Mammalian organ regeneration in spiny mice.

Fibrosis-driven solid organ failure is a major world-wide health burden with few therapeutic options. Spiny mice (genus: Acomys) are terrestrial mammals that regenerate severe skin wounds without fibrotic scars to evade predators. Recent studies have shown that spiny mice also regenerate acute ischemic and traumatic injuries to kidney, heart, spinal cord, and skeletal muscle. A common feature of this evolved wound healing response is a lack of formation of fibrotic scar tissue that degrades organ function, inhibits regeneration, and leads to organ failure. Complex tissue regeneration is an extremely rare property among mammalian species. In this article, we discuss the evidence that Acomys represents an emerging model organism that offers a unique opportunity for the biomedical community to investigate and clinically translate molecular mechanisms of scarless wound healing and regeneration of organ function in a mammalian species.

Utility of the 2018 revised ISN/RPS thresholds for glomerular crescents in childhood-onset lupus nephritis: a Pediatric Nephrology Research Consortium study.

BACKGROUND: The revised 2018 ISN/RPS Classification System for lupus nephritis (LN) includes calculations for both activity index (A.I.) and chronicity index (C.I.). Unchanged were the thresholds of < 25%, 25-50%, and > 50% crescents to distinguish between mild, moderate, and severe activity/chronicity. We aimed to evaluate these thresholds for percent crescents in childhood-onset LN. METHODS: Eighty-six subjects < 21 years of age were enrolled from the Pediatric Glomerulonephritis with Crescents Registry, a retrospective multi-center cohort sponsored by the Pediatric Nephrology Research Consortium. Thresholds of 10%, 25%, and 50% for both cellular/fibrocellular and fibrous crescents were interrogated for primary outcomes of kidney failure, eGFR, and eGFR slope. RESULTS: Median age at time of initial biopsy was 14 years (range 1-21). Median follow-up time was 3 years (range 1-11). Cumulative incidence of kidney failure was 6% at 1 year and 10% at latest follow-up. Median eGFR slope was - 18 mL/1.73 m2/min (IQR - 51 to + 8) at 1 year and - 3 mL/min/1.73 m2/year (IQR - 19 to + 6) at latest follow-up. We found no difference in kidney failure at the proposed < 25% and 25-50% cellular crescents thresholds, and thus added a new provisional threshold of 10% that better predicted outcomes in children. Moreover, use of 10% and 25% thresholds for fibrous crescents showed a fourfold and sevenfold increase in risk of kidney failure. CONCLUSIONS: In children with crescentic LN, use of 10% and 25% thresholds for cellular crescents better reflects disease activity, while these thresholds for fibrous crescents better discriminates kidney disease outcomes. A higher resolution version of the Graphical abstract is available as Supplementary information.

Principles of pediatric lupus nephritis in a prospective contemporary multi-center cohort.

Lupus nephritis (LN) is a life-threatening manifestation of systemic lupus erythematosus (SLE) and is more common in children than adults. The epidemiology and management of childhood-onset SLE (cSLE) have changed over time, prompting the need to reassess expected outcomes. The purpose of this study is to use the Childhood Arthritis and Rheumatology Research Alliance (CARRA) prospective registry to validate historical principles of LN in a contemporary, real-world cohort. After an extensive literature review, six principles of LN in cSLE were identified. The CARRA registry was queried to evaluate these principles in determining the rate of LN in cSLE, median time from cSLE diagnosis to LN, short-term renal outcomes, and frequency of rituximab as an induction therapy. Of the 677 cSLE patients in the CARRA registry, 32% had documented LN. Decline in kidney function was more common in Black cSLE patients than non-Black patients (p = 0.04). Black race was associated with worse short-term renal outcomes. In short-term follow up, most children with LN had unchanged or improved kidney function, and end stage kidney disease (ESKD) was rare. Ongoing follow-up of cSLE patients in the CARRA registry will be necessary to evaluate long-term outcomes to inform risk, management, and prognosis of LN in cSLE.

