comprised of 1) a blood filter apparatus known as the glomerulus and 2) a tubule with discrete functional regions known as segments that selectively modify the filtrate by secreting or reabsorbing specific molecules.¹ For example, the proximal convoluted tubule is responsible for secreting ammonia, which adjusts filtrate pH, and the distal segments facilitate the reabsorption of ions such as sodium and chloride. Ultimately, waste is excreted into collecting ducts which transport the urine to the bladder. During kidney development, vertebrate species can possess up to three kidney structures of increasing complexity – the pronephros, mesonephros, and metanephros – which appear progressively during embryogenesis and then degrade when the next most complex structure is formed. Each kidney structure is comprised of nephrons but the architectural arrangements vary. Animals with more simple nephron arrangements have been useful models to study the mechanisms of nephrogenesis because these functional units are relatively conserved with humans.²

Kidney diseases affect millions of people worldwide and arise from problems that block normal function or development of nephrons. Acute kidney injury (AKI) is defined as a rapid loss of renal function and can occur from exposure to drugs like aminoglycoside antibiotics or cisplatin, an ischemic incident, or infection. Patients that recover from AKI have an increased risk of chronic kidney disease (CKD), or the progressive loss of kidney function over time that culminates with organ failure, known as end stage renal disease (ESRD). Humans and other mammalians exhibit limited capacity to repair or regenerate nephrons after AKI or during CKD. Further, the progressive scarring of the organ due to fibrosis in CKD is too catastrophic to be repaired. At present, renal replacement therapies for ESRD are limited to dialysis or organ transplantation. Formulating regeneration-based medical treatments has been of paramount interest for decades, but has not yet yielded new clinical approaches.³

In contrast to humans, other animals exhibit robust abilities to regenerate kidney tissues.³ Understanding these kidney regeneration mechanisms could be leveraged to formulate strategies to treat kidney disease in patients. For example, the zebrafish (Danio rerio) possesses extraordinary regenerative characteristics, being able to replace nephron epithelial populations after AKI and the ability to grow entirely new nephrons as well, known as neonephrogenesis.⁴ Another emerging model to study kidney regeneration is the spiny mouse (Acomys cahirinus) which is a mammalian “regeneration competent” model that fully regenerates after a bout of renal ischemia and fails to develop fibrosis.^5,6

Now, in a new exciting study that is the first of its kind, Chen et al. have reported that the axolotl (Ambystoma mexicanum) is another vertebrate species that can be used to study kidney regeneration (Figure 1a).⁷ The axolotl has the ability to regrow multiple body parts including limbs, the tail, liver, heart and the brain, but no previous studies had been conducted to determine whether the axolotl kidney was capable of regeneration after injury.⁸ First, the authors elucidated that the axolotl had remarkable structural and functional similarities with the mammalian kidney.⁷ They examined axolotl kidney composition using a variety of techniques, ranging from histology to transmission electron microscopy and immunofluorescence. The axolotl kidney gross anatomy can be described as pear shaped, and the pair of kidney organs is located at the dorsal side of the body cavity (Figure 1b). Found within the axolotl kidney were nephrons with glomerular and tubular features resembling the mammalian kidney (Figure 1c). Many ultrastructural features and molecular markers were also conserved. For example, the proximal tubule contained brush borders of microvilli, with a regular lumen in the distal tubule. Proteins like Megalin and Calbindin were highly expressed in the proximal tubules and distal tubes, respectively, where previous studies have demonstrated their crucial roles in the reabsorption of substances. Furthermore, the authors found that the axolotl kidney could filter and reabsorb small substances but exclude large ones, consistent with a size-based glomerular filtration process.

Figure 1.

Axolotl kidney regeneration. (a) the axolotl kidney is located adjacent to the dorsal body wall and when dissected, the morphology is revealed to (b) consist of two pear-shaped organs.⁷ (c) histological analysis with hematoxylin and eosin staining revealed the glomerular (G) features of the Bowman’s capsule (BC), glomerular tubular junction (GTJ), proximal tubule (PT) and distal tubule (DT).⁷ (d) the proximal tubule undergoes regeneration following gentamicin exposure within approximately 13 weeks. (e) glomerular injury following doxorubicin exposure is fully regenerated within approximately 8 weeks. Created with Biorender.com.

Next, the authors created an AKI model in the axolotl by using gentamicin, an aminoglycoside antibiotic that is both ototoxic and nephrotoxic, and specifically results in cell death of proximal tubule cells within nephrons.⁷ After applying gentamicin to the axolotl, they performed histological analysis to examine the endogenous response (Figure 1d). After 7 days where the injury was the most severe, there was a massive disorganization of proximal tubules which caused numerous cell aggregates to form between the injured tubules at 14 days post injury (dpi). By 30 dpi, the cellular aggregates showed faint lumen morphology and was elongated into immature tubules at 60 dpi. These proximal tubules that are newly regenerated would even contain brush borders by 90 dpi. The authors also characterized cell death and proliferation using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and 5 ethynyl-2’-deoxyuridine (EdU+) Pulse chase studies, chronicling the dynamics of each process. These studies nicely document the progression of cellular changes within the regenerating axolotl kidney.

Another aspect that the authors explored was to examine the transcriptional response to gentamicin induced kidney injury.⁷ Their transcriptome expression profiles showed 2,444 upregulated genes and 2,668 downregulated genes. Some key genes that were downregulated included both Lrp2 and Slc12a3 that play a role in tubular function. They also discovered important nephrogenesis related genes including Ccnd1, Top2a, Sox9, Bmp7 and Wtn4 that were significantly upregulated after the gentamicin injection. These nephrogenesis-related genes could play an important role to kidney regeneration in axolotls after a severe injury, but further studies are needed to explore such mechanisms.

Finally, the authors decided to induce glomerular injury with doxorubicin (Figure 1e).⁷ In the first week of the injury, the authors noticed the glomeruli to be separated from the Bowman’s capsules, with some protein casts accumulating in the lumen. The most severe damage was during the second week, when the interstitium matrix was thicker than the control group. The newly regenerated glomerulus was smaller than the control group up for the first four weeks, before reaching a similar size 8 weeks after injury. The axolotl was also able to recover the glomerular filtration function in addition to the morphology.

In conclusion, Chen et al. have highlighted how the axolotl can be characterized to have similar functions to the mammalian kidney, and that the nephrons could regenerate after severe renal injury.⁷ These findings reveal that the axolotl can now serve as another vertebrate model organism for studying kidney regeneration after injury. Future implications could be to identify different pathways and repair mechanisms for renal regeneration, with a lot of potential to study the molecular mechanisms that drive kidney regeneration, and hopefully contribute to the research efforts of the greater nephrology community in the quest to unlock ways to mediate kidney regeneration in humans.

Funding Statement

This work was supported by funds from the College of Science to R.A.W., and the family of Elizabeth & Michael Gallagher in support of stem cell and developmental biology research.

Disclosure statement

No potential conflict of interest was reported by the author(s).