A Gel Coating Your Hairy Endothelium

To follow up on one of Nate’s posts from 2010, hair grows not only on your skin. He describes a glomerular capillary to be hairy. Glycocalyx, a hairy structure attached to the glomerular endothelium, is a mixture of glycosaminoglycans and proteoglycans. The picture of glycocalyx accompanying that post is quite impressive.

Now we know these hairs are coated with another gel matrix called endothelial surface layer (ESL). ESL, together with glycocalyx, is believed to function as a barrier to prevent protein passage from blood to urine. A recent article addressing this topic was published in JASN.

By using an animal model, the authors showed that loss of ESL increases the sieving co-efficient for albumin and that the degree of albuminuria correlates with the degree of ESL loss (by the way their confocal microscopy images of ESL are pretty cool).

The implication of the study is that it’s not just podocyte or GBM that are responsible for the development of proteinuria; ESL, glycocalyx and endothelium appear to play an important role as well. For example, loss of ESL has been reported in patients with diabetes. Maybe it’s not just on the skin where hair loss occurs.

For those who are interested in this topic, there is a nice review article for further reading.  

Posted by Tomoki Tsukahara

Mg & K

It is well known that hypokalemia does not correct easily if it is accompanied by hypomagnesemia. A medical student I met looked into this topic and found a “Science in Renal Medicine” article. According to this article, one of the mechanisms through which hypokalemia occurs in a hypomagnesemic state is through renal potassium wasting. Several observations have shown that magnesium infusion decreases renal K secretion in the distal nephron.

A study from Nature found that ROMK (aka Kir), one of two potassium channels in the distal nephron, is responsible for the distal renal K wasting in hypomagnesemia. The mechanism is that the intra-cellular free Mg blocks the pore of the ROMK channel and limits potassium secretion in a concentration-dependent manner; therefore low intracellular Mg level increases potassium secretion.

Some renal Mg wasting disorders (e.g. Mg channel TRPM6 mutation) do not always present with hypokalemia. Why is that? The reason is that you need 2 components for potassium excretion. One is increased K permeability of the ROMK, and the other is a driving force to secrete K like increased distal Na delivery or an elevated aldosterone level (via enhanced Na reabsorption in the distal nephron). It seems in these disorders you don’t have the second determinant for K secretion.

Another fascinating renal physiology article! Now we are still confused but have a better understanding of hypomagnesemia in a case of hypokalemia. 

Posted by Tomoki Tsukahara

A unified view of abnormal sodium regulation in the nephron

Summer has come to the northern hemisphere and it is time to praise our kidneys for all the hard work of preserving the intravascular volume. Although only 30% of Na reabsorption takes place in the distal nephron, this is the place for most common genetic diseases causing abnormal sodium handling:

What are the responsible proteins for these diseases?

1. Bartter syndrome: Na reabsorption decreases via NKCC2 due to defects in NKCC2, ROMK and basolateral Cl channels or from an overactive basolateral CaSR.

2. Gitelman syndrome: Na reabsorption decreases due to a reduced number of NCCs on the luminal membrane.

3. Pseudohypoaldosteronism type 1: Na reabsorption decreases due to the defects in ENaC or mineralocorticoid receptor.

4. Liddle syndrome: Na reabsorption increases due to a higher number of ENaCs on the luminal membrane (they are spared from ubiquitination).

5. Gordon syndrome: Na reabsorption increases due to a higher concentration of NCCs on the luminal membrane (defects in WNK4 or overactive WNK1). More recently, mutations were discovered in the genes encoding KLHL3 and CUL3 proteins.

CUL3 is a component of ubiquitin ligase and KLHL3 is its partner (expressed in the DCT). It is speculated that the defects in these proteins may change the distribution of NCC.

Is this clinically relevant?

In Gordon syndrome, these mutations can occur de novo and are encountered more frequently (47% for KLHL3 and 32% for CUL3) than those involving the WNK kinases (13%). It appears that they are under-diagnosed and some of the cases of RTA type 4 with hypertension could be caused by them!

50 years after Bartter syndrome was described, the work on mechanisms responsible for abnormal Na regulation in the nephron continues…

Posted by Tomoki Tsukahara MD 

 

Fractional Excretion

The fractional excretion of sodium (FeNa) is a test that is often used in the setting of acute renal failure to help distinguish between pre-renal and intra-renal causes that has been mentioned in previous blog posts. In general, a FeNa of <1% suggests pre-renal disease, between 1-2% is indeterminate and >2% suggests ATN. There are some exceptions to this but overall, the specificity of this test is more than 80% and this increases if it is used in combination with the fractional excretion of urea.

