Chapter 3

Looking deeper: The X factor

A mysterious substance in cyst fluid

So we have looked at injury of kidney cells earlier in this book (page 5) and we have concluded that injuring a kidney cell in a PKD patient increases the chances of this cell becoming cystic as it switches to fermentation. The next question would be: is this always the case? Or is there an instance where injuring cells over and over does not lead to cyst growth and instead the cells retain the ability to heal and revert back to their healthy state? Yes, such cases have been described in several studies and they are highly intriguing. The question then becomes, what differentiates a cell that reacts to injury with uncontrolled growth from a cell that does not?

To elucidate this, I will take you down the path of some exciting research. As long ago as 1970, Darmady and colleagues had hypothesized that the expression of cysts in PKD might be connected to a specific toxin since the changes in renal structure were found at points where toxins were 'maximally concentrated'.1 This was followed in 1995 by experiments using extracted cyst fluid from PKD patients, applied to healthy non-PKD tissue. The researchers found that this fluid stimulated 'fluid secretion, cyclic adenosine monophosphate accumulation, and cell proliferation', which are hallmarks of PKD, even though there was no mutation present. The toxins themselves were enough to exhibit the same effect.2

In the same year, a group of researchers led by JJ Grantham tried to dig deeper and used mass spectrometry to narrow the constituents of the cyst fluid down to a single substance that actually was responsible for triggering cyst growth. While they were able to concentrate the fluid to a point where its secretory activity was 48-fold above that of the original substance, meaning its ability to produce fluid that could fill a cyst was 48 times higher, they could not narrow it down to a single substance and described it as a fraction of lipids enriched in monoglycerides.3

Nine years before that, Avner had led a study in which he showed that complete regression of cystic changes was possible, after only 120 hours, when he removed the cell culture from its environment, thereby removing all of the present toxins as well.4

Endotoxin

Even though the mass spectrometry study didn't yield conclusive results and bears repeating now that our technology has evolved further, we might still be able to look at other analyses of cyst fluid from PKD kidneys to shed more light on the issue.

Miller-Hjelle and colleagues in 1997 found the substance lipopolysaccharide (LPS), also known as 'endotoxin', or its remnants in all samples of cyst fluid retrieved from human PKD kidneys. LPS, or endotoxin, is part of the outer shell of gram-negative bacteria, which shed it either chronically while they're alive or in a single large bolus upon cell death.5

To understand cyst growth better, we'll take a look at what researchers usually do to create cystic tissue to study. The injury that researchers need to start cyst growth is sometimes triggered by using a chemical such as NDGA (nordihydroguaiaretic acid, a kidney-toxic chemical compound). In 1987, Gardner and colleagues found that to provoke cyst growth with NDGA, they had to add endotoxin.6 Without endotoxin, NDGA injured the kidney cells, but no cyst growth was observed.

Another breadcrumb was added to this mystery in 1990, when Miller and colleagues stated that 73% of PKD patients tested positive for endotoxiuria, meaning endotoxin excretion in urine, which 'raise[d] the possibility that endotoxin is available intrarenally to promote cystogenesis' - meaning, endotoxin is present inside the kidneys to stimulate the development of cysts.7

It's starting to look like there is a connection between cysts and endotoxin. Is this a PKD thing? Or is this a general cyst thing? What about other cystic diseases? Could this be the X-factor that is necessary for cyst growth and PKD progression? As it happens, both polycystic ovary syndrome (PCOS, where the cysts actually are fluid-filled follicles) and even cystic acne have been hypothesized to be connected with the presence of endotoxin.

Now, to repeat what I said above, endotoxin comes from gram-negative bacteria. But these bacteria shouldn't even be inside our kidneys or ovaries or skin in meaningful amounts, should they? Where are they coming from?

There was a paper on PCOS in 2012 by Tremellen and Pearce that hypothesized 'disturbances in bowel bacterial flora ("Dysbiosis of Gut Microbiota") brought about by a poor diet creates an increase in gut mucosal permeability, with a resultant increase in the passage of lipopolysaccharide (LPS/ endotoxin) from gram-negative colonic bacteria into the systemic circulation. [.] Thus, the Dysbiosis of Gut Microbiota (DOGMA) theory of PCOS can account for [.] the development of multiple small ovarian cysts.'8

So, in PCOS it might be that a poor diet gives you leaky gut and, in turn, endotoxin can permeate through the gut wall into the bloodstream and finally end up in the ovaries. Of course, this mechanism might easily translate to PKD. It might even be more likely; kidneys are our main filtration system after all.

Even worse, as we now know, the PKD mutation makes our tight junctions in the gut, which are responsible for holding in what needs to stay in and letting out what needs to get out, more permeable. The mutation gives it more elasticity, which means there is a higher amount of anything going through from the get-go.

Now, what does endotoxin bind to in our bloodstream? How do we usually mitigate this issue?

One of the molecules that endotoxin binds to in our bodies is an enzyme called argininosuccinate synthase (AS). This is one of our endotoxin shields. It's an enzyme that we need for proper excretion of excess nitrogen from protein intake, as well as for synthesizing adequate levels of the amino acids argininosuccinate and, indirectly, arginine. As it turns out, PKD patients have extremely reduced levels of this enzyme. In one rat PKD model study,9 at 21 days old the rats already had their AS levels reduced by a staggering 90%. That's about six years old in human years. So adding insult to injury (pun intended), PKD patients don't just seem to have increased endotoxin; they also have very low levels of this enzyme needed to inactivate it, maybe because it's being used up so quickly?

So, the hypothesis would be:

• bacteria produce endotoxin, which then
• permeates through the bloodstream, where it
• can't be inactivated because of low levels of AS and
• ends up in the kidneys, causing cysts,
• triggered or potentiated by additional injury?

Maybe at least part of this has already been studied somewhere?

Luckily, there is a mouse model of germ-free mice. These mice have been bred to have a sterile intestinal tract (poor mice) and therefore no gut flora. When in 1986 Gardner and colleagues used germ-free CFWwd mice, which are used to model human PKD, and fed them a microbe-free diet, virtually none of them developed cystic kidney disease. Since they do not possess any gut microbes, there's no source of endotoxin in these mice. Once removed from their sterile environment and given the chance to seed a gut microbial population, they began to develop cysts.10

Huh. This is telling us something.

While this hypothesis might be new in PKD, a very similar hypothesis has actually been well established since 2010 for liver cirrhosis. James P Nolan, in 'The role of intestinal endotoxin in liver injury' wrote: 'The overarching concept is the universality of the role of enteric endotoxin in liver injury from toxic agents. [.] eliminating or reducing the enteric endotoxin pool protects the liver from injury by all such agents.'11

Now, why might the body react to endotoxin this way? Obviously, growing cysts in response to endotoxin is not a specific property of the PKD mutation. We discussed earlier how we have found research that shows cyst fluid can trigger cysts in non-mutated tissue.2 There is even research that shows other kinds of cyst can be triggered by endotoxin.12 So why does the body decide to make cysts when it comes into contact with endotoxin at injured cell sites? Is it possible that this is an ingenious form of 'the solution to pollution is dilution'? Is it possible that a cyst is a way to mitigate damage when the ability to detox endotoxin is impaired?

If that were the case, any future medication that would block cyst formation without taking care of the functional defects in the kidney cells could potentially lead to unwanted effects of toxicity or even sepsis.

The ketone connection

We have looked at the capacity of ketones to modulate PKD cyst growth already, but this might not get to the root of the issue just yet. Nolan in...