Mitochondria Transplantation Mitigates Damage in an In Vitro Model of Renal Tubular Injury and in an Ex Vivo Model of DCD Renal Transplantation.

OBJECTIVES: To test whether mitochondrial transplantation (MITO) mitigates damage in 2 models of acute kidney injury (AKI). BACKGROUND: MITO is a process where exogenous isolated mitochondria are taken up by cells. As virtually any morbid clinical condition is characterized by mitochondrial distress, MITO may find a role as a treatment modality in numerous clinical scenarios including AKI. METHODS: For the in vitro experiments, human proximal tubular cells were damaged and then treated with mitochondria or placebo. For the ex vivo experiments, we developed a non-survival ex vivo porcine model mimicking the donation after cardiac death renal transplantation scenario. One kidney was treated with mitochondria, although the mate organ received placebo, before being perfused at room temperature for 24 hours. Perfusate samples were collected at different time points and analyzed with Raman spectroscopy. Biopsies taken at baseline and 24 hours were analyzed with standard pathology, immunohistochemistry, and RNA sequencing analysis. RESULTS: In vitro, cells treated with MITO showed higher proliferative capacity and adenosine 5'-triphosphate production, preservation of physiological polarization of the organelles and lower toxicity and reactive oxygen species production. Ex vivo, kidneys treated with MITO shed fewer molecular species, indicating stability. In these kidneys, pathology showed less damage whereas RNAseq analysis showed modulation of genes and pathways most consistent with mitochondrial biogenesis and energy metabolism and downregulation of genes involved in neutrophil recruitment, including IL1A, CXCL8, and PIK3R1. CONCLUSIONS: MITO mitigates AKI both in vitro and ex vivo.

Amniotic fluid cells: current progress and emerging challenges in renal regeneration.

Amniotic fluid (AF) contains a heterogeneous population of cells that have been identified to possess pluripotent and progenitor-like characteristics. These cells have been applied in various regenerative medicine applications ranging from in vitro cell differentiation to tissue engineering to cellular therapies for different organs including the heart, the liver, the lung, and the kidneys. In this review, we examine the different methodologies used for the derivation of amniotic fluid stem cells and renal progenitors, and their application in renal repair and regeneration. Moreover, we discuss the recent achievements and newly emerging challenges in our understanding of their biology, their immunoregulatory characteristics, and their paracrine-mediated therapeutic potential for the treatment of acute and chronic kidney diseases.

Amniotic fluid stem cells prevent ß-cell injury.

BACKGROUND AIMS: The contribution of amniotic fluid stem cells (AFSC) to tissue protection and regeneration in models of acute and chronic kidney injuries and lung failure has been shown in recent years. In the present study, we used a chemically induced mouse model of type 1 diabetes to determine whether AFSC could play a role in modulating ß-cell injury and restoring ß-cell function. METHODS: Streptozotocin-induced diabetic mice were given intracardial injection of AFSC; morphological and physiological parameters and gene expression profile for the insulin pathway were evaluated after cell transplantation. RESULTS: AFSC injection resulted in protection from ß-cell damage and increased ß-cell regeneration in a subset of mice as indicated by glucose and insulin levels, increased islet mass and preservation of islet structure. Moreover, ß-cell preservation/regeneration correlated with activation of the insulin receptor/Pi3K/Akt signaling pathway and vascular endothelial growth factor-A expression involved in maintaining ß-cell mass and function. CONCLUSIONS: Our results suggest a therapeutic role for AFSC in preserving and promoting endogenous ß-cell functionality and proliferation. The protective role of AFSC is evident when stem cell transplantation is performed before severe hyperglycemia occurs, which suggests the importance of early intervention. The present study demonstrates the possible benefits of the application of a non-genetically engineered stem cell population derived from amniotic fluid for the treatment of type 1 diabetes mellitus and gives new insight on the mechanism by which the beneficial effect is achieved.

The spatially resolved transcriptome signatures of glomeruli in chronic kidney disease.