The macrophage phagocytic receptor CD36 promotes fibrogenic pathways on removal of apoptotic cells during chronic kidney injury.

The removal of apoptotic cells is an innate function of tissue macrophages; however, its role in disease progression is unclear. The present study was designed to investigate the role of macrophage CD36, a recognized receptor of apoptotic cells and oxidized lipids, in two models of kidney injury: unilateral ureteral obstruction (UUO) and ischemia reperfusion. To differentiate the macrophage CD36-specific effects in vivo, we generated CD36 chimeric mice by bone marrow transplantation and evaluated the two models. Fibrosis severity was substantially decreased after UUO with a corresponding decrease in matrix synthesis in macrophage CD36-deficient mice. Despite a reduction in fibrosis severity, a 56% increase in apoptotic cells was found without an increase in apoptotic effectors. In addition, a substantial reduction was observed in tumor necrosis factor-α and transforming growth factor-ß1 mRNA levels and intracellular bioactive oxidized lipid levels in CD36-deficient macrophages. To validate the functional role of macrophage CD36, we performed unilateral ischemia reperfusion, followed by contralateral nephrectomy. Similarly, we found that the severity of fibrosis was reduced by 55% with a corresponding improvement in kidney function by 88% in macrophage CD36-deficient mice. Taken together, these data suggest that macrophage CD36 is a critical regulator of oxidative fibrogenic signaling and that CD36-mediated phagocytosis of apoptotic cells may serve as an important pathway in the progression of fibrosis.

Cysteamine modulates oxidative stress and blocks myofibroblast activity in CKD.

Therapy to slow the relentless expansion of interstitial extracellular matrix that leads to renal functional decline in patients with CKD is currently lacking. Because chronic kidney injury increases tissue oxidative stress, we evaluated the antifibrotic efficacy of cysteamine bitartrate, an antioxidant therapy for patients with nephropathic cystinosis, in a mouse model of unilateral ureteral obstruction. Fresh cysteamine (600 mg/kg) was added to drinking water daily beginning on the day of surgery, and outcomes were assessed on days 7, 14, and 21 after surgery. Plasma cysteamine levels showed diurnal variation, with peak levels similar to those observed in patients with cystinosis. In cysteamine-treated mice, fibrosis severity decreased significantly at 14 and 21 days after unilateral ureteral obstruction, and renal oxidized protein levels decreased at each time point, suggesting reduced oxidative stress. Consistent with these results, treatment of cultured macrophages with cysteamine reduced cellular generation of reactive oxygen species. Furthermore, treatment with cysteamine reduced α-smooth muscle actin-positive interstitial myofibroblast proliferation and mRNA levels of extracellular matrix proteins in mice and attenuated myofibroblast differentiation and proliferation in vitro, but did not augment TGF-ß signaling. In a study of renal ischemia reperfusion, cysteamine therapy initiated 10 days after injury and continued for 14 days decreased renal fibrosis by 40%. Taken together, these data suggest previously unrecognized antifibrotic actions of cysteamine via TGF-ß-independent mechanisms that include oxidative stress reduction and attenuation of the myofibroblast response to kidney injury and support further investigation into the potential benefit of cysteamine therapy in the treatment of CKD.

Mannose receptor 2 attenuates renal fibrosis.

