Globally 2M people are being treated for dialysis or kidney transplants, yet this only represents 10% of people who need it. The kidney in particular is the most in-demand organ for transplants, representing a whopping 87% of all transplant lists in the US.
Apart from it being a sad problem, it's also an expensive problem. In 2018 medicare related spending for patients with end-stage renal failure totaled ~$50B (US).
Solutions of Today
A kidney donation from a living person remains the best option for those with end-stage kidney failure, however, it currently faces the following issues:
Low supply of transplantable kidneys
Long waiting list times
If you are over 50 you are likely not a priority for Transplant, even though you are the most likely to have end-stage renal failure
Dialysis is used whilst waiting for a kidney, however, the average life span is ~10 years
Short life expectancy with transplantable kidneys (with it varying depending on who the donor is and if they were alive/deceased)
At best 10-20 years, so you will need another transplant (if that option is available to you)
High maintenance of transplantable kidneys
Lifelong immunosuppressants with strong side effects
Solutions of Tomorrow
The above criteria are written in the order in which most research prioritizes which problems to solve. The ideal solution would address all of these issues, however, since the world is less than ideal sacrifices must be made.
As a kidney patient myself in need of a transplant, here is my preferred order of priority:
High maintenance of transplantable organs - No pills needed. You should be able to live your life pretty normally, apart from watching your diet (just like any normal person should)
Short life expectancy with transplantable kidneys - Assuming we are pushing for progress to be made within a 20-year timeline, I would be okay with only a slight increase in life expectancy (2-5 years). This would push the upper bound average of 20 to ~25 years, making it a full “lifetime”.
Low supply of transplantable kidneys - If you are a young person (younger than ~40) then you are pretty much-guaranteed kidney. Older than that, then the future can be grim. I am against this sort of prioritization, as I don’t think anyone should decide who lives or dies. My thinking here is that if we find a way to create a small supply of bioartificial kidneys then we should be able to free up the current supply of deceased donor kidneys and prioritize them for older patients.
Most people will probably be surprised that I prioritized quality of life over a significantly longer life expectancy. I can get into that in another post, but for now, this is how we will be assessing current solutions moving forward.
Researchers are currently exploring different ways of creating bioartificial kidneys that can be used for transplants. The most popular examples are bioprinting, xenotransplantation (when you transplant organs and tissues from one species to another, e.g from a pig to a human), and growing them (as you would a plant). So far none have been successful. At best we have only been able to make miniature kidneys that can survive a few weeks (called organoids). Bioprinting and growing kidneys have failed to do so because they can’t create the blood vessels needed to help them grow, and ultimately, stay alive. Xenotransplantation has failed because the human immune system isn’t built to handle another species' cells, so it creates a very very bad immune response (think of it as a deadly allergic reaction).
To get around the problems each solution presents, an alternative approach is now being explored, porcine decell-recell. This is when you take kidneys from pigs, get rid of their cells, and replace them with human cells.
In theory, this approach should solve the following problems:
High maintenance of transplantable organs - If the pig kidney is now wrapped in your own human cells, then your body should recognize the kidney as your own. Therefore there would be no need for immunosuppressant drugs.
Short life expectancy with transplantable kidneys - This problem actually comes from the fact that your body is still attacking your transplanted organ because it sees it as a foreign object. The immunosuppressants just slow down the rate and intensity at which your organ is attacked. In this solution, the theory here is that because the pig kidney is wrapped in your own cells your body won’t attack it at all, and therefore will survive for a much longer time.
Low supply of transplantable kidneys - Pigs are regularly slaughtered in the masses, predominantly for food purposes. The hypothesis here is that since the kidney is discarded during slaughter, we should have a guaranteed supply of cheap, readily available pig kidneys.
The use of pig kidneys in particular is an intentional decision, one made for the following reasons:
Ethical issues around organ harvesting are lower than that of other animals because we slaughter them so much already.
We have studied and learned so much about pigs already, that we have a good set of knowledge to build on top of. A lot of human therapeutics originate from pigs, such as insulin and pig heart valves.
Pigs breed rapidly, contributing to the much-needed large supply of them. It takes ~6 months to mature and develop adult-size organs.
In theory, it should be cheap, as you would be using the discarded part of the organ - no additional work needed.
The slaughtering of pigs is a well-established and large market.
We have similar-sized kidneys and they operate in a similar fashion
For example, normal blood pressure in human adults is ~120/80, with pigs operating in similar ranges (systolic 112–139 and 72–98 mmHg)
So far, we have had a lot of success stripping the cells from a pig’s kidney, leaving just its scaffold behind, and some success putting human cells on mouse kidneys. However, we have yet to build a fully functional organ that could one day be transplanted into a human. Assuming decellularization methods work as intended, then the major remaining problem is recellularization. However, I argue that part of the problem is that we are making that assumption in the first place.