Here, we used digital spatial profiling (DSP) to describe the glomerular transcriptomic signatures that may characterize the complex molecular mechanisms underlying progressive kidney disease in Alport syndrome, focal segmental glomerulosclerosis, and membranous nephropathy. Our results revealed significant transcriptional heterogeneity among diseased glomeruli, and this analysis showed that histologically similar glomeruli manifested different transcriptional profiles. Using glomerular pathology scores to establish an axis of progression, we identified molecular pathways with progressively decreased expression in response to increasing pathology scores, including signal recognition particle-dependent cotranslational protein targeting to membrane and selenocysteine synthesis pathways. We also identified a distinct signature of upregulated and downregulated genes common to all the diseases investigated when compared with nondiseased tissue from nephrectomies. These analyses using DSP at the single-glomerulus level could help to increase insight into the pathophysiology of kidney disease and possibly the identification of biomarkers of disease progression in glomerulopathies.

C3aR-initiated signaling is a critical mechanism of podocyte injury in membranous nephropathy.

The deposition of antipodocyte autoantibodies in the glomerular subepithelial space induces primary membranous nephropathy (MN), the leading cause of nephrotic syndrome worldwide. Taking advantage of the glomerulus-on-a-chip system, we modeled human primary MN induced by anti-PLA2R antibodies. Here we show that exposure of primary human podocytes expressing PLA2R to MN serum results in IgG deposition and complement activation on their surface, leading to loss of the chip permselectivity to albumin. C3a receptor (C3aR) antagonists as well as C3AR gene silencing in podocytes reduced oxidative stress induced by MN serum and prevented albumin leakage. In contrast, inhibition of the formation of the membrane-attack-complex (MAC), previously thought to play a major role in MN pathogenesis, did not affect permselectivity to albumin. In addition, treatment with a C3aR antagonist effectively prevented proteinuria in a mouse model of MN, substantiating the chip findings. In conclusion, using a combination of pathophysiologically relevant in vitro and in vivo models, we established that C3a/C3aR signaling plays a critical role in complement-mediated MN pathogenesis, indicating an alternative therapeutic target for MN.

Injection of amniotic fluid stem cells delays progression of renal fibrosis.

Injection of amniotic fluid stem cells ameliorates the acute phase of acute tubular necrosis in animals by promoting proliferation of injured tubular cells and decreasing apoptosis, but whether these stem cells could be of benefit in CKD is unknown. Here, we used a mouse model of Alport syndrome, Col4a5(-/-) mice, to determine whether amniotic fluid stem cells could modify the course of progressive renal fibrosis. Intracardiac administration of amniotic fluid stem cells before the onset of proteinuria delayed interstitial fibrosis and progression of glomerular sclerosis, prolonged animal survival, and ameliorated the decline in kidney function. Treated animals exhibited decreased recruitment and activation of M1-type macrophages and a higher proportion of M2-type macrophages, which promote tissue remodeling. Amniotic fluid stem cells did not differentiate into podocyte-like cells and did not stimulate production of the collagen IVa5 needed for normal formation and function of the glomerular basement membrane. Instead, the mechanism of renal protection was probably the paracrine/endocrine modulation of both profibrotic cytokine expression and recruitment of macrophages to the interstitial space. Furthermore, injected mice retained a normal number of podocytes and had better integrity of the glomerular basement membrane compared with untreated Col4a5(-/-) mice. Inhibition of the renin-angiotensin system by amniotic fluid stem cells may contribute to these beneficial effects. In conclusion, treatment with amniotic fluid stem cells may be beneficial in kidney diseases characterized by progressive renal fibrosis.

Identification and Characterization of the Wilms Tumor Cancer Stem Cell.

A nephrogenic progenitor cell (NP) with cancer stem cell characteristics driving Wilms tumor (WT) using spatial transcriptomics, bulk and single cell RNA sequencing, and complementary in vitro and transplantation experiments is identified and characterized. NP from WT samples with NP from the developing human kidney is compared. Cells expressing SIX2 and CITED1 fulfill cancer stem cell criteria by reliably recapitulating WT in transplantation studies. It is shown that self-renewal versus differentiation in SIX2+CITED1+ cells is regulated by the interplay between integrins ITGß1 and ITGß4. The spatial transcriptomic analysis defines gene expression maps of SIX2+CITED1+ cells in WT samples and identifies the interactive gene networks involved in WT development. These studies define SIX2+CITED1+ cells as the nephrogenic-like cancer stem cells of WT and points to the renal developmental transcriptome changes as a possible driver in regulating WT formation and progression.

