Polycystic kidney disorder

From Biol557

• started here on 04/14/10.

• We'll do PCKD first.
  • It is a crossover between kidney and liver.
  • We won't have liver or bile background but she'll fill us in as we go.

Contents 1 Polycystic kidney disease 1.1 Background 1.2 Current treatments 1.3 Types 1.4 Liver disease aspect 1.5 What do we know about PCKD? 1.6 PCKD as a ciliopathy 1.7 Micrograph 1.8 Research questions 1.9 A simpler research question 1.9.1 Analysis of differences in ciliar length 1.10 Other aspects to cyst growth 1.11 What do we know so far 1.12 Questions 1.13 Stimulation of anion secretion 1.14 Does human renal cyst fluid contain material that stimulates ion transprot in the MPK cell line? 1.14.1 Short circuit current electrophysiology 1.14.2 Chloride secretion model 1.15 What is it in the fluid that cuases increased secretion 1.16 Stimulation of anion secretion 1.17 Conclusion 1.18 Treatment options 1.19 Nature medicine paper 1.20 The V2 receptor antagonists are now in human clinical trials (phase 3) 1.21 Other drugs in development 1.22 Etc.

[edit] Polycystic kidney disease

• It is a genetic disease just like CF and hypertension.
  • We know less about PKD than the others.
• There are fluid filled cysts that develop in the kidney.
• they develop all over the kidney.
• They are gross.
• They are slow growing.
• Most don't know they have the disease until their early 30s.
• The autosomal dominant version is slowly growing.
• It is more common than CF, about 1 in 500.
• Fourth leading cause of renal failure; around 55.
• Sticks around because people procreate before they know they have it.

[edit] Background

• There are other diseases that manifest cystic kidneys, too.
• In PKCD, other organs (esp. the liver and pancreas) turn cystic. So it isn't limited to the kidney but because that's what causes death, we name it such.
• Crazy picture!
• You can either get a transplant, be on dialysis, or die once the kidneys fail.
• Kidneys become enormous and develop fluid filled cysts with either clear liquid or blood-filled liquid.
• In liver cystosis, it isn't the hepatocytes that turn cystic, it is the bile duct epithelial cells.
• Renal function does not decline until 40, years after cysts began forming.

[edit] Current treatments

• Aspiration of the largest cyst is done to eleviate pain.
• Organ transplantation.
• We'll talk about some of the drugs being developed.
  • However, the major problem is the time course of the disease: how do you measure effect of drug on course of disease when it takes 30 years.
  • Renal function doesn't change until the 40s (good for the patient, bad for the trials).
  • Ideally one treats in the adolescence.
  • However, we don't have any biomarkers for measuring efficacy (which is a requirement for FDA approval).

[edit] Types

• Dominant
  • progresses slowly
  • common form
• Auto recessive
  • Causes problems in early life.
  • Open cysts in the kidney.
  • Closed cysts in the liver.
  • Therefore, liver is the problem if the child lives through early health problems.

[edit] Liver disease aspect

• Pain is a major issue.
• Hepatocytes aren't affected so organ function isn't compromised.
  • therefore liver transplant is unnecessary.
• It is about pain management.
• The liver epithelial cells (cholangiocytes) look just like the epithelial cells of the kidney.
  • they have transporters similar to those in the distal nephron.

[edit] What do we know about PCKD?

• We know exactly which proteins are mutated.
• ARPKD (autosomal recessive polycystic kidney disease) -> PKHD1 = fibrocystin.
• ADPKD -> PKD1 or PKD2, called polycystin 1 and 2.
  • These each cause the disease but of slightly different severity.
• These are found in many places of the cell, most interstingly is the primary cilium.
  • Epithelial and endothelial cells have these. It is a single, long cillia on the surface of the cell.
• PKD1 codes for a mechanosensitive Ca channel.
• We don't have to know all of the parts from the Torres figure.
  • The point is that PKD1 and 2 are in complex.
  • They are always found together.
  • PKD2 is the channel and PKD1 is thought to regulate the channel.
  • Fibrocystin is thought to be part of regulating the channel, too, but this is not well established.
• Second image from Torres, 2009
  • We've found twice as many intermediate steps in these pathways over the last year.
  • The primary cilium is on the apical membrane.
  • It is probably a flow censor.
  • Our three proteins are in the cilium.
  • When it is bent by flow (and we're talking wildtype here), ca comes into the cell, and that may trigger ca release from the ER.
  • this increases the ca levels in the cell.
  • This triggers phosphodiesterase which breaks down cAMP.
  • The ca will also block adenylyl cyclase, which starts the cAMP-PKA pathway.
    • This also decreases cAMP.
  • cAMP activates CFTR which puts Cl in the lumen and increases fluid movement into the lumen.
  • So we turn on the turn off pathwSay when bent so that we don't secrete stuff.
  • this is all normal.
  • When mutated:
    • There is no way to turn off the secretory pathway.
  • What about proliferation and why do we care?
    • When building huge cysts, you have to have lots of epithelial prolif.
    • PKA also controls cellular proliferation. We don't need to memorize this. Only that PKA increases cellular proliferation. So when broken we don't damp down cell proliferation as we should.

