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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.

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.

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 ITGβ1 and ITGβ4. 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., (© 2023 The Authors. Advanced Science published by Wiley-VCH GmbH.)

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.

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., (© 2016 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.)

Amniotic fluid is in continuity with multiple developing organ systems, including the kidney. Committed, but still stem-like cells from these organs may thus appear in amniotic fluid. We report having established for the first time a stem-like cell population derived from human amniotic fluid and possessing characteristics of podocyte precursors. Using a method of triple positive selection we obtained a population of cells (hAKPC-P) that can be propagated in vitro for many passages without immortalization or genetic manipulation. Under specific culture conditions, these cells can be differentiated to mature podocytes. In this work we compared these cells with conditionally immortalized podocytes, the current gold standard for in vitro studies. After in vitro differentiation, both cell lines have similar expression of the major podocyte proteins, such as nephrin and type IV collagen, that are characteristic of mature functional podocytes. In addition, differentiated hAKPC-P respond to angiotensin II and the podocyte toxin, puromycin aminonucleoside, in a way typical of podocytes. In contrast to immortalized cells, hAKPC-P have a more nearly normal cell cycle regulation and a pronounced developmental pattern of specific protein expression, suggesting their suitability for studies of podocyte development for the first time in vitro. These novel progenitor cells appear to have several distinct advantages for studies of podocyte cell biology and potentially for translational therapies.

Introduction: Risk of testicular germ cell tumors (TGCT) is consistently associated with a history of cryptorchidism (CO) in epidemiologic studies. Factors modifying the association may provide insights regarding etiology of TGCT and suggest a basis for individualized care of CO. To identify modifiers of the CO-TGCT association, we conducted a comprehensive, quantitative evaluation of epidemiologic data., Materials and Methods: Human studies cited in PubMed or ISI Web of Science indices through December 2011 and selected unpublished epidemiologic data were reviewed to identify 35 articles and one unpublished dataset with high-quality data on the CO-TGCT association. Association data were extracted as point and 95% confidence interval estimates of odds ratio (OR) or standardized incidence ratio (SIR), or as tabulated data. Values were recorded for each study population, and for subgroups defined by features of study design, CO and TGCT. Extracted data were used to estimate summary risk ratios (sRR) and evaluate heterogeneity of the CO-TGCT association between subgroups., Results: The overall meta-analysis showed that history of CO is associated with four-fold increased TGCT risk [RR = 4.1(95% CI = 3.6-4.7)]. Subgroup analyses identified five determinants of stronger association: bilateral CO, unilateral CO ipsilateral to TGCT, delayed CO treatment, TGCT diagnosed before 1970, and seminoma histology., Conclusions: Modifying factors may provide insight into TGCT etiology and suggest improved approaches to managing CO. Based on available data, CO patients and their parents or caregivers should be made aware of elevated TGCT risk following orchidopexy, regardless of age at repair, unilateral vs. bilateral non-descent, or position of undescended testes.

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.

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.

Acute Tubular Necrosis (ATN) causes severe damage to the kidney epithelial tubular cells and is often associated with severe renal dysfunction. Stem-cell based therapies may provide alternative approaches to treating of ATN. We have previously shown that clonal c-kit(pos) stem cells, derived from human amniotic fluid (hAFSC) can be induced to a renal fate in an ex-vivo system. Herein, we show for the first time the successful therapeutic application of hAFSC in a mouse model with glycerol-induced rhabdomyolysis and ATN. When injected into the damaged kidney, luciferase-labeled hAFSC can be tracked using bioluminescence. Moreover, we show that hAFSC provide a protective effect, ameliorating ATN in the acute injury phase as reflected by decreased creatinine and BUN blood levels and by a decrease in the number of damaged tubules and apoptosis therein, as well as by promoting proliferation of tubular epithelial cells. We show significant immunomodulatory effects of hAFSC, over the course of ATN. We therefore speculate that AFSC could represent a novel source of stem cells that may function to modulate the kidney immune milieu in renal failure caused by ATN.

A new source of stem cells has recently been isolated from amniotic fluid; these amniotic fluid stem cells have significant potential for regenerative medicine. These cells are multipotent, showing the ability to differentiate into cell types from each embryonic germ layer. We investigated the ability of human amniotic fluid stem cells (hAFSC) to integrate into murine lung and to differentiate into pulmonary lineages after injury. Using microinjection into cultured mouse embryonic lungs, hAFSC can integrate into the epithelium and express the early human differentiation marker thyroid transcription factor 1 (TTF1). In adult nude mice, following hyperoxia injury, tail vein-injected hAFSC localized in the distal lung and expressed both TTF1 and the type II pneumocyte marker surfactant protein C. Specific damage of Clara cells through naphthalene injury produced integration and differentiation of hAFSC at the bronchioalveolar and bronchial positions with expression of the specific Clara cell 10-kDa protein. These results illustrate the plasticity of hAFSC to respond in different ways to different types of lung damage by expressing specific alveolar versus bronchiolar epithelial cell lineage markers, depending on the type of injury to recipient lung. Disclosure of potential conflicts of interest is found at the end of this article.

A rising number of patients with acute and chronic renal failure worldwide have created urgency for clinicians and investigators to search out alternative therapies other than chronic renal dialysis and/or organ transplantation. This review focuses on the recent achievements in this area, and discusses the various approaches in the development of bioengineering of renal tissue including recent discoveries in the field of regenerative medicine research and stem cells. A variety of stem cells, ranging from embryonic, bone marrow, endogenous, and amniotic fluid, have been investigated and may prove useful as novel alternatives for organ regeneration both in vitro and in vivo. Tissue engineering, developmental biology, and therapeutic cloning techniques have significantly contributed to our understanding of some of the molecular mechanisms involved in renal regeneration and have demonstrated that renal tissue can be generated de novo with similar physiologic functions as native tissue. Ultimately all of these emerging technologies may provide viable therapeutic options for regenerative medicine applications focused on the bioengineering of renal tissue for the future.

Objectives: The role of stem cells in regenerative medicine is evolving rapidly. Here, we describe the application, for kidney regeneration, of a novel non-genetically modified stem cell, derived from human amniotic fluid. We show that these pluripotent cells can develop and differentiate into de novo kidney structures during organogenesis in vitro., Materials and Methods: Human amniotic fluid-derived stem cells (hAFSCs) were isolated from human male amniotic fluid obtained between 12 and 18 weeks gestation. Green fluorescent protein and Lac-Z-transfected hAFSCs were microinjected into murine embryonic kidneys (12.5-18 days gestation) and were maintained in a special co-culture system in vitro for 10 days. Techniques of live microscopy, histology, chromogenic in situ hybridization and reverse transcriptase polymerase chain reaction were used to characterize the hAFSCs during their integration and differentiation in concert with the growing organ., Results: Green fluorescent protein and Lac-Z-transfected hAFSCs demonstrated long-term viability in organ culture. Histological analysis of injected kidneys revealed that hAFSCs were capable of contributing to the development of primordial kidney structures including renal vesicle, C- and S-shaped bodies. Reverse transcriptase polymerase chain reaction confirmed expression of early kidney markers for: zona occludens-1, glial-derived neurotrophic factor and claudin., Conclusions: Human amniotic fluid-derived stem cells may represent a potentially limitless source of ethically neutral, unmodified pluripotential cells for kidney regeneration.