Generation of a Glomerular Filtration Barrier on a Glomerulus-on-a-Chip Platform.

Despite an enormous investment of clinical and financial resources, chronic kidney disease (CKD) remains a global health threat. The lack of reliable in vitro systems that can efficiently mimic the renal and glomerular environment has hampered our ability to successfully develop novel and more renal specific drugs. Even though some success in generating in vitro tubule analogues and kidney organoids has been described, a major challenge remains for the in vitro assembly of the filtration unit of the kidney, the glomerulus. We have recently developed a novel glomerulus-on-a-chip system that mimics the characteristic and functionality of the glomerular filtration barrier, including its response to injury. This system recapitulates the functions and structure of the in vivo glomerulus, including permselectivity; indeed, we have confirmed free diffusion of insulin as well as impermeability to physiological concentrations of albumin. Exposure to nephrotoxic agents like puromycin aminonucleoside leads to a significant increase in albumin leakage. When exposed to sera from patients with anti-podocyte autoantibodies, the chip shows albumin leakage to an extent proportional to in vivo clinical data, phenomenon not observed with sera from either healthy controls, confirming functional response to injury. We describe here the detailed procedure to obtain a glomerulus-on-a-chip system that replicates both phenotypically and functionally the in vivo glomerular microenvironment.

Intravital imaging reveals glomerular capillary distension and endothelial and immune cell activation early in Alport syndrome.

Alport syndrome (AS) is a genetic disorder caused by mutations in type IV collagen that lead to defective glomerular basement membrane, glomerular filtration barrier (GFB) damage, and progressive chronic kidney disease. While the genetic basis of AS is well known, the molecular and cellular mechanistic details of disease pathogenesis have been elusive, hindering the development of mechanism-based therapies. Here, we performed intravital multiphoton imaging of the local kidney tissue microenvironment in a X-linked AS mouse model to directly visualize the major drivers of AS pathology. Severely distended glomerular capillaries and aneurysms were found accompanied by numerous microthrombi, increased glomerular endothelial surface layer (glycocalyx) and immune cell homing, GFB albumin leakage, glomerulosclerosis, and interstitial fibrosis by 5 months of age, with an intermediate phenotype at 2 months. Renal histology in mouse or patient tissues largely failed to detect capillary aberrations. Treatment of AS mice with hyaluronidase or the ACE inhibitor enalapril reduced the excess glomerular endothelial glycocalyx and blocked immune cell homing and GFB albumin leakage. This study identified central roles of glomerular mechanical forces and endothelial and immune cell activation early in AS, which could be therapeutically targeted to reduce mechanical strain and local tissue inflammation and improve kidney function.

The milieu of damaged alveolar epithelial type 2 cells stimulates alveolar wound repair by endogenous and exogenous progenitors.

Alveolar epithelial integrity is dependent upon the alveolar milieu, yet the milieu of the damaged alveolar epithelial cell type 2 (AEC2) has been little studied. Characterization of its components may offer the potential for ex vivo manipulation of stem cells to optimize their therapeutic potential. We examined the cytokine profile of AEC2 damage milieu, hypothesizing that it would promote endogenous epithelial repair while recruiting cells from other locations and instructing their engraftment and differentiation. Bronchoalveolar lavage and lung extract from hyperoxic rats represented AEC2 in vivo damage milieu, and medium from a scratch-damaged AEC2 monolayer represented in vitro damage. CINC-2 and ICAM, the major cytokines detected by proteomic cytokine array in AEC2 damage milieu, were chemoattractive to normoxic AECs and expedited in vitro wound healing, which was blocked by their respective neutralizing antibodies. The AEC2 damage milieu was also chemotactic for exogenous uncommitted human amniotic fluid stem cells (hAFSCs), increasing migration greater than 20-fold. hAFSCs attached within an in vitro AEC2 wound and expedited wound repair by contributing cytokines migration inhibitory factor and plasminogen activator inhibitor 1 to the AEC2 damage milieu, which promoted wound healing. The AEC2 damage milieu also promoted differentiation of a subpopulation of hAFSCs to express SPC, TTF-1, and ABCA3, phenotypic markers of distal alveolar epithelium. Thus, the microenvironment created by AEC2 damage not only promotes autocrine repair but also can attract uncommitted stem cells, which further augment healing through cytokine secretion and differentiation.

