New Model for Kidney Regeneration

Harvard Stem Cell Institute Kidney Diseases Program Leader Benjamin Humphreys has examined tissue regeneration in the kidney. His interest in kidney regeneration has occupied a major part of his career, but some of his more recent work resulted from his skepticism of a particular theory of kidney regeneration.

The kidney stem cell repair model postulates that scattered throughout the kidney are small stem cell populations and are activated after the kidney is injured to repair it. This theory, however, conflicts with another view of kidney regeneration. Namely that after injury, the cells of the kidney dedifferentiate into more primordial versions of themselves and proliferate, after which they differentiate into the various tissues of the kidney.

Humphreys and his colleagues now have evidence that strongly suggests that all the cells of the kidney have the capacity to divide after injury and contribute to kidney regeneration.

Their evidence comes in the form of experiments in mice in which the cells of the kidney were genetically tagged, and then the kidneys were injured to determine what cells contributed to the regeneration of the kidney.

The tagging in this experiment is complicated, but quite technically brilliant. The kidney is composed of myriads of tiny functional units called nephrons. Each nephron is fed by a tiny knot of blood vessels called a glomerulus.  The structure of a nephron is shown below.  


The blood supply to the kidney comes from branches off the descending aorta knows as renal arteries.  After entering the kidneys, the renal arteries branch multiple times until they become tiny vessels that feed into each nephron known as afferent arterioles.  The afferent arterioles forms a dense network of knot-like vessels that form the glomerulus and the portion of the nephron that interacts with the glomerulus is known as the Bowman’s capsule..  The blood vessels of the glomerulus are very special because they are exceptionally porous.  However, the Bowman’s capsule has a series of cells with foot-like extensions that coat the glomerulus called “podocytes.”  An especially beautiful picture of podocytes wrapped around a glomerular vessel is shown below.


The podocytes cover the pores of the glomerulus and only allows water and things dissolved in water through the pores.  Proteins do not make it through – they are too heavily charged.  Cells also do not make it through – they are too big.  But water, sodium ions, potassium ions, hydrogen ions, some drugs, metabolites, waste products, and things like that all make it through the podocyte-guarded pores.  For this reason, if you have excessive protein or some blood cells in your urine, it is usually an indication that something is wrong.  

Now, rest of the tubing attached to the nephron serve to reabsorb all the things you do not want to get rid of and not absorb all the things you do want to get rid of.  The amount of water you eliminate depends on your degree of hydration and is controlled by a hormone called antidiuretic hormone, which is release by the posterior lobe of your pituitary gland when you are dehydrated.  In the presence of ADH, the posterior tubing reabsorbs more water, and in lower concentrations of this hormone, it reabsorbs far less.  

Now that we know something about the kidney, here’s how Humphreys and others genetically marked the kidneys of their mice.  The sodium-dependent inorganic phosphate transporter (SLC34a1) is only expressed in mature proximal tubule cells.  Tetsuro Kusaba, the first author on this paper, and his colleagues inserted a CreERT2 cassette into this gene.  If you are lost at this point all you need to remember is this: the CreERT2 cassette is inserted into a gene that is ONLY expressed in specific kidney cells.  The Cre gene encodes a recombinase that clips out specific bits of DNA from a chromosome.  Kusaba and others crossed these engineered mice with another strain of mice that had the gene for a bright red dye inserted into another gene, but this dye could not be expressed because another piece of DNA was in the way.  When these hybrid mice were fed a drug called tamoxifen, it activated the expression of the Cre protein, but only in the proximal tubule cells of the kidney and this Cre protein clipped out the piece of DNA that was preventing the red dye gene from being expressed.  Therefore, these mice had a particular part of their nephrons, the proximal tubules glowing bright red.  This is a stroke of shear genius and it genetically marks these cells specifically and strongly.  

Next, Kusaba and colleagues used unilateral ischemia reperfusion injury (IRI) to damage the kidneys.  In IRI, the blood supply is stopped to one kidney but not the other for a short period of time (26 minutes).  This causes cell death and kidney damage.  The other kidney is not damaged and serves as a control for the experiment.  