By definition, the FeNa is the ratio between the quantity of Na excreted in the urine relative to the amount filtered at the glomerulus. So how can we make this calculation with a spot sample without reference to volume of filtrate or urine? Given that Na is freely filtered at the glomerulus, this means that:

Filtered Na = Plasma Na x GFR

And:

Excreted Na = Urine Na x urine flow rate

Thus:

FeNa = Excreted Na

Filtered Na

= urine Na x urine flow

plasma Na x GFR

Because the creatinine clearance is a surrogate for the GFR:

FeNa = urine Na x urine flow

(plasma Na x urine Cr x urine flow)/plasma Cr

= urine Na x plasma Cr

Plasma Na x urine Cr

Or to make it easier to recall:

= Two smaller numbers

Two larger numbers

It is important to remember to take the serum sample at the same time as the urine sample because if the GFR is changing, this will affect the results. Remember also that the test can be unreliable in the setting of a near-normal GFR and with the use of diuretics, which is why combining it with the fractional excretion of urea can be useful.

This derivation can be used to determine the fractional excretion of any substance that is freely filtered at the glomerulus. However, remember that if the substance that you are trying to calculate is partially bound to albumin, an adjustment will have to be made. For example, Mg is about 30% albumin bound so that the plasma level would need to be multiplied by 0.7 for an accurate result.

Thanks to Dr Seifter for his help with this.

Surviving Stress in the Kidney Medulla

Hypertonicity in kidney medullary interstitium is essential for urinary concentration. Osmolality in the papillary interstitium can reach up to 1200 mOsm/kg. How do cells in the renal medullary interstitium survive this hypertonic stress?

Virtually all cells respond to hypertonicity by the accumulation of organic osmolytes (i.e. myo-inositol, betaine, taurine, glycerophosphorylcholine, sorbitol) and renal medullary interstitial cells are not the exception.

Hypertonicity upregulates the transcription of certain genes involved in organic osmolyte transport or synthesis such as SMIT (sodium-myo-inositol cotransporter), BGT1 (sodium chloride/betaine cotransporter), TauT (sodium chloride/taurine cotransporter), NTE (neuropathy target esterase, enzyme responsible for the synthesis of glycerophosphorylcholine) and AR (aldose reductase, enzyme responsible for the synthesis of sorbitol).

This increase in gene transcription is mediated by TonE (Tonicity-responsive Enhancer) located in the promoter region of these osmoprotective genes. TonEBP (Tonicity-responsive Enhancer Binding Protein), also known as NFAT5, has been identified as the transcription factor that binds TonE and stimulate transcription. Severe atrophy of renal medulla was observed in TonEBP knockout mice.

Urea is one of the main solutes responsible for renal medullary hypertonicity. However, high urea concentrations have been shown to cause cell cycle delay and apoptosis in renal medullary cells. HSP70 is a heat-shock protein found to play an important role in the protection against the deleterious effects of urea. TonEBP has also been shown to increase the expression of HSP70.

In summary, TonEBP is the master regulator of renal medulla for cellular protection against hypertonicity and high urea concentrations via accumulation of organic osmolytes and increased expression of protective heat shock proteins.

Mind the Gap

As Nate mentioned in a previous post, the urinary anion gap is helpful in differentiating whether a non-gap acidosis is of renal or extra-renal origin.

Urinary Anion Gap = Na + K – Cl

Because the major cation in the urine is NH4, this gives you a rough estimate of the NH4 level. In the setting of a distal RTA, the urine NH4 should be low and therefore there should be a positive anion gap. The problem with this test is that if there is some other unmeasured anion (e.g. ketoacids or hippurate following glue-sniffing) or even if the patient’s diet leads to significant changes in PO4 or SO4 excretion, it can be very inaccurate. One alternative suggested by Mitch Halperin is to measure the urinary osmolar gap. This is more useful because it detects the NH4 excretion regardless of the anion that is excreted along with it.

Urine Osmolar Gap = measured Uosm – calculated Uosm

Calculated Uosm = 2(Na + K) + Urea (mmol) + Glucose (mmol)

Because the other major cation in the urine is NH4 and this must be matched by an accompanying anion, most of the gap is therefore made up of NH4, giving you this formula.