Amniotic fluid as a source of pluripotent and multipotent stem cells for organ regeneration.

PURPOSE OF REVIEW: Amniotic fluid, due to its contact to the fetus during development, is considered an important diagnostic tool to evaluate the health status of the fetus during pregnancy. However, amniotic fluid also contains a heterogeneous cellular population that can be safely collected by amniocentesis and easily cultured. Many different cell types have been found within amniotic fluid and currently some of them are being tested for their possible use for cellular therapy. RECENT FINDINGS: Potential of pluripotent and multipotent cells isolated from the amniotic fluid has been tested and in-vitro differentiations toward various cell types have been successfully performed. Furthermore, in-vivo studies are highlighting the benefits and mechanisms of amniotic fluid cells for therapy, with particular focus on kidney and lung diseases. SUMMARY: Amniotic fluid may represent a precious source for easily and safely retrievable cell types that may be used for regenerative medicine purposes.

Emerging Technologies to Study the Glomerular Filtration Barrier.

Kidney disease is characterized by loss of glomerular function with clinical manifestation of proteinuria. Identifying the cellular and molecular changes that lead to loss of protein in the urine is challenging due to the complexity of the filtration barrier, constituted by podocytes, glomerular endothelial cells, and glomerular basement membrane. In this review, we will discuss how technologies like single cell RNA sequencing and bioinformatics-based spatial transcriptomics, as well as in vitro systems like kidney organoids and the glomerulus-on-a-chip, have contributed to our understanding of glomerular pathophysiology. Knowledge gained from these studies will contribute toward the development of personalized therapeutic approaches for patients affected by proteinuric diseases.

Human amniotic fluid as a potential new source of organ specific precursor cells for future regenerative medicine applications.

PURPOSE: Human amniotic fluid contains multiple cell types, including pluripotent and committed progenitor cells, and fully differentiated cells. We characterized various cell populations in amniotic fluid. MATERIALS AND METHODS: Optimum culture techniques for multiple cell line passages with minimal morphological change were established. Cell line analysis and characterization were done with reverse transcriptase and real-time polymerase chain reaction. Immunoseparation was done to distinguish native progenitor cell lines and their various subpopulations. RESULTS: Endodermal and mesodermal marker expression was greatest in samples of early gestational age while ectodermal markers showed a constant rate across all samples. Pluripotent and mesenchymal cells were always present but hematopoietic cell markers were expressed only in older samples. Specific markers for lung, kidney, liver and heart progenitor cells were increasingly expressed after 18 weeks of gestation. We specifically focused on a CD24+OB-cadherin+ population that could identify uninduced metanephric mesenchyma-like cells, which in vivo are nephron precursors. The CD24+OB-cadherin+ cell line was isolated and subjected to further immunoseparation to select 5 distinct amniotic fluid kidney progenitor cell subpopulations based on E-cadherin, podocalyxin, nephrin, TRKA and PDGFRA expression, respectively. CONCLUSIONS: These subpopulations may represent different precursor cell lineages committed to specific renal cell fates. Committed progenitor cells in amniotic fluid may provide an important and novel resource of useful cells for regenerative medicine purposes.

Loss of decay-accelerating factor triggers podocyte injury and glomerulosclerosis.

