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Crescentic glomerulonephritis is characterized by vascular necrosis and parietal epithelial cell hyperplasia in the space surrounding the glomerulus, resulting in the formation of crescents. Little is known about the molecular mechanisms driving this process. Inducing crescentic glomerulonephritis in two Pax2Cre reporter mouse models revealed that crescents derive from clonal expansion of single immature parietal epithelial cells. Preemptive and delayed histone deacetylase inhibition with panobinostat, a drug used to treat hematopoietic stem cell disorders, attenuated crescentic glomerulonephritis with recovery of kidney function in the two mouse models. Three-dimensional confocal microscopy and stimulated emission depletion superresolution imaging of mouse glomeruli showed that, in addition to exerting an anti-inflammatory and immunosuppressive effect, panobinostat induced differentiation of an immature hyperplastic parietal epithelial cell subset into podocytes, thereby restoring the glomerular filtration barrier. Single-cell RNA sequencing of human renal progenitor cells in vitro identified an immature stratifin-positive cell subset and revealed that expansion of this stratifin-expressing progenitor cell subset was associated with a poor outcome in human crescentic glomerulonephritis. Treatment of human parietal epithelial cells in vitro with panobinostat attenuated stratifin expression in renal progenitor cells, reduced their proliferation, and promoted their differentiation into podocytes. These results offer mechanistic insights into the formation of glomerular crescents and demonstrate that selective targeting of renal progenitor cells can attenuate crescent formation and the deterioration of kidney function in crescentic glomerulonephritis in mice.

BACKGROUND AND AIMS Crescentic glomerulonephritis (crescentic GN) encompasses a group of diverse disorders characterized by the presence of massive hyperplasia of parietal epithelial cells (PEC) as the main histopathological lesion at kidney biopsy. It is associated with a rapid decline in kidney function. Typically, crescent formation is the consequence of diverse upstream pathomechanisms involving the specific activation of PEC. PEC normally reside peacefully along Bowman capsule and represent in part renal progenitor cells (RPC). Previous studies observed RPC markers in crescents from patients with different types of glomerulonephritis. Similarities between stem cell niches of bone marrow and kidney, prompted us to hypothesized that crescents result from monoclonal expansion of a single RPC clone conceptually similar to monoclonal diseases originating from hematopoietic stem cells. According to this analogy, we further hypothesized that drugs known to cure monoclonal disease of the hematopoietic stem cells by enforcing their terminal differentiation could also attenuate crescentic glomerulonephritis. METHOD To address this hypothesis, we established a crescentic GN disease model in a conditional transgenic mouse based on the mT/mG and the Confetti reporter that allows lineage tracing and clonal analysis of RPCs. Animals were treated with known pharmacological inhibitors of clonal stem cell proliferation in myeloproliferative disorders. Crescentic lesions were characterized by super-resolution STED microscopy. Finally, we employed single cell RNA sequencing of human renal progenitor cultures to identify the immature progenitor subset-generating crescent in human to identify putative new biomarkers of crescentic GN to validate in biopsy of patients. RESULTS We observed that the crescentic lesions originated from the clonal expansion of single RPC, thus suggesting a clonal stem cell disorder. Therefore, we administrated a series of drugs known to ameliorates myeloproliferative neoplasms to our crescentic GN mouse model as potential therapeutic agents. Treatment with one of the compounds induced a reduction in both proteinuria and crescent formation. STED super-resolution imaging of glomeruli showed that this compound turned the uncontrolled hyperplasia of a specific immature PEC subset into a controlled differentiation into podocytes thereby restoring the injured glomerular filtration barrier. Moreover, delayed drug administration still induced proteinuria remission and avoided long-term development of chronic kidney disease (CKD), an effect associated to a continued generation of new podocytes and crescent regression over time. Single cell RNA sequencing of human RPC identified a new marker of the crescent-generating progenitor cells. Expression of this marker in biopsies of patients with crescentic GN associated with progression toward end stage kidney disease. Treatment of human PEC with the drug that in in vivo experiments showed a therapeutic effect on crescentic GN reduced proliferation of the immature progenitor subset promoting their differentiation into podocytes. CONCLUSION These results demonstrate that glomerular hyperplastic lesions derive from clonal amplification of a RPC subset and that shifting proliferation to podocyte differentiation reverses improves clinical outcome and avoid CKD.

