#NephMadness 2018: Animal House Region

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“Saying the product of the kidneys is urine is like saying the product of a factory is pollution.  Urine is a by-product. The product is homeostasis.”

Nephrologists are exceedingly proud of the fact that the kidney is the organ primarily responsible for maintaining homeostasis in the body. Not enough water? ADH reabsorbs it in the collecting duct. Too much potassium? Aldosterone kicks it out of the principal cell. The fact that no normal values exist for urine sodium, osmolality, potassium, etc show that no matter the surroundings, the kidney is making adjustments to maintain homeostasis.

This region of NephMadness 2018 highlights some of the amazing physiology that exists in the animal kingdom outside of the human kidney. Yet the remarkable physiology displayed by these animals all share the same goal of the human kidney in maintaining homeostasis, be it osmolarity, pH, electrolyte, or water balance. We will look at two fascinating matchups – osmolality balance in the ocean, and water storage in desert. First, let’s meet our selection expert!

Selection Committee Member for the Animal House Region:

Mark Zeidel, MD

Dr. Zeidel is the Herman L. Blumgart Professor of Medicine at Harvard Medical School and Chairman of Medicine at Beth Israel Deaconess Medical Center. His research has focused on mechanisms of water flow across biological membranes, and his observations defined the role of atrial peptides in renal salt excretion and characterized the biophysical function of aquaporin channels.


 Competitors for the Animal House Region

Shark Osmolarity vs Salmon Osmolarity

Camel Water Storage vs Toad Water Storage

Shark Maintenance of Osmolarity

A brief backstory is necessary here as an introduction. In August 2017, a series of tweets caught the eye of many nephrologists:

Although the Greenland shark is not actually made of pee, there is truth to the fact that sharks in general maintain extremely high levels of urea. Some of these earliest observations of shark osmolality come from the grandfather of kidney physiology himself, Homer Smith. Many of his experiments were performed on fish in a laboratory at Mount Desert Island (MDI), Maine (the same MDI that holds the annual “Origins of Renal Physiology” course).

In the late 1920s, Homer Smith recognized that urea was retained in sharks as an “osmoregulatory ballast” to raise body fluid osmolality to be slightly hyperosmotic to the ocean the shark swims in. This is particularly impressive as the shark typically maintains an osmolality of 900 – 1000 mOsm/kg. Other marine animals such as bony fish (eg salmon) and mammals (eg whales) maintain lower serum osmolalities near 300 mOsm/kg, with their osmolality made up predominantly of sodium salts.

Earlier observations noted high concentrations of urea, but hypothesized that its presence was similar in function to mammals – a waste product of nitrogen metabolism to be eliminated by the kidneys. Smith rightly saw urea as a metabolite specifically retained for its osmotic pressure, and demonstrated that even if you observe fasting sharks (a seemingly treacherous activity), the high levels of urea persisted. In Homer Smith’s seminal book From Fish to Philosopher, he notes:

Urea also has an obvious role in the human kidney – particularly with regard to maintenance of the concentrated interstitium and countercurrent multiplication. In the shark, urea’s presence in all bodily fluids act as an organic osmolyte. In humans, urea is a tiny portion of osmolality (BUN/2.8 if using mg/dL – in healthy individuals, only 5/290, or 1.7% of total osmolality). In shark serum, urea is measured at levels of 1300 mg/dL, making up roughly 30% of their osmolality.

Interestingly, to protect the tissue from the effects of such a high concentration of urea, sharks also maintain high levels of free amino acids and methylamines to counter the effects of urea on protein and enzyme function. To prevent passive loss of urea across the gills, sharks have a specialized back-transporter on the basolateral membrane to return urea into the bloodstream and the apical membrane of the gill is relatively impermeable to urea. In addition to countering loss at the gills, the shark kidney reabsorbs up to 99% of filtered urea (against a concentration gradient) via active Na/urea cotransport.

In addition to the role of urea in osmolality, another remarkable Homer Smith prediction pertained to the rectal gland of the shark. Also discovered at the MDI Lab, the rectal gland functions to secrete a hypertonic sodium chloride solution up to 1.33 mL/kg/h (an amount that exceeds urine flow in the shark). As Smith observed that shark kidneys were not capable of producing hypertonic urine, he inferred that the “elasmobranchs excrete by some extrarenal mechanism a solution of NaCl that is hypertonic to their plasma.” Because of their osmoregulatory strategy of using urea, levels of sodium and chloride in blood are distinctly lower than the surrounding seawater. As a result, there is passive movement of NaCl into the body, and the shark excretes this excess via the rectal gland.