Urinary NH4 = Urinary Osmolar Gap/2

The osmolar gap must be divided by two in order to account for the anion being excreted with NH4. Of course, this would all be easier if we could measure the NH4 directly. In our hospital, the assay for measuring NH4 is an enzymatic method using glutamate dehydrogenase. The lab was not able to give me a specific answer as to why they could not use this test on urine but looking around the net, it appears that it is not useful for measuring large quantities of NH4. The normal value in the serum is <35 µmol/L while in the setting of a metabolic acidosis, urine levels should be >200 mmol/day, orders of magnitude higher. So it seems for the moment that we are stuck with the osmolar gap as the best estimate in many hospitals.

Ref: Fluid, Electrolyte and Acid-Base Physiology, Halperin 2010

Pendrin – role in distal bicarbonate secretion

There are two major types of intercalated cell in the collecting duct of the nephron – the alpha and beta intercalated cells. We are probably most familiar with the alpha intercalated cells in terms of acid-base handling by the kidney. They have a luminal H+ATPase enzyme and a basolateral anion exchanger. This polarity allows them to pump H+ into the lumen, facilitating acid excretion, which is equivalent to reabsorption of bicarbonate.

The beta-intercalated cells essentially have a reversed polarity – they have a protein called pendrin in their luminal membrane. This protein is a chloride-bicarbonate exchanger, facilitating chloride entry to cells in exchange for bicarbonate excretion.

As mentioned in previous posts, mutations in pendrin can cause Pendred syndrome – characterized by sensorineural deafness, hypothyroidism and goitre.

It appears that pendrin is critical for bicarbonate excretion during a metabolic alkalosis, being up-regulated during this process and down-regulated by potassium depletion (an effect that could serve as a maintenance factor in a metabolic alkalosis). Pendrin also appears to play an improtant role in chloride reabsorption in the distal tubule, with increased distal chloride delivery being associated with decreased expression of pendrin and vice-versa. Furthermore, pendrin deficient mice appear to be somewhat resistant to NaCl induced hypertension.

An interesting case report highlighted the development of a profound metabolic alkalosis in a child with Pendred syndrome, after being treated with a thiazide diuretic. Inhibition of the NaCl exchanger in the distal tubule by the thiazide would have lead to increased luminal chloride. Presumably whatever small residual amount of functioning pendrin was completely shut off by the presence of high luminal chloride. This would have limited bicarbonate excretion, facilitating development of the alkalosis.

The Bezold-Jarisch reflex

This intriguing set of clinical signs was discovered by von Bezold and Hirt in 1867 – they found that injection of a veratrum alkaloid caused bradycardia, hypotension and apnea.

In the 1930’s Jarisch and Richter were performing similar experiments in cats to see what effect interruption of the cardiac branches of the vagus nerves would have. They were able to prove that the hypotensive effect described by von Bezold was reflex in origin. Many years later, Dawes was able to prove that the reflex apnoea occurred by a separate mechanism to the haemodynamic changes.

Today the Bezold-Jarisch reflex (BJR) refers to the discoveries of Dawes in 1947 and describes the triad of bradycardia, hypotension and vasodilation that occurs upon stimulation of cardiac receptors.

So, how does all this relate to nephrology? Well, this reflex is an important entity to be aware of when in the haemodialysis unit. To understand why, we need to delve a little bit further into the physiology of the reflex.

Mechanosensitive and chemosensitive receptors in the walls of the ventricles send afferent fibres through the vagus nerve to the vasomotor centres of the brainstem. The basal output from the vasomotor centre is mainly sympathetic, which keeps vessels partially constricted, thereby maintaining blood pressure - this is known as vasomotor tone. The afferent fibres of the BJR have a tonic inhibitory effect on the vasomotor centre, but have a very low rate of basal firing. Upon stimulation, they cause profound inhibition of the vasomotor centre, resulting in decreased sympathetic outflow, bradycardia, hypotension and vasodilation.

Now think of the haemodialysis patient who is 4 or 5 Kg up from their estimated dry weight. As we try to ultrafiltrate the patient, if the rate of fluid removal is greater than their rate of vascular refilling, then there may be trouble ahead. As early hypovolaemia develops, the baroreceptor reflex kicks in and simultaneously the BJR fibres decrease firing – the result is increased sympathetic outflow, increased heart rate and initially, preservation of blood pressure. However, as hypovolaemia becomes more severe, the ventricles contract more vigorously around a poorly filled LV cavity – this is thought to allow the BJR fibres to become paradoxically more active. At this stage the BJR overrides the baroreceptor reflex, causing vasomotor inhibition and the resulting bradycardia, hypotension and vasodilation.

