Endocycle-related tubular cell hypertrophy and progenitor proliferation recover renal function after acute kidney injury.

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.

Podocyte Regeneration Driven by Renal Progenitors Determines Glomerular Disease Remission and Can Be Pharmacologically Enhanced.

Podocyte loss is a general mechanism of glomerular dysfunction that initiates and drives the progression of chronic kidney disease, which affects 10% of the world population. Here, we evaluate whether the regenerative response to podocyte injury influences chronic kidney disease outcome. In models of focal segmental glomerulosclerosis performed in inducible transgenic mice where podocytes are tagged, remission or progression of disease was determined by the amount of regenerated podocytes. When the same model was established in inducible transgenic mice where renal progenitors are tagged, the disease remitted if renal progenitors successfully differentiated into podocytes, while it persisted if differentiation was ineffective, resulting in glomerulosclerosis. Treatment with BIO, a GSK3s inhibitor, significantly increased disease remission by enhancing renal progenitor sensitivity to the differentiation effect of endogenous retinoic acid. These results establish renal progenitors as critical determinants of glomerular disease outcome and a pharmacological enhancement of their differentiation as a possible therapeutic strategy.

Human Urine-Derived Renal Progenitors for Personalized Modeling of Genetic Kidney Disorders.

The critical role of genetic and epigenetic factors in the pathogenesis of kidney disorders is gradually becoming clear, and the need for disease models that recapitulate human kidney disorders in a personalized manner is paramount. In this study, we describe a method to select and amplify renal progenitor cultures from the urine of patients with kidney disorders. Urine-derived human renal progenitors exhibited phenotype and functional properties identical to those purified from kidney tissue, including the capacity to differentiate into tubular cells and podocytes, as demonstrated by confocal microscopy, Western blot analysis of podocyte-specific proteins, and scanning electron microscopy. Lineage tracing studies performed with conditional transgenic mice, in which podocytes are irreversibly tagged upon tamoxifen treatment (NPHS2.iCreER;mT/mG), that were subjected to doxorubicin nephropathy demonstrated that renal progenitors are the only urinary cell population that can be amplified in long-term culture. To validate the use of these cells for personalized modeling of kidney disorders, renal progenitors were obtained from (1) the urine of children with nephrotic syndrome and carrying potentially pathogenic mutations in genes encoding for podocyte proteins and (2) the urine of children without genetic alterations, as validated by next-generation sequencing. Renal progenitors obtained from patients carrying pathogenic mutations generated podocytes that exhibited an abnormal cytoskeleton structure and functional abnormalities compared with those obtained from patients with proteinuria but without genetic mutations. The results of this study demonstrate that urine-derived patient-specific renal progenitor cultures may be an innovative research tool for modeling of genetic kidney disorders.

Functional avidity-driven activation-induced cell death shapes CTL immunodominance.

Immunodominance is a complex phenomenon that relies on a mere numerical concept, while being potentially influenced at every step of the immune response. We investigated the mechanisms leading to the establishment of CTL immunodominance in a retroviral model and found that the previously defined subdominant Env-specific CD8(+) T cells are endowed with an unexpectedly higher functional avidity than is the immunodominant Gag-recognizing counterpart. This high avidity, along with the Env Ag overload, results in a supraoptimal TCR engagement. The overstimulation makes Env-specific T lymphocytes more susceptible to apoptosis, thus hampering their expansion and leading to an unintentional "immune kamikazing." Therefore, Ag-dependent, hyperactivation-induced cell death can be regarded as a novel mechanism in the establishment of the immunodominance that restrains and opposes the expansion of high-avidity T cells in favor of lower-affinity populations.

Proteinuria impairs podocyte regeneration by sequestering retinoic acid.