Mannose receptor 2 (Mrc2) expresses an extracellular fibronectin type II domain that binds to and internalizes collagen, suggesting that it may play a role in modulating renal fibrosis. Here, we found that Mrc2 levels were very low in normal kidneys but subsets of interstitial myofibroblasts and macrophages upregulated Mrc2 after unilateral ureteral obstruction (UUO). Renal fibrosis and renal parenchymal damage were significantly worse in Mrc2-deficient mice. Similarly, Mrc2-deficient Col4α3(-/-) mice with hereditary nephritis had significantly higher levels of total kidney collagen, serum BUN, and urinary protein than Mrc2-sufficient Col4α3(-/-) mice. The more severe phenotype seemed to be the result of reduced collagen turnover, because procollagen III (α1) mRNA levels and fractional collagen synthesis in the wild-type and Mrc2-deficient kidneys were similar after UUO. Although Mrc2 associates with the urokinase receptor, differences in renal urokinase activity did not account for the increased fibrosis in the Mrc2-deficient mice. Treating wild-type mice with a cathepsin inhibitor, which blocks proteases implicated in Mrc2-mediated collagen degradation, worsened UUO-induced renal fibrosis. Cathepsin mRNA profiles were similar in Mrc2-positive fibroblasts and macrophages, and Mrc2 genotype did not alter relative cathepsin mRNA levels. Taken together, these data establish an important fibrosis-attenuating role for Mrc2-expressing renal interstitial cells and suggest the involvement of a lysosomal collagen turnover pathway.

ECM-Focused Proteomic Analysis of Ear Punch Regeneration in Acomys Cahirinus.

In mammals, significant injury is generally followed by the formation of a fibrotic scar which provides structural integrity but fails to functionally restore damaged tissue. Spiny mice of the genus Acomys represent the first example of full skin autotomy in mammals. Acomys cahirinus has evolved extremely weak skin as a strategy to avoid predation and is able to repeatedly regenerate healthy tissue without scar after severe skin injury or full-thickness ear punches. Extracellular matrix (ECM) composition is a critical regulator of wound repair and scar formation and previous studies have suggested that alterations in its expression may be responsible for the differences in regenerative capacity observed between Mus musculus and A. cahirinus , yet analysis of this critical tissue component has been limited in previous studies by its insolubility and resistance to extraction. Here, we utilize a 2-step ECM-optimized extraction to perform proteomic analysis of tissue composition during wound repair after full-thickness ear punches in A. cahirinus and M. musculus from weeks 1 to 4 post-injury. We observe changes in a wide range of ECM proteins which have been previously implicated in wound regeneration and scar formation, including collagens, coagulation and provisional matrix proteins, and matricryptic signaling peptides. We additionally report differences in crosslinking enzyme activity and ECM protein solubility between Mus and Acomys. Furthermore, we observed rapid and sustained increases in CD206, a marker of pro-regenerative M2 macrophages, in Acomys, whereas little or no increase in CD206 was detected in Mus. Together, these findings contribute to a comprehensive understanding of tissue cues which drive the regenerative capacity of Acomys and identify a number of potential targets for future pro-regenerative therapies.

Single cell spatial transcriptomic profiling of childhood-onset lupus nephritis reveals complex interactions between kidney stroma and infiltrating immune cells.

Children with systemic lupus erythematosus (SLE) are at increased risk of developing kidney disease, termed childhood-onset lupus nephritis (cLN). Single cell transcriptomics of dissociated kidney tissue has advanced our understanding of LN pathogenesis, but loss of spatial resolution prevents interrogation of in situ cellular interactions. Using a technical advance in spatial transcriptomics, we generated a spatially resolved, single cell resolution atlas of kidney tissue (>400,000 cells) from eight cLN patients and two controls. Annotated cells were assigned to 35 reference cell types, including major kidney subsets and infiltrating immune cells. Analysis of spatial distribution demonstrated that individual immune lineages localize to specific regions in cLN kidneys, including myeloid cells trafficking to inflamed glomeruli and B cells clustering within tubulointerstitial immune hotspots. Notably, gene expression varied as a function of tissue location, demonstrating how incorporation of spatial data can provide new insights into the immunopathogenesis of SLE. Alterations in immune phenotypes were accompanied by parallel changes in gene expression by resident kidney stromal cells. However, there was little correlation between histologic scoring of cLN disease activity and glomerular cell transcriptional signatures at the level of individual glomeruli. Finally, we identified modules of spatially-correlated gene expression with predicted roles in induction of inflammation and the development of tubulointerstitial fibrosis. In summary, single cell spatial transcriptomics allows unprecedented insights into the molecular heterogeneity of cLN, paving the way towards more targeted and personalized treatment approaches.