[edit] PCKD as a ciliopathy

• It is a ciliopathy because the defective proteins are found in the cilia.
• Meckel-Gruber syndrome
  • Brain on outside of skull.
• Bardet-Biedl syndrome
• Nephronophthsis
• These all cause cyst formation.
• These all have mutations in the cilia.
• These are all very rare.
• So we came up with the term ciliopathy.
• So we believe that the cilia plays an important role in proliferaiton and secretion.

[edit] Micrograph

• Here we see a micrograph of a primary cilia.
• What does it do?
  • It is involveed in dorsal-ventral patterning during embryonic development.
    • This is sometimes found in BB disorder patients.
  • Thought to sense flow of fluid in lumen of kidney and bile duct.
  • Thought to be a signal sensor for secretory events, like we saw int he Torres diagrams.
  • It is thought to be signal sensor to limit proliferation.
  • Might be important in growth arrest.
  • Might be important for cell polarity signaling.
• These could all be correct or none be correct; only proposed.

[edit] Research questions

• How do mutations cause cyst formation?
  • Maybe because of excess cell proliferation. If you have a small cyst, does it grow because you have cellular proliferation or does it grow because you're secreeting fluid and increase in fluid causes cyst formation. Chicken or the egg?
  • Excess secretion into tubular lumen.
  • Mis-localizaiton of ion transproters to the wrong membrane. Maybe if the cilia is messed up the targeting of transporters doesn't work.
  • Could be none or all of the above.

[edit] A simpler research question

• Is the liver cyst formation the same mechanism as the renal cyst formation?
• There are some drugs in pilot tests.
  • Can we make a drug that equally affects kidney and liver cyst formation.
• The liver cyst formation is established: secretion via TGFR.
• Investigation work with IUSM's Gattone.
• He has a mouse model of the recessive PCKD.
• There is a protein called cystin that is found in the primary cilia.
• Mouse homozygotes die within weeks of birth; heterozygotes live 12-18 months have huge cysts.
• They harvested the cyst and the epithelial cell wall around it.
• they used lab techniques to look at ion transprot across the epithelia.
• they also looked at histochemical stuff in the diseased state.
  • This showed us a single layer of epithelial cells and lots of fibrosis under the epitheila layer. hence we call it fibrosis.
• Looking at bile ducts in wt, hetero, and homozygous, we see that the bile ducts are small, medium, and large, respectively, even before the cyst forms. And we go on to show that the epithelial cell layer is not stopping its dividing in the hetero and homozygotes.
• They looked, too, at the bile duct primary cilia.
  • Wildtype had short primary cilia whereas in hetero and homozygotes there are much longer primary cillia.
  • This is via EM.
• Looking at the cysts,
  • We see that there are very long cilia.
  • Some of the epithelial cells have no cilia or very short cilia.
  • Upon measuring the primary cilia:
    • WT: 90 percent were within 1-4 micrometers
    • Cystic epithelia: lots with long and some with very short or no cilia.

[edit] Analysis of differences in ciliar length

• Upon cell division, primary cilia are absorbed and then each daughter regenerates its own.
• So when there is lots of division we will see some short ones.
• But why are there long ones?
  • Is it because they are in a cyst and there is no flow?

[edit] Other aspects to cyst growth

• Growth is due to electrolyte secretion with water following.
• Most of the work in this area has been done in kidney cells.
• cAMP does seem to be involved.
  • Recall that ADH stimulates the cAMP pathway which leads to insertion of water channels and activation of na and ca channels.
• Normally:
  • ADH binds which activates adenylyl cyclase, cAMP, PKA, ENaC / CFTR / AQP2 activation.
    • ENaC brings in Na.
    • CFTR secretes cl- into the lumen. So this is the one we suspect is not working properly and this has been shown.

• But what is going on with the bile duct epithelial cells.
  • We put them in the eucine chamber.
  • Forskolin constitutively turns on adenylyl cyclase.
  • this generates a current because Na is moving in and Cl is moving out (because cAMP levels high which turns on ENaC and CFTR).
    • One paper was published saying it was all ENaC. But this doens't make sense.
  • But upon treatment with amiloride there was no change in current so it must not be ENaC that is causing the current.
  • So they applied NPPB (which inhibits the CFTR channel) and they found a drop in current. So we know that it is the cloride channel that is causing the current.

[edit] What do we know so far

• Spent less than 1.4 seconds here.

[edit] Questions

• What is stimulating ion secretion?
• Are there components in cyst fluid that modulate cilliary length?
• Are there components in the cyst fluid that contribute to the epithelial proliferation.

[edit] Stimulation of anion secretion

• Background:
  • Took renal epithelial cells from dog.
  • Grew them in an auger matrix instead of on plastic.
  • In this way, they formed a long, thin tubule.
  • If you add renal cyst fluid to the cells, they will form cysts.
  • So we think there is a signal in the cyst fluid that causes cyst formation.