Overall the BJR is felt to be cardioprotective – by causing bradycardia and afterload reduction via peripheral vasodilation, the workload of the heart is reduced and hopefully ischaemia is avoided. See here and here for more information.

I think this is an interesting physiological principle to be aware of in the dialysis unit, where the prevalence of LVH and multiple cardiovascular risk factors is so common. We must closely watch the heart rate and blood pressure during the treatment. Often, tachycardia precedes the precipitous drop in blood pressure – therefore those patients may need closer monitoring and titration of the UF rate.

Hot peppers for hypertension?

An interesting article was published in the August 4th edition of Cell Metabolism. I'm always intrigued when common food ingredients are used to modulate biological systems. This article explores the effect of capsaicin on blood vessel tone. Capsaicin (or 8-methyl-N-vanillyl-6-nonenamide) is what makes "hot peppers" taste hot. This is achieved by activating a particular TRP channel (see Lisa's or Nate's prior post on TRP channels) called the TRPV1 channel. Capsaicin activates the TRPV1 channel and leads to an increase in intracellular calcium. This, in turn, causes the release of several neuropeptides such as substance P or calcitonin gene-related peptide. When a hot pepper in eaten, these changes in sensory nerves leads to the sensation of pain and local heat production.

What other tissue types express TRPV1 channels? And what role do these receptors play in patho- and physio-logical states? It has been previously demonstrated that TRPV1 channels are present in blood vessels. This study demonstrates the presence of TRPV1 in cultures endothelial cells as well as expression in the endothelial layer of an intact vessel. However, there have been a few often contradictory articles published about the acute actions of capsaicin on the blood pressure. Several investigators have shown a relaxing effect of capsaicin in various isolated vessels of pigs and rats. On the other hand, it has also been shown that TRPV1 activation causes vasoconstiction in vessels.

This study demonstrated that chronic TRPV1 activation by capsaicin led to enhanced production of the vasodilatory substance nitric oxide by endothelial cells. After performing several in vitro studies to verify this finding, the investigators fed spontaneous hypertensive rats a diet rich in capsaicin for 7 months and measured blood pressure. This led to a 15-20mmHg decrease in blood pressure as measured by radiotelemetry. However, this decrease in blood pressure was only evident after 4 months of treatment. An excellent editorial written by Dr. Sessa goes into more detail about this study. In conclusion, this is an interesting and provocative study. Much more research is needed before we can offer capsaicin as a therapy for hypertension. But, this could potentially lead to novel therapies aimed at activating the TRPV1 channel. It is still unclear exactly how capsaisin lowers blood pressure in SHR rats. It is likely that other systems are affected by chronic TRPV1 activation that can affect blood pressure. Such as the sympathetic nervous system or the renin-angiotensin system. For now, I'll keep enjoying my yearly hot pepper garden. I'll be interested to see where this research goes from here.

Structure & Function of Other Animals' Kidneys

Here's a very brief overview of the kidneys of animals (other than humans). All vertebrates have kidneys and the functional unit of the kidney in all species is the nephron.  Freshwater fish--like the zebrafish--have a kidney which is kind of like a long tube.  In zebrafish embryos (as discussed previously in another post) the kidney starts out as a structure called the pronephric duct, which consists of a single nephron, and as the fish becomes an adult develops into the adult mesonephros which contains multiple nephrons.  The function of the freshwater fish kidney is predominantly to create a dilute urine that osmolar homeostasis. 

The frog kidney must be able to adapt to both freshwater and land conditions--thus the frog kidney must be able to create both a dilute and a concentrated urine, depending on the environment to which the frog is exposed.

Snakes live in a dry environment and therefore their kidneys must retain water.  Snakes metabolize nitrogenous wastes into uric acid, which can be excreted in an insoluble form using very small amounts of water.  

The bird kidney also eliminates waste via uric acid.  The whitish pasty component of birdshit is in fact a uric acid paste that represents the renal waste product, which is usually mixed in with the brown stuff (the feces from the GI tract).  

Mouse kidneys are similar to human kidneys in that they use soluble urea, rather than insoluble uric acid, as the waste product.