Genome Report: chromosome-scale genome assembly of the African spiny mouse (Acomys cahirinus).

There is increasing interest in the African spiny mouse (Acomys cahirinus) as a model organism because of its ability for regeneration of tissue after injury in skin, muscle, and internal organs such as the kidneys. A high-quality reference genome is needed to better understand these regenerative properties at the molecular level. Here, we present an improved reference genome for A. cahirinus generated from long Nanopore sequencing reads. We confirm the quality of our annotations using RNA sequencing data from 4 different tissues. Our genome is of higher contiguity and quality than previously reported genomes from this species and will facilitate ongoing efforts to better understand the regenerative properties of this organism.

GENOME REPORT: Chromosome-scale genome assembly of the African spiny mouse ( Acomys cahirinus ).

There is increasing interest in the African spiny mouse ( Acomys cahirinus ) as a model organism because of its ability for regeneration of tissue after injury in skin, muscle, and internal organs such as the kidneys. A high-quality reference genome is needed to better understand these regenerative properties at the molecular level. Here, we present an improved reference genome for A. cahirinus generated from long Nanopore sequencing reads. We confirm the quality of our annotations using RNA sequencing data from four different tissues. Our genome is of higher contiguity and quality than previously reported genomes from this species and will facilitate ongoing efforts to better understand the regenerative properties of this organism.

Investigating mechanisms of chronic kidney disease in mouse models.

Animal models of chronic kidney disease (CKD) are important experimental tools that are used to investigate novel mechanistic pathways and to validate potential new therapeutic interventions prior to pre-clinical testing in humans. Over the past several years, mouse CKD models have been extensively used for these purposes. Despite significant limitations, the model of unilateral ureteral obstruction (UUO) has essentially become the high-throughput in vivo model, as it recapitulates the fundamental pathogenetic mechanisms that typify all forms of CKD in a relatively short time span. In addition, several alternative mouse models are available that can be used to validate new mechanistic paradigms and/or novel therapies. Here, we review several models-both genetic and experimentally induced-that provide investigators with an opportunity to include renal functional study end-points together with quantitative measures of fibrosis severity, something that is not possible with the UUO model.

Wound healing and regeneration in spiny mice (Acomys cahirinus).

The winds of Patagonia are referred to by locals as "The Broom of God" because they sweep away the less fit species that cannot survive there. Fitness as an evolutionary trait has been considered as fundamental for many aspects of morphogenesis and behavior in metazoans. Yet, it has not received much attention in the area of wound healing, despite the obvious relevance of this polygenic trait to an organism's survival in nature. In this chapter, we review the evidence that the rodent species Acomys cahirinus is an emerging mammalian model system that has evolved a non-typical (for mammals) wound healing response that offers unique opportunities for the study of organ regeneration without fibrosis in an adult mammalian species.

Galectin-3 preserves renal tubules and modulates extracellular matrix remodeling in progressive fibrosis.

Renal tubular cell apoptosis is a critical detrimental event that leads to chronic kidney injury in association with renal fibrosis. The present study was designed to investigate the role of galectin-3 (Gal-3), an important regulator of multiple apoptotic pathways, in chronic kidney disease induced by unilateral ureteral obstruction (UUO). After UUO, Gal-3 expression significantly increased compared with basal levels reaching a peak increase of 95-fold by day 7. Upregulated Gal-3 is predominantly tubular at early time points after UUO but shifts to interstitial cells as the injury progresses. On day 14, there was a significant increase in TdT-mediated dUTP nick end labeling-positive cells (129%) and cytochrome c release (29%), and a decrease in BrdU-positive cells (62%) in Gal-3-deficient compared with wild-type mice. The degree of renal damage was more extensive in Gal-3-deficient mice at days 14 and 21, 35 and 21% increase in total collagen, respectively. Despite more severe fibrosis, myofibroblasts were significantly decreased by 58% on day 14 in the Gal-3-deficient compared with wild-type mice. There was also a corresponding 80% decrease in extracellular matrix synthesis in Gal-3-deficient compared with wild-type mice. Endo180 is a recently recognized receptor for intracellular collagen degradation that is expressed by interstitial cells during renal fibrogenesis. Endo180 expression was significantly decreased by greater than 50% in Gal-3-deficient compared with wild-type mice. Taken together, these results suggested that Gal-3 not only protects renal tubules from chronic injury by limiting apoptosis but that it may lead to enhanced matrix remodeling and fibrosis attenuation.