Kidney glomerulosclerosis commonly progresses to end-stage kidney failure, but pathogenic mechanisms are still poorly understood. Here, we show that podocyte expression of decay-accelerating factor (DAF/CD55), a complement C3 convertase regulator, crucially controls disease in murine models of adriamycin (ADR)-induced focal and segmental glomerulosclerosis (FSGS) and streptozotocin (STZ)-induced diabetic glomerulosclerosis. ADR induces enzymatic cleavage of DAF from podocyte surfaces, leading to complement activation. C3 deficiency or prevention of C3a receptor (C3aR) signaling abrogates disease despite DAF deficiency, confirming complement dependence. Mechanistic studies show that C3a/C3aR ligations on podocytes initiate an autocrine IL-1ß/IL-1R1 signaling loop that reduces nephrin expression, causing actin cytoskeleton rearrangement. Uncoupling IL-1ß/IL-1R1 signaling prevents disease, providing a causal link. Glomeruli of patients with FSGS lack DAF and stain positive for C3d, and urinary C3a positively correlates with the degree of proteinuria. Together, our data indicate that the development and progression of glomerulosclerosis involve loss of podocyte DAF, triggering local, complement-dependent, IL-1ß-induced podocyte injury, potentially identifying new therapeutic targets.

Author Correction: A glomerulus-on-a-chip to recapitulate the human glomerular filtration barrier.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A glomerulus-on-a-chip to recapitulate the human glomerular filtration barrier.

In this work we model the glomerular filtration barrier, the structure responsible for filtering the blood and preventing the loss of proteins, using human podocytes and glomerular endothelial cells seeded into microfluidic chips. In long-term cultures, cells maintain their morphology, form capillary-like structures and express slit diaphragm proteins. This system recapitulates functions and structure of the glomerulus, including permselectivity. When exposed to sera from patients with anti-podocyte autoantibodies, the chips show albuminuria proportional to patients' proteinuria, phenomenon not observed with sera from healthy controls or individuals with primary podocyte defects. We also show its applicability for renal disease modeling and drug testing. A total of 2000 independent chips were analyzed, supporting high reproducibility and validation of the system for high-throughput screening of therapeutic compounds. The study of the patho-physiology of the glomerulus and identification of therapeutic targets are also feasible using this chip.

Circulating B Cells With Memory and Antibody-Secreting Phenotypes Are Detectable in Pediatric Kidney Transplant Recipients Before the Development of Antibody-Mediated Rejection.

Development of anti-human leukocyte antigen donor-specific antibodies (DSAs) is associated with antibody-mediated rejection (AMR) and reduced allograft survival in kidney transplant recipients. Whether changes in circulating lymphocytes anticipate DSA or AMR development is unclear. METHODS: We used time-of-flight mass cytometry to analyze prospectively collected peripheral blood mononuclear cells (PBMC) from pediatric kidney transplant recipients who developed DSA (DSA-positive recipients [DSAPOS], n = 10). PBMC were obtained at 2 months posttransplant, 3 months before DSA development, and at DSA detection. PBMC collected at the same time points posttransplant from recipients who did not develop DSA (DSA-negative recipients [DSANEG], n = 11) were used as controls. RESULTS: DSAPOS and DSANEG recipients had similar baseline characteristics and comparable frequencies of total B and T cells. Within DSAPOS recipients, there was no difference in DSA levels (mean fluorescence intensity [MFI]: 13 687 ± 4159 vs 11 375 ± 1894 in DSAPOSAMR-positive recipients (AMRPOS) vs DSAPOSAMR-negative recipients (AMRNEG), respectively; P = 0.630), C1q binding (5 DSAPOSAMRPOS [100%] vs 4 DSAPOSAMRNEG [80%]; P = 1.000), or C3d binding (3 DSAPOSAMRPOS [60%] vs 1 DSAPOSAMRNEG [20%]; P = 0.520) between patients who developed AMR and those who did not. However, DSAPOS patients who developed AMR (n = 5; 18.0 ± 3.6 mo post-DSA detection) had increased B cells with antibody-secreting (IgD-CD27+CD38+; P = 0.002) and memory (IgD-CD27+CD38-; P = 0.003) phenotypes compared with DSANEG and DSAPOSAMRNEG recipients at DSA detection. CONCLUSIONS: Despite the small sample size, our comprehensive phenotypic analyses show that circulating B cells with memory and antibody-secreting phenotypes are present at DSA onset, >1 year before biopsy-proven AMR in pediatric kidney transplant recipients.