If the thick ascending limb of the loop of Henle is in the tournament, then the shark rectal gland is the #1 seed to beat. The membranes of shark rectal gland are incredibly dense – they contain 50 times the number of Na/K/2Cl cotransporters of the human kidney (the most of these transporters of any known tissue). Additionally, the gland is highly accessible via a single artery and vein, and contains a single type of tubule with the sole physiologic function of excreting salt. Together, these attributes make the shark rectal gland particularly conducive to study. As it mimics the thick ascending limb of the mammalian kidney (an area difficult to access even with micropuncture), we have learned a great deal about loop diuretic pharmacology, properties of Na/K/2Cl transport, and even natriuretic peptides from the shark model.  

Figure from MDI Laboratories comparing the shark rectal gland to the thick ascending limb. The rectal gland secretes chloride and sodium while the thick ascending limb absorbs these ions, with the relevant transporters placed on different membranes. However, the transporters function in the same way in both tissues, which is why the rectal gland is such a good model for the thick ascending limb.

Observations of the shark by Homer Smith (and colleagues since) have laid a strong foundation of kidney physiology. Next, let’s look at the salmon!


Salmon Maintenance of Osmolarity

If the shark is incredible for its ballast of urea and the ability to secrete salt from its rectum, salmon are equally impressive in their ability to adapt from freshwater to salt water and back while maintaining osmotic equilibrium. To understand how remarkable this is, we first need to understand the life cycle of a wild salmon.

Salmon belong to the subclass of teleost fish. The female’s nest of eggs is fertilized in freshwater. In springtime, the fish hatch into this freshwater environment where the osmolality of their surroundings is 0 mOsm/kg. Depending on the species, they spend their first several months in this water but gradually move downstream via estuaries towards the ocean; the osmolality of their environment also gradually increases along the way. Eventually, they transition entirely to ocean life, where salt can increase the osmolality to well over 1000 mOsm/kg. They spend years in this salty environment before journeying back to freshwater streams to spawn the next generation of fish.

To sum this up, a salmon needs to maintain a constant internal osmotic pressure irrespective of their presence in both hypotonic and hypertonic environments. In freshwater, it must counteract passive loss of ions to the environment. In seawater, it must counteract passive loss of water. Somehow during the transition from freshwater to seawater, its gills must transform in function from a salt preserving to a salt secreting organ. How does this occur?

The gills are traditionally thought of as the fish equivalent of the human lung – necessary for oxygenation. They function well in this capacity, and are able to extract an adequate amount despite the low oxygen tension by moving large quantities of water past the gill. However, gas exchange is only one of many physiological processes that are mediated by the fish gill. Because of the large surface area of the gill epithelium, this organ is also the site of passive movement of salt and water down ionic gradients between the fish plasma and seawater. Just like the human kidney, fish gills maintain osmolality, balance pH, and remove nitrogenous waste products. Let’s focus on how this develops.

Teleost fish, like the salmon, use osmosensors to detect changes in their surroundings. In freshwater, salmon drink only when necessary, much like humans. They passively lose salt into their environment and minimize excess water intake in addition to making a maximally dilute urine. As adult salmon move towards the ocean they acclimate by gradually moving into saltier environments. During this phase, their kidneys adapt to create a more concentrated urine.

Once fully transitioned to life in the hypertonic ocean, two processes put the fish at risk for salt overload. Water passively diffuses out of their bodies and they also increase ingestion of the hypertonic seawater. To counter this process, the function of their gills must change. Na/K ATPase and Na/K/2Cl transporters in the gills are massively upregulated to actively pump out concentrated NaCl through their gills (the exact mechanism here is complicated but involve calcium sensing receptor activation with subsequent pathway signals leading to upregulation of NaCl transporters). Like the rectal gland, the gills secrete chloride through apical chloride channels. Like both the rectal gland and the thick limb, sodium can cross the epithelium via paracellular pathways. Also, like the thick ascending limb, but unlike the rectal gland, in gills the movement of NaCl from inside the fish to the sea is not accompanied by water, because the apical membranes of these cells are water-tight. Upon returning to freshwater to lay their eggs and die, salmon again acclimate gradually to less brackish waters with downregulation of salt excretion across their gills as their environment becomes more dilute.