Acute kidney injury (AKI) is characterized by a rapid deterioration of kidney function, representing a global healthcare concern. In addition, AKI survivors frequently develop chronic kidney disease (CKD), contributing to a substantial proportion of disease burden globally. Yet, over the past 30 years, the burden of CKD has not declined to the same extent as many other important non-communicable diseases, implying a substantial deficit in the understanding of the disease progression. The assumption that the kidney response to AKI is based on a high proliferative potential of proximal tubular cells (PTC) caused a critical confounding factor, which has led to a limited development of strategies to prevent AKI and halt progression toward CKD. In this review, we discuss the latest findings on multiple mechanisms of response related to cell cycle behavior of PTC upon AKI, with a specific focus on their biological relevance. Collectively, we aim to (1) provide a new perspective on interpreting cell cycle progression of PTC in response to damage and (2) discuss how this knowledge can be used to choose the right therapeutic window of treatment for preserving kidney function while avoiding CKD progression.

Acute kidney injury (AKI) is frequent, often fatal and, for lack of specific therapies, can leave survivors with chronic kidney disease (CKD). We characterize the distribution of tubular cells (TC) undergoing polyploidy along AKI by DNA content analysis and single cell RNA-sequencing. Furthermore, we study the functional roles of polyploidization using transgenic models and drug interventions. We identify YAP1-driven TC polyploidization outside the site of injury as a rapid way to sustain residual kidney function early during AKI. This survival mechanism comes at the cost of senescence of polyploid TC promoting interstitial fibrosis and CKD in AKI survivors. However, targeting TC polyploidization after the early AKI phase can prevent AKI-CKD transition without influencing AKI lethality. Senolytic treatment prevents CKD by blocking repeated TC polyploidization cycles. These results revise the current pathophysiological concept of how the kidney responds to acute injury and identify a novel druggable target to improve prognosis in AKI survivors.

Background and Aims Acute kidney injury (AKI) is a global health concern with an incidence of 13.3 million patients per year, and increasing. AKI is recognized as an important risk factor for the development of chronic kidney disease (CKD). A crucial aspect for successful renal recovery after AKI is an efficient proliferative response of surviving tubular epithelial cells (TECs). Recently, we established a murine model in which the functional and histological recovery of a single kidney, injured by ischemia, is enhanced by removal of the unharmed contralateral kidney; a phenomenon termed nephrectomy-induced recovery. The renal epithelial reparative response in this unique physiological model has not been investigated, yet can provide new insights in unlocking the inherent regenerative potential of the renal epithelium. Method AKI was induced in R26RtdTomato and PAX2/Confetti mice by left unilateral ischemia/reperfusion (UIRI) for 21 min at 34°C, after which either right nephrectomy (Nx) or no Nx was performed 3 days later. Mice were euthanized 6 weeks and 28 days after UIRI, respectively. At week 6, kidneys were weighted and renal function was assessed by serum creatinine. At 28 days, renal tissue of Pax2/Confetti mice was collected to perform renal progenitor cell lineage tracing experiments by immunofluorescence and confocal microscopy. Results When nephrectomy was performed after UIRI, left kidney-to-body weight ratio did not change significantly over time, whereas, when no nephrectomy was performed, left kidney-to-body weight ratio gradually declined from 7,84 ± 0,48 mg/dl at day 3 till 3,26 ± 0,51 mg/dl at week 6, indicating severe atrophy in the injured left kidney. This loss of renal mass was associated with a significant increase in serum creatinine (1,76 ± 0,13 mg/dl) as compared to control (0,21 ± 0,12 mg/dl), whereas with nephrectomy, renal function fully restored. Clonal analysis in PAX2/Confetti mice revealed that nephrectomy after UIRI led to a significant increase in proliferating (i.e. clonogenic) Pax2+ progenitor cells, resulting in more multicellular clones as compared to un-nephrectomized controls. Conclusion Nephrectomy after UIRI overcomes chronic loss of renal mass and function within the investigated 6-week time frame. This study is the first to demonstrate that nephrectomy stimulates clonal expansion of renal progenitor cells in an injured kidney, beyond that observed for spontaneous repair after UIRI. Insight in the signaling mechanisms may reveal new therapeutic approaches to incite the inherent renal regeneration potential.