So, let’s investigate.
Decellularization
There are a number of ways to decellularize a pig’s kidney. You can use physical pressure, chemical detergents, heat etc. However, the most accepted method in recent times has been chemical detergents, so we will focus on that.
Below is a list of supporting materials showcasing how pig kidneys (and other animal kidneys) can be decellularized. Most mention the use of sodium dodecyl sulfate and Trition X-100 (a type of detergent).
Using a chemical solution of sodium dodecyl sulfate, Triton X-100 (a detergent), and phosphate buffer saline, it takes ~5 days for full decellularization
A monkey’s kidney was decellularized using 1% Triton X-100 and SDS @ 4 degrees celsius. It showed that glycoproteins such as fibronectin remained in their original place.
Both pig and human kidneys can be decellularized using 1% Triton X-100 and SDS at a constant pressure of 40mmHg with ~10% DNA material left over. All blood vessels were left intact.
Decellularized ECMs have been shown to influence cell differentiation and growth
.
The above demonstrates that it is possible to:
Strip a pig kidney of at least 90% of its DNA
It is possible to strip a kidney of its cell content and keep the scaffold intact
Maintain the location of different biochemicals the kidney had when it was “alive”
The pig’s scaffold plays some sort of role in differentiating cells, and therefore has the potential to “regenerate” the scaffold using stem cells
Although these signs are promising, there are a ton of assumptions made that it doesn’t address, the most important being the benchmark for an effective decellularization method.
Most of the above papers compare the biochemical locations and concentrations before and after treatment to determine whether the method was successful. However, since our goal is determining if these scaffolds are suitable for organ regeneration within humans - and not just creating a scaffold for its own sake - then I am not entirely sure this is the right benchmark.
Current methodologies (as far as I can tell) do not address if:
Biochemicals are damaged
The sensitivity of any potential changes to chemical structures
If these concentrations are the right dosage for human stem cell growth
If these biochemical locations are suitable for human stem cell growth
If porcine scaffold biochemicals are compatible with human stem cells in the first place
Then there is the issue of actually reseeding these scaffolds (depositing cells on the scaffold). Assuming we can verify the above assumptions and can create a pig scaffold that can support organ regeneration, we need to figure out how to get many millions of cells in the right place, performing the right function.
Reseeding
We have seen some reseeding success with smaller tissues such as heart valves, but none with full-on organs, especially complicated organs like kidneys. This is because organs, unline tissues, are much bigger in volume and have a larger variety of cells (the kidney has over ~25 different types of cells vs the heart valve which has ~4 types of cells . )
Below are some reseeding experiments that have had varying levels of success (not limited to kidneys):
The bladder has been successfully transplanted into a small number of patients, who are all still alive 10 years post-operation, no medication or additional surgery needed.
In rats, reseeding has been done on the liver (another complex organ) but it was only functional for only a few hours
Skin organ regeneration has been done and in clinical use https://sci-hub.wf/10.1016/j.jcyt.2015.04.003
The heart has been reseeded, but the pumping efficacy was only 2% of a human adult heart
In rat kidneys, cell perfusion was performed through the artery or ureter, with the glomeruli being successfully reseeded
Although the seeded cells reached the glomeruli capillaries, it did not reach the tubular capillaries
The reseeded kidney lowered creatine levels and produce some urine, however, it wasn’t clear if this was due to inertia / mechanical forces
Different levels of kidney function shown in reseeded rat kidney
8mm acellular monkey biopsies reseeded with human umbilical vein endothelial cells
Reseeding decellularized organs can be broken down into 4 steps:
Finding a cell source (cell source)
Distributing the cells within the organ (cell distribution)
Getting the cells to stick to the organ (cell adhesion)
Having the cells perform the right function (cell differentiation)
Cell Source
For complex organs, like the kidney, figuring out a good cell source is pretty important. The discovery of induced pluripotent stem cells (already formed cells that have been re-programmed to become any type of cell in the body) offers a potential solution, however, we still do not know enough about them in this sort of setting to call them a reliable cell source.
Cell Source - Experiments
Most experimentation around cell source has been around human umbilical cord stem cells, iPSCs and mesenamych stem cells. In short, not a lot of research has gone into this, however, I don’t think this is the biggest of problems right now. It will become more of a concern when these organs are in production (figuring out how the source will fit into different supply chains will not be straightforward forward.)
Cell Source - Questions
How can 1 billion cells be manufactured on demand?
How much would it cost to manufacture 1 billion + cells?
Do iPSC cause an immune response?
Cell Distribution
Getting cells to the right location is an important step in organ regeneration. If the cells aren’t in the right place, or there aren’t enough cells deposited in the right place, then the organ won’t function properly (so it is assumed).
