Mitochondrial transplants recover damaged kidneys

Every year, thousands of patients die while waiting for a kidney transplant. The overwhelming demand for donated kidneys has pushed medical professionals to expand the donation criteria to injured kidneys and kidneys donated after cardiac death (DCD). Still, up to a quarter of donated kidneys are too damaged for patients to receive.

“We need to render transplantable untransplantable organs,” said Giuseppe Orlando, a transplant surgeon at Wake Forest University School of Medicine. He and his collaborators recently demonstrated a potential new way to do that: injecting donor kidneys with healthy mitochondria (1).

Giuseppe Orlando smiles while wearing glasses, a blue-and-white pin-stripe shirt, a dark blue tie, and a white lab coat.

Giuseppe Orlando studies regenerative therapies and bioengineering transplantable organs at Wake Forest University School of Medicine. He is also a practicing surgeon.

credit: Giuseppe Orlando

The team used a protocol for mitochondrial transplantation (MITO) developed by James McCully, a coauthor of the new study at Boston Children’s Hospital (2). The method originally came to Orlando’s attention when he received a call from his long-time collaborator Benedetta Bussolati at the University of Turin. Bussolati’s PhD student Andrea Rossi, the study’s first author, had been using MITO in kidney cells and wanted Orlando’s help to develop a more clinically-relevant animal model for improving kidney transplant. “I read the literature and [thought] it was pretty cool,” said Orlando. “I smelled the potential.”

For their initial in vitro studies, they extracted mitochondria from a model kidney cell line (cIPTECs). To confirm that these isolated mitochondria were functioning properly, the researchers used luminescence and fluorescence assays to show that they were producing ATP and that their membranes were intact. The team then confirmed through fluorescent tagging that the mitochondria could be successfully transplanted into new cIPTECs.

A common issue for donated organs is ischemia reperfusion injury. When organs get cut off from the donor blood stream (ischemia), they can incur even further damage when blood begins to flow through them again (reperfusion). As a model for this phenomenon, Orlando’s team subjected ciPTECs to antimycin A, an ATP production inhibitor, and 2-deoxyglucose, a glycolysis inhibitor. Predictably, this led to a depletion of ATP production and cell proliferation along with an increase in cell death, but the team found that MITO was largely able to reverse this. MITO also alleviated oxidative damage and recovered Krebs Cycle enzyme activity.

“There's an adage that just says that everything works in mice,” said Orlando, who instead chose what he described as a more clinically relevant porcine model for their ex vivo animal studies. To mimic DCD conditions, they extracted the kidneys and subjected them to warm ischemia, storing them in a 37°C bath without blood supply for 30 minutes. The researchers then treated one kidney from each animal with MITO and left the other untreated before placing both of them in separate reperfusion machinery for 24 hours. MITO kidneys consistently exhibited fewer signs of apoptosis and structural tissue damage than the untreated control kidneys.

Lastly, the team collected the external blood flow from each kidney’s reperfusion. As a way of characterizing gene expression, the team used Raman spectroscopy to examine the molecules shed by the control and MITO kidneys during the 24-hour period. “What we found was very interesting,” said Orlando. “The control and the study groups basically localized to two extremes of the spectrum.” Whereas MITO kidneys activated the expression of several genes related to regeneration and repair, the control kidneys did not. MITO kidneys also shed fewer molecules overall, which Orlando explained was a sign of less damage. RNA sequencing supported this finding; dozens of transcripts were upregulated in MITO that were not in the controls. The researchers further investigated these upregulated transcripts and found that they were largely involved in key signaling pathways, both within the mitochondria and in the surrounding cell.

We need to render transplantable untransplantable organs.
- Giuseppe Orlando, Wake Forest University School of Medicine

“I don't believe any of this in this field, I'm afraid,” said Mike Murphy, a mitochondrial redox biologist at the University of Cambridge who was not part of the study. Murphy was not convinced that the mitochondria integrated and were active inside the cell. He argued that the changes in gene expression between MITO and control kidneys could have been the result of cells generally protecting themselves from stress or damage. “The assumption is that mitochondria go in there, doing stuff inside the cell that is making it a better place,” he said. “There are no papers that I'm aware of that show that convincingly.”

“There are still many questions to be answered,” Orlando acknowledged. His team is in talks with the Food and Drug Administration and the Wake Forest Institutional Review Board to move forward to clinical trials. He also hopes to optimize the MITO protocol and to identify a source for more durable mitochondria, as isolated mitochondria decay within an hour or two of extraction. “The holy grail in the field would be the ability to isolate mitochondria that stay viable,” he said.

However, Murphy recommended going back to the basics: “This field, they’ve sort of jumped ahead quite a bit,” he said. “The positive side is we're getting interesting effects, protective things going on. Adding anything that does these things is really interesting, but how does it do it?”

Murphy suggested a series of controls to clarify the mechanisms of MITO, for example, injecting individual components of mitochondria such as the DNA or cardiolipin, a uniquely mitochondrial phospholipid. He also suggested testing for the same effect with aged or denatured mitochondria, which do not perform active respiration, and other cellular components altogether. “If you can show for certain that you really were enhancing the mitochondria, that will be very important,” said Murphy.

Orlando remained hopeful about the potential of MITO technology, which he believed will have many applications across most if not all fields of health sciences. “We are still at the dawn of this mitochondrial transplant era,” said Orlando. “The next couple of decades will be very interesting.”