Another point that should be made about maintenance of salmon osmolality pertains to their gastrointestinal tract. The consumption of hypertonic seawater poses unique challenges for the GI tract outside of digestion and nutrient absorption. Their GI epithelium is lined with H+ pumps and carbonic anhydrase (similar to our proximal tubule), Na/K/2Cl transporters (similar to our loop), Na/Cl co-transporters (similar to our distal convoluted tubule) and Na/H antiporters (similar to our intercalated cell). See figure below:

Digestion is a completely separate process – ingested food is shoved into blind pouches beyond the stomach (called pyloric caeca) which are closed off so that the remainder of the intestine can focus on ion transport and homeostasis.

These physiologic observations have resulted in very real clinical implications. The earliest experiments on tissue that show fluid movement against an osmotic gradient via active Na/Cl transport were performed in teleost intestine. These mechanisms in animals have obvious similarities to humans and have informed much of what we know about kidney epithelial water and ion transport. As an added bonus, this topic will almost certainly spark a lively dinner conversation the next time you are enjoying sake nigiri.


Camel Water Storage

Most kindergarteners know that camels drink a lot of water and can survive in the desert for days (even weeks) without drinking water. A common misconception is that water is stored in the camel’s hump. In truth, the camel’s hump stores fat, which is metabolized into water as the fat is burned.  

The word “dehydration” is frequently misused by clinicians when in fact they mean “volume depletion.” Saying “she looks dehydrated” in a patient with a systolic blood pressure of 80 and orthostatic symptoms will make your nephrology colleagues cringe because what you actually mean is “she is volume depleted,” or, more precisely, that the patient lacks adequate preload, leading to inadequate cardiac output. Dehydrate literally means to lose water, which results in serum concentration and subsequent hyperosmolarity. As camels can go days (sometimes even weeks, depending on conditions) without drinking water, they truly get dehydrated. When they are given access to water, a camel that weighs 400 kg can drink over 100 L of water in less than 5 minutes. What happens to their osmolarity during these drastically different periods?

Like humans, camels maintain an osmolality of roughly 300 mOsm/kg. Camel husbandry practices from Ethiopia show that during dry season, a camel can go without water for 2 weeks and up to 2 months during rainy season (due to increased moisture content of plants that are grazed). During water deprivation, the camel has developed multiple mechanisms to conserve insensible losses of water including:

  • Body temperature maintenance (fluctuates between 35-41oC to minimize evaporative losses)
  • Desaturation of exhaled air (via heat exchange between inhaled air and the surface of nasal passages)
  • Concentration of urine (a “dilute” urine for a camel is 1000 Osm/L).
  • Reduction in feeding to reduce water consumed in metabolism, plus metabolism of the fat in the hump, which generates free water.  

Despite these adaptive responses, as camels dehydrate over their 2 weeks without water, their Na rises from 154 to 191 mEq/L, with corresponding serum osmolality rising from 304 to 406 mOsm/kg. They lose 30% of their body weight and their hemoglobin doubles due to water loss.  Despite this change, they have no alterations in their mentation or functional ability. When given access to water, they re-hydrate rapidly; they lower their serum Na and osmolality to baseline levels within hours. And again, despite this massive shift, their mentation and neural function remains intact. Remarkably, these large swings in serum osmolality do not alter the camel’s neurological functioning. The camel here is the exception to the rule of homeostasis, but this aspect is also what makes its physiology so remarkable!

These massive shifts in osmolality would lead to potentially devastating consequences in humans. The question then becomes: “How does a camel tolerate dramatic dehydration/rehydration?”  The remainder of this section is open for discussion as very little has been written about this unique perturbation in osmolarity. Maybe the camel brain is sealed separate from serum osmolality to prevent brain cell swelling. Or perhaps their blood/brain barrier is impermeable to water. More appealing to me personally is the possibility that camels have developed a refined cellular mechanism to deal with osmoregulatory changes (expelling and retrieving organic osmolytes) to prevent water shifts between cells. All water-stressed organisms accumulate organic osmolytes such as free amino acids, urea, methylamines, etc, to minimize cellular swelling during osmolar shifts. Camels may simply do this better than humans due to evolutionary necessity over many generations.

Next time you’re out on the basketball court with sweat pouring off your head in 90° heat, just remember that no matter how thirsty you may feel, you will never be as dipsogenic as a dromedary!