Examination of the damaged kidneys showed that  red-glowing cells were found in areas other than the proximal tubules.  The only way these cells could have ended up in these places was if the differentiated cells divided and helped repair the damaged parts of the nephrons.  

Other research groups have seen similar results, but interpreted them as evidence of stem cell populations in the kidney.  However, Humphreys groups discovered something even more fascinating.  These “stem cell-markers” in the kidney are actually markers of kidney damage and regeneration and all cells in the kidney express them.  In Humphreys words, “What was really interesting is when we looked at the appearance and expression patterns of these differentiated cells, we found that they expressed the exact same ‘stem cell markers’ that these other groups claimed to find in their stem cell populations.  And so, if a differentiated cell is able to express a ‘stem cell marker’ after injury, then what our work shows is that that’s an injury marker – is doesn’t define a stem cell.”  

Indeed, several genes that have been taken to be signs of a kidney stem cell population (CD133, CD24, vimentin, and KIM-1) were expressed in red-glowing cells.  A stem cell population should not be fully differentiated and therefore, should not be able to express the red dye.  However, red-glowing cells clearly expressed these found genes after injury.  This rather definitely shows that it is the fully differentiated cells that are doing the regeneration in the kidney and not a resident stem cell population.  This does not prove that there is no resident stem cell population in the kidney, but only that the lion’s share of the regeneration is done by differentiated cells, and that under these conditions, no stem cell population was detected.  

This new interpretation of kidney repair suggests that cells can reprogram themselves in a way that resembles the way mature cells are chemically manipulated to revert to an induced pluripotent state.  

See Tetsuro Kusaba, Matthew Lalli, Rafael Kramann, Akio Kobayashi, and Benjamin D. Humphreys. Differentiated kidney epithelial cells repair injured proximal tubule. PNAS (October 14, 2013); doi:10.1073/pnas.1310653110.  

Knee Plica Surgeries

The Regenexx blog has a very interesting article on knee plica surgeries. Knee plicas refer to collisions between the knee cap (patella) and the nearby synovial membrane, which surrounds the joint. The pinching of the synovial membrane irritates it and generates swelling and pain. The common surgical procedure to treat knee plica is to extirpate the irritated synovial membrane. Centeno points out that this portion of the synovial membrane houses a robust stem cell population that helps heal knee problems. Therefore, this procedure might not be the best choice for knee plica. Centeno suggests that the knee is not aligned properly and that realignment of the knee cap could solve the problem without surgery. Read his blog post here and see what you think.

Severe Shoulder Rotator Cuff Tear treated with Stem Cells

Shoulder rotator-cuff tears are painful and usually require surgery. Can stem cells treat such a condition? Regenexx has attempted to do just that with surprising success.

One patient who is called JS had a particularly bad rotator cuff tear that included a retraction of the rotator cuff muscle. Stem cell treatments might not help heal this tear since the two ends of the tendon or muscle need to be surgically pulled together for the stem cells to heal the tendon. When the tendon experiences a retracted tear of the rotator cuff muscle the two sides of the tear pull back like a rubber band. The tendon bunches up on either side of the tear, and it is difficult to envision how stem cells might heal such a tear, since tears where the two ends of the tear are close together can be filled in with stem cells that are precisely applied to the tear.

All of this changed, however, with the treatment of a patient known as JS. He had a 1.5 cm retracted tear from a weight-lifting injury. After reluctantly agreeing to treat him with a mesenchymal stem cell application, they placed the shoulder in a splint to bring the two ends of the tear closer together. Then JS received a Regenexx-SD procedure that was followed up with a Regenexx-SCP procedure. The Regenexx-SD procedure takes mesenchymal and hematopoietic stem cells from a bone marrow aspiration and then mixes them with a preparation of platelets taken from peripheral blood. Regenexx-SCP treatment uses a specific stem cell population from bone marrow (CD66e+ cells) that are mixed with a platelet-rich mixture from blood serum and injected into joints.