Adaptations in Hippo-Yap signaling and myofibroblast fate underlie scar-free ear appendage wound healing in spiny mice.

Spiny mice (Acomys cahirinus) are terrestrial mammals that evolved unique scar-free regenerative wound-healing properties. Myofibroblasts (MFs) are the major scar-forming cell type in skin. We found that following traumatic injury to ear pinnae, MFs appeared rapidly in both Acomys and mouse yet persisted only in mouse. The timing of MF loss in Acomys correlated with wound closure, blastema differentiation, and nuclear localization of the Hippo pathway target protein Yap. Experiments in vitro revealed an accelerated PP2A-dependent dephosphorylation activity that maintained nuclear Yap in Acomys dermal fibroblasts (DFs) and was not detected in mouse or human DFs. Treatment of Acomys in vivo with the nuclear Yap-TEAD inhibitor verteporfin prolonged MF persistence and converted tissue regeneration to fibrosis. Forced Yap activity prevented and rescued TGF-ß1-induced human MF formation in vitro. These results suggest that Acomys evolved modifications of Yap activity and MF fate important for scar-free regenerative wound healing in vivo.

Spiny mice activate unique transcriptional programs after severe kidney injury regenerating organ function without fibrosis.

Fibrosis-driven solid organ failure is an enormous burden on global health. Spiny mice (Acomys) are terrestrial mammals that can regenerate severe skin wounds without scars to avoid predation. Whether spiny mice also regenerate internal organ injuries is unknown. Here, we show that despite equivalent acute obstructive or ischemic kidney injury, spiny mice fully regenerate nephron structure and organ function without fibrosis, whereas C57Bl/6 or CD1 mice progress to complete organ failure with extensive renal fibrosis. Two mechanisms for vertebrate regeneration have been proposed that emphasize either extrinsic (pro-regenerative macrophages) or intrinsic (surviving cells of the organ itself) controls. Comparative transcriptome analysis revealed that the Acomys genome appears poised at the time of injury to initiate regeneration by surviving kidney cells, whereas macrophage accumulation was not detected until about day 7. Thus, we provide evidence for rapid activation of a gene expression signature for regenerative wound healing in the spiny mouse kidney.

CD36 regulates oxidative stress and inflammation in hypercholesterolemic CKD.

Scavenger receptors play a central role in atherosclerosis by processing oxidized lipoproteins and mediating their cellular effects. Recent studies suggested that the atherogenic state correlates with progression of chronic kidney disease (CKD); therefore, scavenger receptors are candidate mediators of renal fibrogenesis. Here, we investigated the role of CD36, a class B scavenger receptor, in a hypercholesterolemic model of CKD. We placed CD36-deficient mice and wild-type male mice on a high-fat Western diet for 7 to 8 wk and then performed either sham or unilateral ureteral obstruction surgery. CD36-deficient mice developed significantly less fibrosis compared with wild-type mice at days 3, 7, and 14 after obstruction. Compared with wild-type mice, CD36-deficient mice had significantly more interstitial macrophages at 7 d but not at 14 d. CD36-deficient mice exhibited reduced levels of activated NF-kappaB and oxidative stress (assessed by measuring fatty acid-derived hydroxyoctadecadienoic acid and protein carbonyl content) and decreased accumulation of interstitial myofibroblasts compared with wild-type mice. These data suggest that CD36 is a key modulator of proinflammatory and oxidative pathways that promote fibrogenesis in CKD.

