OBJECTIVE: Diabetic nephropathy is a major consequence of inflammation developing in type 1 diabetes, with interleukin-8 (IL-8)-CXCR1/2 axis playing a key role in kidney disease progression. In this study, we investigated the therapeutic potential of a CXCR1/2 non-competitive allosteric antagonist (Ladarixin) in preventing high glucose-mediated injury in human podocytes and epithelial cells differentiated from renal stem/progenitor cells (RSC) cultured as nephrospheres.
METHODS: We used human RSCs cultured as nephrospheres through a sphere-forming functional assay to investigate hyperglycemia-mediated effects on IL-8 signalling in human podocytes and tubular epithelial cells.
RESULTS: High glucose impairs RSC self-renewal, induces an increase in IL-8 transcript expression and protein secretion and induces DNA damage in RSC-differentiated podocytes, while exerting no effect on RSC-differentiated epithelial cells. Accordingly, the supernatant from epithelial cells or podocytes cultured in high glucose was able to differentially activate leucocyte-mediated secretion of pro-inflammatory cytokines, suggesting that the crosstalk between immune and non-immune cells may be involved in disease progression in vivo.
CONCLUSIONS: Treatment with Ladarixin during RSC differentiation prevented high glucose-mediated effects on podocytes and modulated either podocyte or epithelial cell-dependent leucocyte secretion of pro-inflammatory cytokines, suggesting CXCR1/2 antagonists as possible pharmacological approaches for the treatment of diabetic nephropathy.

We reasoned that unraveling the dynamic changes in accessibility of genomic regulatory elements and gene expression at single-cell resolution will inform the basic mechanisms of nephrogenesis.
We performed single-cell ATAC-seq and RNA-seq both individually (singleomes; Six2GFP cells) and jointly in the same cells (multiomes; kidneys) to generate integrated chromatin and transcriptional maps in mouse embryonic and neonatal nephron progenitor cells.
We demonstrate that singleomes and multiomes are comparable in assigning most cell states, identification of new cell type markers, and defining the transcription factors driving cell identity. However, multiomes are more precise in defining the progenitor population. Multiomes identified a \"pioneer\" bHLH/Fox motif signature in nephron progenitor cells. Moreover, we identified a subset of Fox factors exhibiting high chromatin activity in podocytes. One of these Fox factors, Foxp1, is important for nephrogenesis. Key nephrogenic factors are distinguished by strong correlation between linked gene regulatory elements and gene expression.
Mapping the regulatory landscape at single-cell resolution informs the regulatory hierarchy of nephrogenesis. Paired single-cell epigenomes and transcriptomes of nephron progenitors should provide a foundation to understand prenatal programming, regeneration after injury, and ex vivo nephrogenesis.

There is intense interest in replacing kidneys from stem cells. It is now possible to produce, from embryonic or induced pluripotent stem cells, kidney organoids that represent immature kidneys and display some physiologic functions. However, current techniques have not yet resulted in renal tissue with a ureter, which would be needed for engineered kidneys to be clinically useful.
We used a published sequence of growth factors and drugs to induce mouse embryonic stem cells to differentiate into ureteric bud tissue. We characterized isolated engineered ureteric buds differentiated from embryonic stem cells in three-dimensional culture and grafted them into ex fetu mouse kidney rudiments.
Engineered ureteric buds branched in three-dimensional culture and expressed Hoxb7, a transcription factor that is part of a developmental regulatory system and a ureteric bud marker. When grafted into the cortex of ex fetu kidney rudiments, engineered ureteric buds branched and induced nephron formation; when grafted into peri-Wolffian mesenchyme, still attached to a kidney rudiment or in isolation, they did not branch but instead differentiated into multilayer ureter-like epithelia displaying robust expression of the urothelial marker uroplakin. This engineered ureteric bud tissue also organized the mesenchyme into smooth muscle that spontaneously contracted, with a period a little slower than that of natural ureteric peristalsis.
Mouse embryonic stem cells can be differentiated into ureteric bud cells. Grafting those UB-like structures into peri-Wolffian mesenchyme of cultured kidney rudiments can induce production of urothelium and organize the mesenchyme to produce rhythmically contracting smooth muscle layers. This development may represent a significant step toward the goal of renal regeneration.

Cell-based therapies aimed at replenishing renal parenchyma have been proposed as an approach for treating CKD. However, pathogenic mechanisms involved in CKD such as renal hypoxia result in loss of kidney function and limit engraftment and therapeutic effects of renal epithelial progenitors. Jointly administering vessel-forming cells (human mesenchymal stromal cells [MSCs] and endothelial colony-forming cells [ECFCs]) may potentially result in in vivo formation of vascular networks.
We administered renal tubule-forming cells derived from human adult and fetal kidneys (previously shown to exert a functional effect in CKD mice) into mice, alongside MSCs and ECFCs. We then assessed whether this would result in generation of \"renovascular units\" comprising both vessels and tubules with potential interaction.
Directly injecting vessel-forming cells and renal tubule-forming cells into the subcutaneous and subrenal capsular space resulted in self-organization of donor-derived vascular networks that connected to host vasculature, alongside renal tubules comprising tubular epithelia of different nephron segments. Vessels derived from MSCs and ECFCs augmented in vivo tubulogenesis by the renal tubule-forming cells. In vitro coculture experiments showed that MSCs and ECFCs induced self-renewal and genes associated with mesenchymal-epithelial transition in renal tubule-forming cells, indicating paracrine effects. Notably, after renal injury, renal tubule-forming cells and vessel-forming cells infused into the renal artery did not penetrate the renal vascular network to generate vessels; only administering them into the kidney parenchyma resulted in similar generation of human renovascular units in vivo.
Combined cell therapy of vessel-forming cells and renal tubule-forming cells aimed at alleviating renal hypoxia and enhancing tubulogenesis holds promise as the basis for new renal regenerative therapies.

