Search

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.

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.

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

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.