[edit] Does human renal cyst fluid contain material that stimulates ion transprot in the MPK cell line?

• The previous group showed that it did cause cyst formation and that addition of CFTR inhibitor stopped cyst growth.

[edit] Short circuit current electrophysiology

• The cells are put onto porous structure so that you can provide nutrients from top and bottom.
• This causes better, more normal polarization.
• Then cut out the filter paper and put it in the chamber.
• Electrodes in the back measure potential across the cell membrane. (Measures the net ion transport.)
• We keep them warm and well oxygenated and fed.

[edit] Chloride secretion model

• Ca comes in, stimulates PDE and inhibits adenylyl cyclase which causes decrease in cAMP.
• In abnormal conditions, there is not this decrease in cAMP and therefore CFTR is activated and secrete ions such that water follows.
• Upon adding cyst fluid, there is an increase in ion secretion.
  • Via other inhibitor studies we showed that this increase is due to Cl secretion via CFTR.

[edit] What is it in the fluid that cuases increased secretion

• Whatever it is, is it part of a membrane bound vesicle? We wondered because there was lots of literature that said there might be vesicles budding off from one cell and landing on another.
• So they spun cells out for a long time at high gravity.
• They then added the separate sections to the cells in the electro-thing.
• They found that the unspun cyst fluid and supernatant cause the same peak in ion transport. This tells us the component is soluble.

[edit] Stimulation of anion secretion

• Another group did the same experiments and finally isolated the component of cyst fluid that was causing secretion: forskolin.
  • But forskolin is a plant compound and it isn't usally associated with mammals.
  • These results have not been replicated even though they did mass spec and such.
• Forskolin has a mw of 140 or so and is soluble.
• So we did some size fractionation of the cyst fluid (10K and above or only below 10K).
  • The >10K components caused the spike but less than 10K didn't.
  • This doesn't match the forskolin hypothesis.
• It could, however, still be a soluble protein binding forskolin and causing secretion of ions.
• So they added forskolin to the cells.
  • If you do this and activate cAMP, you find that there is a sustained increase in transport and it is amiloride sensitive. So, it is all ENaC (which has been previously shown to be a non-player in cyst formation.)
  • So, it's not forskolin.

[edit] Conclusion

• Still don't know what it is.
• But what is the molecular weight?
  • Separated into 100K and under 100K.
  • Found that greater than 100K causes the spike in ion transport.
• Why the two peaks? Are they cloride transport?
  • If you pre-treat twith a CFTR blocker (NPPB) you don't get the peak (because it is the CFTR ion transporter that is important) but with amiloride (ENaC blocker) you still get the pieak (because ENaC doesn't matter in cyst formation).

[edit] Treatment options

• We are making some drugs even though the FDA won't approve it.
• One drug targets the ADH receptor with something that blocks the ADH receptor.
• This turns off the whole pathway.
• These kinds of drugs are already FDA approved but not for this disease.
• Because of the delay, we don't know how to measure the efficacy.

[edit] Nature medicine paper

• Gattone and Torres did these original drug studies via rat model.
• So V2R is the ADH receptor and it can be inhibited with the antatgonist OPC.
  • This generates far fewer cysts.
• In the treated animal there are still some cysts but not nearly as much.
• Even if you start after the cysts have formed, the drug is still effective, though there are more cysts.
• The drug also caused decreased kidney weight, creatinine excretion, cyst volume, blood pressure, intracellular cAMP and disease progression.
• This treatment will not affect polycystic liver issues because there are no V2 receptors in the cholangiocytes.

[edit] The V2 receptor antagonists are now in human clinical trials (phase 3)

• There are some problems with these drugs.
• Why don't we use them off label?
  • If you block the ADH receptor you will generate 5-8 liters of urine per day.*
    • Not too convenient for the patient.
  • $250 / day for the drug.
  • Insurance won't pay for this drug because it is cheaper to wait for renal failure and then dialysis.

[edit] Other drugs in development

• CFTR inhibitors are being investigators and would be great if we could get them to the right side of the cell.
  • None approved for other things yet, though, so we can't use them off label.
• We could target the ca release from the ER.
  • These kinds of drugs are in phase I trials.
• For liver disease, somatostatin could be used to stop the adenylyl cyclase pathway.
• We could use chemotherapeutic agents to inhibit cellular proliferation.
  • It better be at low concetrations because the patients will need life-long treatment.
• The MTOR pathway
  • We often target this pathway with immunosuppressive drugs so we might be able to use these kinds of drugs for PCKD treatment.
  • MTOR, in wildtype situations, helps inhibit proliferation.

[edit] Etc.

• There are still lots of questions.
• There is interesting biology.
• The PCKD group is talking to the FDA to find out what they want as a marker.
  • One idea is waist size. Seems like radiology of the kidney would be better.
  • Task force should have a list of what is wanted in the next couple of years.
• This is where the field stands for the moment.

• stopped here on 04/14/10.