Toad Water Storage

The bladder of a desert toad is much cooler than ours. The human bladder is a muscular sac that holds final urine until it can be expelled from the body. It is remarkably impermeable, maintaining enormous gradients for hours of normally permeable substances like water, ammonia, and urea, but it does not otherwise modulate homeostasis. As a nephrologist, I regretfully disregard the bladder unless it is causing a plumbing issue in the ureters or the kidneys above. However, the toad bladder is an all-around player that does much more.

Toads live in freshwater and drink essentially pure water, which their kidneys filter and excrete into their bladder as an extremely dilute urine (50 mOsm/kg) containing nitrogenous waste as its only solute, and their bladder stores this dilute urine. Toads often will leave their watery environment and sit on rocky or grassy surfaces to eat insects or other organisms.

If you caught toads/frogs when you were younger, you may have noticed that their skin is very thin and is permeable to water. As the toad basks in the sun, waiting for the right dragonfly to go by, water is lost through its skin via evaporation with subsequent increase in the amphibian’s osmolality. To counter this, it needs to reabsorb the water it drank earlier from its bladder. If you thought this sounded eerily similar to the work of ADH and the collecting duct of the kidney, you’d be absolutely right. Before we ramble on about toad bladders, think about what happens when you go to bed at night; as you sleep, you experience insensible water loss, and your serum osmolality increases. This is sensed in your hypothalamus osmoreceptors, which promote ADH release. ADH circulates to your distal nephron, where it stimulates reabsorption of free water and excretion of a concentrated urine, correcting the rise in serum osmolality. When you wake up and void, you pass a concentrated, dark yellow urine.

A simple breakdown of ADH and aquaporins at the cellular level is as follows:

  1. In the setting of hyperosmolality, ADH is released from the posterior pituitary.
  2. ADH circulates and binds to a vasopressin receptor on the basolateral side of the collecting duct epithelium.
  3. Intracellular cAMP is increased and protein kinase A is activated with subsequent phosphorylation of aquaporin 2 (AQP2) in endosomes.
  4. Endosomes traffic to and fuse with the apical membrane with insertion of AQP2 water channels, leading to a 100-fold increase in water permeability.

We have only had this knowledge for the past 30 years or so, but as the Homer Smith quote demonstrates, human observations of water transport date much further back.

If you dissect a toad, the hemibladder can be so large that it takes up half of the entire organism. Measuring the contents reveal a dilute fluid with an osmolality of 40 mOsm/kg. Placing an enclosed bladder alone in a hypertonic solution does not result in any water flux across the membrane. However, Bentley and colleagues observed in 1958 that neurohypophysial extract stimulated water transport across the toad bladder when the sac was filled with a hypotonic fluid. They also observed that the vasopressor fraction from the posterior pituitary (aka ADH) was much more active in this process than the oxytocic one. Additional observations showed that hormone withdrawal led to removal of water permeability, raising the question of water channels moving to and from the cell surface. The term “shuttle hypothesis” was coined in 1981 by Wade et al to theorize the concept of channel insertion.

Eventually the channel protein was discovered on red blood cells and initially named “CHIP28” (channel forming integral membrane protein of 28 kDa) before being named “aquaporin.” The discovery netted the 2003 Nobel Prize in Chemistry for Peter Agre when he again harnessed another unique animal physiology model; in vitro transcribed CHIP28 RNA was injected into Xenopus oocytes which were then placed in hypotonic buffer. In control eggs, there was no movement and the cell did not swell. Eggs injected with the aquaporin RNA rapidly swelled and exploded when placed in the same solution. The effect was reversed by mercury chloride which is a known inhibitor of aquaporin.

It is indeed incredible that our understanding of water handling in the human collecting duct was observed, studied, and translated directly from the bladder and eggs of toads and frogs. In a fascinating demonstration of the adage “necessity is the mother of invention,” Aboriginal Australians have been known to traverse incredible distances in arid environments without carrying water. To survive, they learned to drink the dilute urine of the toad bladder.

 Post written by Timothy Yau (@Maximal_Change)

How to Claim CME
US-based physicians can earn 1.0 CME credit for reading this region. Please register/log in at the NKF PERC portal. Click on “Continue,” click on the “Animal House Region,” then click on “Continue” to access the evaluation. You’ll need to click on “Continue” again to complete the evaluation, after which you can claim 1.0 credit and print your certificate. The CME activity will expire on June 15th, 2018.

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