In theory, cell distribution sounds like it should be easy (just pump some cells inside the organs right?) but it seems much harder to do in reality. An effective cell distribution method would need to ensure that no part of the organ gets damaged during the process, cells are distributed evenly (to avoid clots or malfunctions), and that the right amount of cells are in the right place. Basically, not so straightforward.
Cell Distribution (not limited to kidneys) - Experiments
It has been shown that the access line used for injecting the cells has a major impact on cell distribution.
Multi-step infusion through different portals has been shown to be effective at generating different types of cells in different places [I can’t find the original paper as the scihub link is not working, but once found I will cite it]
Mechanical conditioning (varying the pressure) has been shown to impact cell distribution
Dynamic rotating bioreactors have been shown to improve cell distribution
Cell Distribution - Questions
Is it easier to distribute organoid tissue than stem cells? If so, why?
What is the maximum pressure that can be used before the internal structure of a kidney will be damaged?
Do different distribution methods work better for different parts of a kidney? If so, why?
Does the size of the human kidney cells matter when distributing it in a pig’s ECM?
Cell Adhesion
One of the biggest problems with decell-recell is the thrombosis (blood clotting) caused during the reseeding process. This occurs because the cells used to line the inside of blood vessels have not yet differentiated and stuck to the wall. These cells are important because they allow for the smooth passage of blood through these vessels.
Cell Adhesion - Experiments (not limited to kidneys)
Coat cells in polyethylene glycol to increase the absorption of cell nutrients. This has been shown to work with acellular heart valves transplanted into goat hearts, decreasing thrombosis
What is the immune response of polyethylene glycol?
Fibronection and different growth factors showed good endothelial cell adhesion
What are the necessary growth factors that go with fibronectin for kidney regeneration?
Biotin, avidin, and biotinylated anti-rat CD90 applied sequentially have been shown to increase cell adhesion of rat bone mesenchymal stem cells on porcine heart valves by trapping and immobilizing them
Cell Adhesion - Questions
What causes cell adhesion in cells naturally? Is this the same for pig cells?
Does cell adhesion work the same for all types of cells?
Is it possible to stick cells on a fully formed ECM?
Does the size of the human kidney cells matter when sticking to a pig’s ECM?
Cell Differentiation
A scaffold without the right cells performing its function correctly is pretty useless (in terms of transplantation), so figuring out how to induce stem cells to become the right differentiated cell is very important.
Cell Differentiation - Experiments (not limited to the kidney)
VEGF with growth factor PDGF-BB (delivered in alginate) was shown to stimulate the growth of endothelial cells and smooth muscles (in heart cells)
Will human-sourced VEGF work within a pig environment?
Does porcine VEGF work with human cells?
In pig kidney ECMs it was found that a number of proteins unique to the scaffold remained such as keratins, antimicrobials, growth factors including TGF-β and EGF-7 and proteins that stimulate cell growth and differentiation [I can’t find the paper anymore but once I do I will be sure to link it!]
Are these proteins compatible with human cells?
What are the known cell signalling pathways for the different kidney cells, and do these exist in porcine structures?
Evidence of cell differentiation into endothelial cells in rat kidney
4-step strategy to differentiate stem cells into kidney progenitor cells
The presence of endothelial cells made a huge difference in the generation of other kidney cells (in mice)
A high dosage of VEGF was given
The kidney was reseeded and monitored for 12 weeks
Vascularisation (the creation of blood vessels) was observed
It detected 70% of urea
It detected 75% of the creatine (a type of protein)
ECM impacts cell growth and differentiation
Are the proteins found in porcine ECM the same as in human ECM? Do they have the same concentrations, same placements etc
Cell Differentiation - Questions
Does cell differentiation occur the same way in pig ECMs the same way it does in human ECMs?
What are the cell signalling pathways for all human kidney cells?
What impact is had if we differentiate cells before insertion vs after?
Does the size of the human kidney cells matter when differentiating in a pig environment?
Immune Response
Despite the reseeding efforts & challenges stated above, there remains an even bigger issue that has yet to be addressed - the immune response of decell-recell.
So far no one really knows if decell-recell will cause an immune response in the kidney recepient. However, some studies have been done on acellular organs (organs that have been decellularised and not reseeded) showing that it does provoke some level of immune response. It is unclear why this is, however most guesses suggest that it is because of the proteins in the ECM (the scaffold).
Immune Response - Experiments (not limited to kidneys)
Are these properties needed in cell adhesion, differentiation and/or distribution?
Some level of immune response seen in decellularized rat livers
Some level of immune response is seen in decellularized rat uterui
Given that the main attraction of decell-recell is an immunotherapy-free transplant, this seems like a pretty major assumption to gloss over.
Immune Response - Questions
Does the decellularization method have an impact on the immune response seen?
How sensitive is the immune system to the level of DNA removal possible?
Is there any overlap between the ECM properties needed for organ regeneration and those that cause an immune response?