The kidney is considered to be a structurally stable organ with limited baseline cellular turnover. Nevertheless, single cells must be constantly replaced to conserve the functional integrity of the organ. PDGF chain B (PDGF-BB) signaling through fibroblast PDGF receptor-β (PDGFRβ) contributes to interstitial-epithelial cell communication and facilitates regenerative functions in several organs. However, the potential role of interstitial cells in renal tubular regeneration has not been examined.
In mice with fluorescent protein expression in renal tubular cells and PDGFRβ-positive interstitial cells, we ablated single tubular cells by high laser exposure. We then used serial intravital multiphoton microscopy with subsequent three-dimensional reconstruction and ex vivo histology to evaluate the cellular and molecular processes involved in tubular regeneration.
Single-tubular cell ablation caused the migration and division of dedifferentiated tubular epithelial cells that preceded tubular regeneration. Moreover, tubular cell ablation caused immediate calcium responses in adjacent PDGFRβ-positive interstitial cells and the rapid migration thereof toward the injury. These PDGFRβ-positive cells enclosed the injured epithelium before the onset of tubular cell dedifferentiation, and the later withdrawal of these PDGFRβ-positive cells correlated with signs of tubular cell redifferentiation. Intraperitoneal administration of trapidil to block PDGFRβ impeded PDGFRβ-positive cell migration to the tubular injury site and compromised the recovery of tubular function.
Ablated tubular cells are exclusively replaced by resident tubular cell proliferation in a process dependent on PDGFRβ-mediated communication between the renal interstitium and the tubular system.

The kidney is a complex organ whose excretory and regulatory functions are vital for maintaining homeostasis. Previous techniques used to study the kidney, including various animal models and 2D cell culture systems to investigate the mechanisms of renal development and regeneration have many benefits but also possess inherent shortcomings. Some of those limitations can be addressed using the emerging technology of 3D organoids. An organoid is a 3D cluster of differentiated cells that are developed ex vivo by addition of various growth factors that result in a miniature organ containing structures present in the tissue of origin. Here, we discuss renal organoids, their development, and how they can be employed to further understand kidney development and disease.

Current therapies for pyelonephritic renal damage have severe limitations; stem cells may offer an exciting potential in regenerating nephrology. We aimed to investigate the feasibility of direct intrarenal injection of autologous renal progenitor cells (RPCs; originated from epithelial cells in Bowman\'s capsule) in chronic pyelonephritis rat model. Twenty-seven rats were divided into three groups. The control group (GI, n = 3) underwent sham subcapsular injection of isotonic saline. Pyelonephritis was induced in the right kidney of the remaining 24 rats and isotonic saline (GII, n = 12) or labeled autologous RPCs, obtained from a biopsy of left kidney (GIII, n = 12), were injected into the subcapsular space 6 weeks later. At 7, 14, 28, and 60 days, dimercaptosuccinic acid scan was performed in three animals of each group at every interval and subsequently renal sections were obtained for the evaluation of tubular and glomerular regeneration and proliferation. Cell transplantation resulted in the reduction of tubular and glomerular atrophy after 2 weeks. The transplanted cells were observed in the reconstructed region of the kidneys as evidenced by the presence of fluorescently labeled cells both in tubules and glomeruli. We also observed significant decrease in interstitial fibrosis in the fourth week and there were higher amount of Ki-67-positive cells in GIII. Notably, the right renal tissue integrity was significantly improved in this group and revealed normal cortical function on day 60. Transplanting RPCs showed the potential for partial augmentation of kidney structure and function in pyelonephritis. Cellular repair was seen predominantly in the proximal tubule, the major site of injury in pyelonephritis. Our findings may pave the way toward the future regeneration of renal scarring of pyelonephritis in children.

Kidney disease is an escalating global health problem, for which the formulation of therapeutic approaches using stem cells has received increasing research attention. The complexity of kidney anatomy and function, which includes the diversity of renal cell types, poses formidable challenges in the identification of methods to generate replacement structures. Recent work using the zebrafish has revealed their high capacity to regenerate the integral working units of the kidney, known as nephrons, following acute injury. Here, we discuss these findings and explore the ways that zebrafish can be further utilized to gain a deeper molecular appreciation of renal stem cell biology, which may uncover important clues for regenerative medicine.

The renal system is vital to maintain homeostasis in the body, where the kidneys contain nephron functional units that remove metabolic waste from the bloodstream, regulate fluids, and balance electrolytes. Severe organ damage from toxins or ischemia that occurs abruptly can cause acute kidney injury (AKI) in which there is a rapid, life-threatening loss of these activities. Humans have a limited but poorly understood ability to regenerate damaged nephrons after AKI. However, researchers studying AKI in vertebrate animal models such as mammals, and more recently the zebrafish, have documented robust regeneration within the nephron blood filter and tubule following injury. Further, zebrafish kidneys contain progenitors that create new nephrons after AKI. Here, we review investigations in zebrafish which have established a series of exciting renal pathology paradigms that complement existing AKI models and can be implemented to discover insights into kidney regeneration and the roles of stem cells.

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