Tipping the redox balance of oxidative stress in fibrogenic pathways in chronic kidney disease.

Patients with moderate to advanced chronic kidney disease or end-stage renal disease have a greatly increased cardiovascular risk that cannot be explained entirely by traditional cardiovascular risk factors. An increase in oxidative stress and inflammation have been proposed as nontraditional cardiovascular risk factors in this patient population. Oxidative stress reflects the redox balance between oxidant generation and antioxidant mechanisms. The generation of reactive oxygen species is not simply a random process that oxidizes nearby macromolecules, but, in many instances, the oxidants target particular amino acid residues or lipid moieties. Oxidant mechanisms are now recognized to be intimately involved in cell signaling and to be vital components of the immune response. This is equally true for antioxidant mechanisms as well. In the progression of chronic kidney disease, the redox balance is not in equilibrium and is tipped toward oxidation, resulting in the dysregulation of cellular process and subsequent tissue injury. In this review we discuss the major oxidant and antioxidant pathways and the biomarkers to assess redox status. We also review the data linking the pathogenesis of oxidative stress, inflammation, and the progressive loss of kidney function in chronic kidney disease.

Developing a Research Mentorship Program: The American Society of Pediatric Nephrology's Experience.

Background: Most pediatric nephrologists work in academia. Mentor-mentee relationships provide support and guidance for successful research career. Mentorship program implementation is valuable in medical fields for providing research opportunities to young faculty. Methods: The American Society of Pediatric Nephrology (ASPN) established a research mentorship program to (a) assist with matching of appropriate mentor-mentee dyads and (b) establish metrics for desirable mentor-mentee outcomes with two independent components: (1) the grants review workshop, a short-term program providing mentor feedback on grant proposals, and (2) the longitudinal program, establishing long-term mentor-mentee relationships. Regular surveys of both mentors and mentees were reviewed to evaluate and refine the program. Results: Twelve mentees and 17 mentors participated in the grant review workshop and 19 mentees were matched to mentors in the longitudinal program. A review of NIH RePORTER data indicated that since 2014, 13 NIH grants have been awarded. Mentees in the longitudinal program reported that the program helped most with identifying an outside mentor, improving grant research content, and with general career development. Mentors perceived themselves to be most helpful in assisting with overall career plans. Email communications were preferred over phone or face-to-face communications. Mentees endorsed strong interest in staying in touch with their mentors and 100% of mentors expressed their willingness to serve in the future. Conclusion: This mentorship program was initiated and supported by a relatively small medical society and has shown early success in cultivating mentoring relationships for a future generation of clinician-scientists.

Distinct patterns of transcriptional and epigenetic alterations characterize acute and chronic kidney injury.

Acute kidney injury (AKI) and chronic kidney disease (CKD) are considered early and late phases of a pathologic continuum of interconnected disease states. Although changes in gene expression patterns have recently been elucidated for the transition of AKI to CKD, the epigenetic regulation of key kidney injury related genes remains poorly understood. We used multiplex RT-qPCR, ChIP-qPCR and integrative analysis to compare transcriptional and epigenetic changes at renal disease-associated genes across mouse AKI and CKD models. These studies showed that: (i) there are subsets of genes with distinct transcriptional and epigenetically profiles shared by AKI and CKD but also subsets that are specific to either the early or late stages of renal injury; (ii) differences in expression of a small number of genes is sufficient to distinguish AKI from CKD; (iii) transcription plays a key role in the upregulation of both AKI and CKD genes while post-transcriptional regulation appears to play a more significant role in decreased expression of both AKI and CKD genes; and (iv) subsets of transcriptionally upregulated genes share epigenetic similarities while downregulated genes do not. Collectively, our study suggests that identified common transcriptional and epigenetic profiles of kidney injury loci could be exploited for therapeutic targeting in AKI and CKD.