Simple Summary Kidney cancer is a frequent malignant tumor that accounts for approximately 5% of all cancer incidences. It affects both males and females, but males are twice as likely to develop kidney cancer than females. Evidence shows that this discrepancy takes root in individual differences, such as genetics or pathologies that affect the patient. It is then reflected in the clinical characteristics of the tumors, as males have larger and more aggressive tumors. Understanding the sex- and gender-based differences in kidney cancer is essential to be able to offer patients individualized medicine that would better cover their needs in terms of prevention, diagnosis and treatment. Abstract Sex and gender disparities have been reported for different types of non-reproductive cancers. Males are two times more likely to develop kidney cancer than females and have a higher death rate. These differences can be explained by looking at genetics and genomics, as well as other risk factors such as hypertension and obesity, lifestyle, and female sex hormones. Examination of the hormonal signaling pathways bring further insights into sex-related differences. Sex and gender-based disparities can be observed at the diagnostic, histological and treatment levels, leading to significant outcome difference. This review summarizes the current knowledge about sex and gender-related differences in the clinical presentation of patients with kidney cancer and the possible biological mechanisms that could explain these observations. Underlying sex-based differences may contribute to the development of sex-specific prognostic and diagnostic tools and the improvement of personalized therapies.

Background and Aims Acute kidney injury (AKI) is a global health concern. If not lethal in the acute phase, AKI is considered reversible based on the capacity of surviving tubular cells (TECs) to re-enter cell cycle. However, even mild AKI episodes carry a substantial risk of developing chronic kidney disease (CKD). The pathophysiological basis for this phenomenon remains unclear. Recently, we demonstrated that tubular epithelial cells (TECs) can undergo endoreplication-mediated hypertrophy after AKI. Endoreplications are incomplete cell cycles that lead to the formation of polyploid cells. As polyploid cells can provide increased cell function without restoring tissue integrity, we hypothesized that this mechanism is essential to survive AKI but it can be potentially maladaptive. Method To address this hypothesis, we employed a series of in vitro and in vivo transgenic models based on the Fluorescence Ubiquitin Cell Cycle Indicator (FUCCI) technology to monitor cell cycle phasing in combination with YAP1 overexpression or downregulation. In the in vivo models, YAP1 overexpressing mice and YAP1 knock-out mice were subjected to unilateral ischemia reperfusion injury (IRI) or glycerol-induced rhabdomyolysis to induce AKI. Polyploid cells have been then characterized by microarray analysis, cell sorting, super-resolution STED microscopy and transmission electron microscopy. Results In vitro, human renal tubular cells undergo polyploidization. The fraction of polyploid cells significantly decreases when YAP1 nuclear translocation is blocked, suggesting a possible involvement of YAP1 in regulating TEC polyploidization. After AKI in mice, the inhibition of YAP1 significantly reduces the number of polyploid cells and worsens kidney function resulting in a dramatic decrease of mouse survival. In contrast, YAP1 overexpression leads to an increase in the number of polyploid cells up to 20% of all TECs, further confirming the role of YAP1 in controlling TEC polyploidization. In YAP1 overexpressing mice, electron microscopy and STED analysis revealed the presence of both mononucleated and binucleated polyploid cells. Strikingly, these mice appear to be more prone to develop tubulointerstitial fibrosis acquiring a marked senescent phenotype along with significant decline in renal function thus suggesting an association between polyploidization and CKD development. Indeed, isolation of polyploid cells proved that these cells actively transcribe and secrete pro-fibrotic and senescent factors confirming their role in CKD progression. Conclusion These data suggest that: 1) polyploidization after AKI is required to preserve renal function in the acute phase of damage and it is essential for survival 2) polyploid cells are pro-fibrotic and senescent leading in the long run to the progression of AKI to CKD.

Insufficient podocyte regeneration after injury is a central pathomechanism of glomerulosclerosis and chronic kidney disease. Podocytes constitutively secrete the chemokine CXCL12, which is known to regulate homing and activation of stem cells; hence we hypothesized a similar effect of CXCL12 on podocyte progenitors. CXCL12 blockade increased podocyte numbers and attenuated proteinuria in mice with Adriamycin-induced nephropathy. Similar studies in lineage-tracing mice revealed enhanced de novo podocyte formation from parietal epithelial cells in the setting of CXCL12 blockade. Super-resolution microscopy documented full integration of these progenitor-derived podocytes into the glomerular filtration barrier, interdigitating with tertiary foot processes of neighboring podocytes. Quantitative 3D analysis revealed that conventional 2D analysis underestimated the numbers of progenitor-derived podocytes. The 3D analysis also demonstrated differences between juxtamedullary and cortical nephrons in both progenitor endowment and Adriamycin-induced podocyte loss, with more robust podocyte regeneration in cortical nephrons with CXCL12 blockade. Finally, we found that delayed CXCL12 inhibition still had protective effects. In vitro studies found that CXCL12 inhibition uncoupled Notch signaling in podocyte progenitors. These data suggest that CXCL12-driven podocyte-progenitor feedback maintains progenitor quiescence during homeostasis, but also limits their intrinsic capacity to regenerate lost podocytes, especially in cortical nephrons. CXCL12 inhibition could be an innovative therapeutic strategy in glomerular disorders., Graphical abstract