Recap: Assumptions & Problems
Decell-Recell would lead to an immunotherapy-free transplant
So far we have not seen any strong evidence of this which is a problem if we aim to significantly increase the quality of life!
We have the right cell sources
It has been shown that iPSC can cause an immune response. If this is the case, then again, this is not aligned with our goals.
We also don’t know enough about iPSC functionality on a long enough time horizon, but in my opinion, this is not an issue that can’t be overcome.
We can distribute cells externally via different access portals
It is not entirely clear if this will eventually be possible, but so far the evidence suggests that its possible
In the case of the kidney, it seems as though the hardest part is getting into multiple intricate places such as the glomeruli.
We can stick foreign cells on a foreign scaffold
The compatibility between foreign proteins and human stem cells has not been studied enough (to the best of my knowledge).
If it turns out the two are actually incompatible then this could potentially be rectified by some level of genetic modification, however, this would significantly increase the price of the kidney and potentially kill the idea of having an immunosuppressant-free transplant
We can differentiate foreign cells within a foreign scaffold
Same issues as mentioned above.
Path Forward
Methodology
Most regenerative studies occur in mice or rats, which at first glance makes sense. They are cheap, highly abundant, low ethical issues and have a similar enough human anatomy. However, in the case of regenerative medicine they are uniquely different in a way that trumps all of this - they are so small! The width of a mouse is ~2cm. At this distance it greatly improves a mouse’s potential to regenerate nearly anything.
Given that the aim of these studies is to test regenerative methods, it seems as though an alternative animal model should be used moving forward. This is easier said than done as I imagine there will be a number of ethical issues associated with using a new animal model, however, the idea deserves to be explored at the very least.
Moving beyond better animal models, we also need an agreed-upon set of “success” metrics. One of the biggest issues within regenerative medicine, particularly in the case of bioartificial kidneys, is that success is measured differently. It should be a requirement that any experiment done in the pursuit of creating a bioartificial kidney, tests the ability to clear creatine, produce erythropoietin, urine and the many other functions a kidney performs at an agreed-upon level.
I suggest that all proteins and growth factors such as fibronectin should be measured and used alongside DNA staining to assess how effective the decellularization process was, as opposed to just the latter.
The relevant concentrations, placements and compatibility with human cells should be measured, not just a before & after measurement of placement and concentration.
What we can learn from the bladder
Could we have kidney augmentation surgery?
What if we shifted the focus (for now) to augmenting stage 2-4 kidney failure patients, where only a part of their kidney needs to be replaced? For this to be a reality we would need to know specifically what parts of their kidney (on average) get damaged at these stages. Is the damage within one area? How many functions are affected?
Can the cell adhesion and growth factor mixture used for bladder augmentation surgery be used for other organs? Or just the bladder?
Additional Questions & Ideas
“Spheroid cells exhibited polarized localization of podocalyxin (PODXL) to the luminal surface, zonula occludens 1 (ZO-1) to apical cell–cell junctions, and β-catenin (βCAT) to primarily basolateral membranes” (The cells that make up spheroids (round balls of cells) had a protein called podocalyxin on their surface, which is used to make tight connections between cells. They also had proteins called zonula occludens 1 and β-catenin in the right places, which help cells to stick together.) Is this a characteristic that could be exploited to increase cell adhesion?
Some tissues were stained for selectins (endothelial cell leucocyte adhesion molecule [ELAM]-1, CD62), integrins (very late antigen [VLA]-1, VLA2, VLA-3, VLA-4, VLA-5, VLA-6), immunoglobulin (Ig) supergene family members (platelet endothelial cell adhesion molecule [PECAM]-1, intercellular adhesion molecule [ICAM]-1, ICAM-2, class I heavy-chain proteins), complement adhesion molecules (CD34, CD44), and von Willebrand factor. Could these be used to increase cell adhesion? [I can’t find the original paper as the sci-hub link is broken, but once I do I will replace it]
When a stem cell is next to an differentiated cell it can differentiate and grow faster
Could we exploit this insight when trying to solve cell differentiation and distribution within pig ECM’s ?
We have done a pretty good job of creating kidney organoids (miniature, less developed kidneys). Given that the biggest issue of organoids are its lack of blood vessel system, and that is a solved problem with decell-recell, could there be a potential overlap between the two solutions? Is it possible to deposit already-grown kidney tissue on a scaffold?
Some early signs of success here with intestine organoids and decellularized scaffolds.
Can you get a tissue to the right place within an ECM?
What makes it so difficult to stick multiple cell types onto a scaffold?
Does the order in which these steps are applied matter? For example, does solving the adhesion problem for endothelial cells increase the likelihood of cell adhesion and differentiation of other cells because of the now good blood supply?
I am not entirely convinced decell-recell as its being pursued today is the solution that I as a patient would prefer. However, I believe if certain issues are better addressed s then it could be.