The results of a study headed by Cedars-Sinai investigators have uncovered why some injured kidneys heal while others develop scarring (fibrosis) that can lead to kidney failure. The team’s research, including tests in mouse models of kidney damage, indicated that after injury, regenerating epithelial cell lineages that can switch off a protein called SOX9 at an appropriate time point heal well, whereas continued SOX9 production leads to fibrosis. The investigators suggest that their finding could lead to the development of noninvasive tests to detect kidney scarring and, potentially, point to the development of new therapies to reverse the condition.

“The key to this discovery was our ability to directly compare injured kidney cells that successfully regenerated with those that did not,” said Sanjeev Kumar, MD, PhD, a nephrologist-scientist in the Board of Governors Regenerative Medicine Institute and the Department of Medicine at Cedars-Sinai and senior author of the study. “Injured cells activate a protein called SOX9 to regenerate themselves. When they have healed, the cells silence this protein. Cells that aren’t able to regenerate leave SOX9 active, and this leads to a type of scarring called fibrosis. But when we deactivate SOX9 in a timely fashion, the scarring literally goes away.”

Kumar and colleagues outlined their findings in Science, in a paper titled “SOX9 switch links regeneration to fibrosis at the single-cell level in mammalian kidneys.” In their report they concluded “Thus, we have uncovered a sensor of epithelial repair status, the activity of which determines regeneration with or without fibrosis.”

The kidneys, two fist-sized organs that filter waste from the blood, can be injured by diabetes and high blood pressure, serious infections such as COVID-19, and overuse of antibiotics and non-steroidal anti-inflammatory pain medications, said Kumar, who is also part of the Department of Biomedical Sciences at Cedars-Sinai. However, as the authors noted in their paper, in mammalian kidneys, some injured tissue will repair well without scarring, whereas other, even adjacent areas may exhibit impaired regeneration and progressive fibrosis. “This spatial heterogeneity in interstitial fibrosis seen across diverse organs is also frequently observed during progression of acute kidney injury (AKI) to chronic kidney disease (CKD),” the team noted. And as they further pointed out, in their accompanying research summary, “The precise mechanism through which the regeneration response culminates in fibrosis while at the same time driving scarless tissue restoration in the same microenvironment is unclear.”

The SOX9 protein plays a major role in organ development but is not active in healthy adult kidneys. In previous work at another institution, Kumar and the team found that when kidneys are injured, surviving proximal tubular epithelial cells (PTEC) cells reactivate SOX9 as part of the healing process. “We hypothesized that the lineages of PTECs reactivating SOX9 would subsequently silence SOX9 upon regeneration, whereas those unable to fully restore the epithelium would maintain SOX9 activity,” the team noted in their summary report. “If confirmed, this would establish a model system to compare regeneration in two initially committed lineages, one efficiently restoring (SOX9on-off) and the other unable to fully restore the epithelium (SOX9on-on) from the onset of a single insult.”

To investigate this further, Kumar and fellow investigators studied kidney damage in laboratory mice. They labeled individual cells at the point of injury, then followed how the cells’ progeny evolved over time. Their results demonstrated that healing without fibrosis was dependent on the cells’ ability to switch off SOX9. “Lineages that regenerated epithelia silenced SOX9 and healed without fibrosis (SOX9on-off). By contrast, lineages with unrestored apicobasal polarity maintained SOX9 activity in sustained efforts to regenerate …”

Kumar explained, “At Day 10, some cells’ descendants were fully healed while others were not. The cell lineage that healed had switched off SOX9 expression, while the unhealed lineage, in a continuing attempt to fully regenerate, maintained SOX9 activity. It’s like a sensor that switches on when cells want to regenerate, and off when they are restored, and we are the first to identify this.” In their paper, the team further noted, “In this study we have shown that the duration of the regeneration response is a key determinant of healing with or without fibrosis.”

Further, the investigators discovered that cells that were unable to regenerate began recruiting proteins called Wnts, another key player in organ development. Over time, this accumulation of Wnts triggered scarring. “These reprogrammed cells generated substantial single-cell WNT activity to provoke a fibroproliferative response in adjacent fibroblasts, driving AKI to chronic kidney disease,” they commented. They further explained, “Single-cell RNA sequencing (scRNA-seq) revealed SOX9on-on cells in a distinct regenerating state demarcated by Cadherin 6 (CDH6). The SOX9on-off lineages healed without fibrosis, whereas adjacent SOX9on-onCDH6+ lineages displayed robust, intimate association with myofibroblasts.” These myofibroblasts, the investigators reported, were identified as injury-induced WNT-responsive cells (WRCs) sited adjacent to the SOX9on-on CDH6+ cells. “Our study revealed that in contrast to SOX9on/SOX9on-off cells, SOX9on-on activity highlighted by SOX9+CDH6+ cell state, during its sustained efforts to regenerate the lineage, generated substantial single-cell WNT activity to activate adjacent fibroblasts, thus driving AKI to CKD. Therefore, we have also identified how WNTs might be spatiotemporally regulated in the damaged tissue microenvironment.”

The team then found that deactivating SOX9 a week after injury promoted kidney recovery. Investigators observed the same process in patient databases from collaborating institutions in Switzerland and Belgium. “We could see that by Day 7, human patients with transplanted kidneys that were slow to begin working also activated SOX9,” Kumar said. “And in our collaborators’ database, we were able to distinguish that patients who had sustained SOX9 activation had lower kidney function and more scarring than those who did not. Human kidneys with cells that maintained SOX9 were also enriched with Wnts and showed increased fibrosis.”

These discoveries provide targets for drug development, as well as for noninvasive biomarker discovery permitting diagnosis of kidney fibrosis through the urine, Kumar suggested. Currently, the only available test for kidney fibrosis is a biopsy, which carries many risks.

The findings could also lead to new treatment options for patients, the researchers suggested. And while, “… a pharmacological approach to perturb the identified pathway remains unidentified,” they noted in their paper, “…our findings lay the ground for drug discovery and precise cell-state–specific genetic perturbation strategies to retard fibrosis.” Added coo-author Clive Svendsen, PhD, executive director of the Board of Governors Regenerative Medicine Institute at Cedars-Sinai, “These findings help us understand for the first time how the kidney’s response to injury sometimes leads to fibrosis. Future work along these lines could also advance our understanding of fibrosis in the heart, lungs and liver.”

Added Paul Noble, MD, chair of the department of medicine and director of the Women’s Guild Lung Institute at Cedars-Sinai and a co-author of the study, “Elucidating the mechanisms of scarless healing versus fibrosis has eluded investigators for decades and has implications beyond the kidney, including for certain cancers.”

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