In acute organ failure, rapid compensation of function loss assures survival. Dedifferentiation and/or proliferation of surviving parenchymal cells could imply a transient (and potentially fatal) impairment of residual functional performance. However, evolution has selected two flexible life-saving mechanisms acting synergistically on organ function recovery. Sustaining residual performance is possible when the remnant differentiated parenchymal cells avoid cell division, but increase function by undergoing hypertrophy via endoreplication, leading to polyploid cells. In addition, tissue progenitors, representing a subset of less-differentiated and/or self-renewing parenchymal cells completing cytokinesis, proliferate and differentiate to regenerate lost parenchymal cells. Here, we review the evolving evidence on polyploidization and progenitor-driven regeneration in acute liver, heart, and kidney failure with evolutionary advantages and trade-offs in organ repair.

Acute kidney injury (AKI) is considered largely reversible based on the capacity of surviving tubular cells to dedifferentiate and replace lost cells via cell division. Here we show by tracking individual tubular cells in conditional Pax8/Confetti mice that kidney function is recovered after AKI despite substantial tubular cell loss. Cell cycle and ploidy analysis upon AKI in conditional Pax8/FUCCI2aR mice and human biopsies identify endocycle-mediated hypertrophy of tubular cells. By contrast, a small subset of Pax2+ tubular progenitors enriches via higher stress resistance and clonal expansion and regenerates necrotic tubule segments, a process that can be enhanced by suitable drugs. Thus, renal functional recovery upon AKI involves remnant tubular cell hypertrophy via endocycle and limited progenitor-driven regeneration that can be pharmacologically enhanced., The recovery of function upon acute kidney injury is thought to involve tubular cell dedifferentiation and proliferation. Here the authors show that Pax2+ progenitors regenerate tubules via cell division while other tubular cells support function recovery by undergoing hypertrophy through endoreplication.

The important achievements in kidney physiological and pathophysiological mechanisms can largely be ascribed to progress in the technology of microscopy. Much of what we know about the architecture of the kidney is based on the fundamental descriptions of anatomic microscopists using light microscopy and later by ultrastructural analysis provided by electron microscopy. These two techniques were used for the first classification systems of kidney diseases and for their constant updates. More recently, a series of novel imaging techniques added the analysis in further dimensions of time and space. Confocal microscopy allowed us to sequentially visualize optical sections along the z-axis and the availability of specific analysis software provided a three-dimensional rendering of thicker tissue specimens. Multiphoton microscopy permitted us to simultaneously investigate kidney function and structure in real time. Fluorescence-lifetime imaging microscopy allowed to study the spatial distribution of metabolites. Super-resolution microscopy increased sensitivity and resolution up to nanoscale levels. With cryo-electron microscopy, researchers could visualize the individual biomolecules at atomic levels directly in the tissues and understand their interaction at subcellular levels. Finally, matrix-assisted laser desorption/ionization imaging mass spectrometry permitted the measuring of hundreds of different molecules at the same time on tissue sections at high resolution. This review provides an overview of available kidney imaging strategies, with a focus on the possible impact of the most recent technical improvements.

Acute tissue injury causes DNA damage and repair processes involving increased cell mitosis and polyploidization, leading to cell function alterations that may potentially drive cancer development. Here, we show that acute kidney injury (AKI) increased the risk for papillary renal cell carcinoma (pRCC) development and tumor relapse in humans as confirmed by data collected from several single-center and multicentric studies. Lineage tracing of tubular epithelial cells (TECs) after AKI induction and long-term follow-up in mice showed time-dependent onset of clonal papillary tumors in an adenoma-carcinoma sequence. Among AKI-related pathways, NOTCH1 overexpression in human pRCC associated with worse outcome and was specific for type 2 pRCC. Mice overexpressing NOTCH1 in TECs developed papillary adenomas and type 2 pRCCs, and AKI accelerated this process. Lineage tracing in mice identified single renal progenitors as the cell of origin of papillary tumors. Single-cell RNA sequencing showed that human renal progenitor transcriptome showed similarities to PT1, the putative cell of origin of human pRCC. Furthermore, NOTCH1 overexpression in cultured human renal progenitor cells induced tumor-like 3D growth. Thus, AKI can drive tumorigenesis from local tissue progenitor cells. In particular, we find that AKI promotes the development of pRCC from single progenitors through a classical adenoma-carcinoma sequence.