Direct Isolation and Characterization of Human Nephron Progenitors.

Mature nephrons originate from a small population of uninduced nephrogenic progenitor cells (NPs) within the cap mesenchyme. These cells are characterized by the coexpression of SIX2 and CITED1. Many studies on mouse models as well as on human pluripotent stem cells have advanced our knowledge of NPs, but very little is known about this population in humans, since it is exhausted before birth and strategies for its direct isolation are still limited. Here we report an efficient protocol for direct isolation of human NPs without genetic manipulation or stepwise induction procedures. With the use of RNA-labeling probes, we isolated SIX2+ CITED1+ cells from human fetal kidney for the first time. We confirmed their nephrogenic state by gene profiling and evaluated their nephrogenic capabilities in giving rise to mature renal cells. We also evaluated the ability to culture these cells without complete loss of SIX2 and CITED1 expression over time. In addition to defining the gene profile of human NPs, this in vitro system facilitates studies of human renal development and provides a novel tool for renal regeneration and bioengineering purposes. Stem Cells Translational Medicine 2017;6:419-433.

A step towards clinical application of acellular matrix: A clue from macrophage polarization.

The outcome of tissue engineered organ transplants depends on the capacity of the biomaterial to promote a pro-healing response once implanted in vivo. Multiple studies, including ours, have demonstrated the possibility of using the extracellular matrix (ECM) of animal organs as platform for tissue engineering and more recently, discarded human organs have also been proposed as scaffold source. In contrast to artificial biomaterials, natural ECM has the advantage of undergoing continuous remodeling which allows adaptation to diverse conditions. It is known that natural matrices present diverse immune properties when compared to artificial biomaterials. However, how these properties compare between diseased and healthy ECM and artificial scaffolds has not yet been defined. To answer this question, we used decellularized renal ECM derived from WT mice and from mice affected by Alport Syndrome at different time-points of disease progression as a model of renal failure with extensive fibrosis. We characterized the morphology and composition of these ECMs and compared their in vitro effects on macrophage activation with that of synthetic scaffolds commonly used in the clinic (collagen type I and poly-L-(lactic) acid, PLLA). We showed that ECM derived from Alport kidneys differed in fibrous protein deposition and cytokine content when compared to ECM derived from WT kidneys. Yet, both WT and Alport renal ECM induced macrophage differentiation mainly towards a reparative (M2) phenotype, while artificial biomaterials towards an inflammatory (M1) phenotype. Anti-inflammatory properties of natural ECMs were lost when homogenized, hence three-dimensional structure of ECM seems crucial for generating an anti-inflammatory response. Together, these data support the notion that natural ECM, even if derived from diseased kidneys promote a M2 protolerogenic macrophage polarization, thus providing novel insights on the applicability of ECM obtained from discarded organs as ideal scaffold for tissue engineering.

Decellularized Renal Matrix and Regenerative Medicine of the Kidney: A Different Point of View.

Over the past years, extracellular matrix (ECM) obtained from whole organ decellularization has been investigated as a platform for organ engineering. The ECM is composed of fibrous and nonfibrous molecules providing structural and biochemical support to the surrounding cells. Multiple decellularization techniques, including ours, have been optimized to maintain the composition, microstructure, and biomechanical properties of the native renal ECM that are difficult to obtain during the generation of synthetic substrates. There are evidences suggesting that in vivo implanted renal ECM has the capacity to induce formation of vasculature-like structures, but long-term in vivo transplantation and filtration activity by these tissue-engineered constructs have not been investigated or reported. Therefore, even if the process of renal decellularization is possible, the repopulation of the renal matrix with functional renal cell types is still very challenging. This review aims to summarize the current reports on kidney tissue engineering with the use of decellularized matrices and addresses the challenges in creating functional kidney units. Finally, this review discusses how future studies investigating cell-matrix interaction may aid the generation of a functional renal unit that would be transplantable into patients one day.