The Regenexx physicians had low expectations of this procedure, but to their surprise, JS reported a 99% improvement over the three months. A follow-up ultrasound demonstrated excellent healing with some fill-in of the retracted gap.  For MRIs for this patient’s shoulder before and after the treatment, see here.

One year after the shoulder stem cell injections, the improvement due to precision injections of the patient’s own stem cells is nothing short of amazing. The large gap in the rotator cuff  is now healed.  For MRI images of JS’s shoulder one year after the Regenexx treatment, see here.  JS did require a specialized treatment and bracing protocol unique to the Regenexx procedures and developed by Dr. Hanson. This is an example of stem-based orthopedic surgery at its best.

Making Older Mice Younger with Stem Cell Injections

University of Pittsburgh scientists have used stem cells derived from younger young mice to revitalize older mice. They used mice that were bred to age quickly, but after these stem cell injections, they seemed to have sipped from the fountain of youth. These stem cells were derived from muscles of young, healthy animals, and instead of becoming infirm and dying early as untreated mice did, the injected animals improved their health and lived two to three times longer than expected. These findings were published in the Jan. 3 edition of Nature Communications.

Previous research has revealed stem cell dysfunction, such as poor replication and differentiation, in a variety of tissues in old age. However it is not clear whether that loss of function contributes to the aging process or is a result of it. Senior investigators in this work were Johnny Huard, Ph.D., professor in the Departments of Orthopaedic Surgery and of Microbiology and Molecular Genetics, Pitt School of Medicine, and director of the Stem Cell Research Center at Pitt and Children’s Hospital of PIttsburgh of UPMC, and Laura Niedernhofer, M.D., Ph.D. associate professor in Pitt’s Department of Microbiology and Molecular Genetics and the University of Pittsburgh Cancer Institute (UPCI).

Niedernhofer explained: “Our experiments showed that mice that have progeria, a disorder of premature aging, were healthier and lived longer after an injection of stem cells from young, healthy animals. That tells us that stem cell dysfunction is a cause of the changes we see with aging.”

The research team examined a stem/progenitor cell population derived from the muscle of mice engineered to suffer from a genetic disease called progeria. Progeria is a genetic disease that causes premature aging. Human patients with progeria age extremely quickly and die at a very young age from old age. Muscle-derived stem cells from progeria mice were fewer in number, did not replicate as often, didn’t differentiate as readily into specialized cells and were impaired in their ability to regenerate damaged muscle in comparison to those found in normal rodents. The same defects were discovered in the stem/progenitor cells isolated from very old mice.

Dr. Huard said: “We wanted to see if we could rescue these rapidly aging animals, so we injected stem/progenitor cells from young, healthy mice into the abdomens of 17-day-old progeria mice. Typically the progeria mice die at around 21 to 28 days of age, but the treated animals lived far longer – some even lived beyond 66 days. They also were in better general health.”

As the progeria mice age, they lose muscle mass in their hind limbs, hunch over, tremble, and move slowly and awkwardly. Affected mice received an injection of stem cells just before showing the first signs of aging were more like normal mice, and they grew almost as large. Closer examination showed new blood vessel growth in the brain and muscle, even though the stem/progenitor cells weren’t detected in those tissues. However, the injected cells didn’t migrate to any particular tissue after injection into the abdomen.

Niedernhofer noted: “This leads us to think that healthy cells secrete factors to create an environment that help correct the dysfunction present in the native stem cell population and aged tissue. In a culture dish experiment, we put young stem cells close to, but not touching, progeria stem cells, and the unhealthy cells functionally improved.”

Animals that age normally were not treated with stem/progenitor cells, but these provocative findings urge further research. They hint that it might be possible one day to forestall the biological declines associated with aging by delivering a shot of youthful vigor, particularly if specific rejuvenating proteins or molecules produced by the stem cells could be identified and isolated.