In CKD, the risk of kidney failure and death depends on the severity of proteinuria, which correlates with the extent of podocyte loss and glomerular scarring. We investigated whether proteinuria contributes directly to progressive glomerulosclerosis through the suppression of podocyte regeneration and found that individual components of proteinuria exert distinct effects on renal progenitor survival and differentiation toward a podocyte lineage. In particular, albumin prevented podocyte differentiation from human renal progenitors in vitro by sequestering retinoic acid, thus impairing retinoic acid response element (RARE)-mediated transcription of podocyte-specific genes. In mice with Adriamycin nephropathy, a model of human FSGS, blocking endogenous retinoic acid synthesis increased proteinuria and exacerbated glomerulosclerosis. This effect was related to a reduction in podocyte number, as validated through genetic podocyte labeling in NPHS2.Cre;mT/mG transgenic mice. In RARE-lacZ transgenic mice, albuminuria reduced retinoic acid bioavailability and impaired RARE activation in renal progenitors, inhibiting their differentiation into podocytes. Treatment with retinoic acid restored RARE activity and induced the expression of podocyte markers in renal progenitors, decreasing proteinuria and increasing podocyte number, as demonstrated in serial biopsy specimens. These results suggest that albumin loss through the damaged filtration barrier impairs podocyte regeneration by sequestering retinoic acid and promotes the generation of FSGS lesions. Our findings may explain why reducing proteinuria delays CKD progression and provide a biologic rationale for the clinical use of pharmacologic modulators to induce regression of glomerular diseases.

Characterization of renal progenitors committed toward tubular lineage and their regenerative potential in renal tubular injury.

Recent studies implicated the existence in adult human kidney of a population of renal progenitors with the potential to regenerate glomerular as well as tubular epithelial cells and characterized by coexpression of surface markers CD133 and CD24. Here, we demonstrate that CD133+CD24+ renal progenitors can be distinguished in distinct subpopulations from normal human kidneys based on the surface expression of vascular cell adhesion molecule 1, also known as CD106. CD133+CD24+CD106+ cells were localized at the urinary pole of Bowman's capsule, while a distinct population of scattered CD133+CD24+CD106- cells was localized in the proximal tubule as well as in the distal convoluted tubule. CD133+CD24+CD106+ cells exhibited a high proliferative rate and could differentiate toward the podocyte as well as the tubular lineage. By contrast, CD133+CD24+CD106- cells showed a lower proliferative capacity and displayed a committed phenotype toward the tubular lineage. Both CD133+CD24+CD106+ and CD133+CD24+CD106- cells showed higher resistance to injurious agents in comparison to all other differentiated cells of the kidney. Once injected in SCID mice affected by acute tubular injury, both of these populations displayed the capacity to engraft within the kidney, generate novel tubular cells, and improve renal function. These properties were not shared by other tubular cells of the adult kidney. Finally, CD133+CD24+CD106- cells proliferated upon tubular injury, becoming the predominating part of the regenerating epithelium in patients with acute or chronic tubular damage. These data suggest that CD133+CD24+CD106- cells represent tubular-committed progenitors that display resistance to apoptotic stimuli and exert regenerative potential for injured tubular tissue.

Notch activation differentially regulates renal progenitors proliferation and differentiation toward the podocyte lineage in glomerular disorders.

Glomerular diseases account for 90% of end-stage kidney disease. Podocyte loss is a common determining factor for the progression toward glomerulosclerosis. Mature podocytes cannot proliferate, but recent evidence suggests that they can be replaced by renal progenitors localized within the Bowman's capsule. Here, we demonstrate that Notch activation in human renal progenitors stimulates entry into the S-phase of the cell cycle and cell division, whereas its downregulation is required for differentiation toward the podocyte lineage. Indeed, a persistent activation of the Notch pathway induced podocytes to cross the G(2)/M checkpoint, resulting in cytoskeleton disruption and death by mitotic catastrophe. Notch expression was virtually absent in the glomeruli of healthy adult kidneys, while a strong upregulation was observed in renal progenitors and podocytes in patients affected by glomerular disorders. Accordingly, inhibition of the Notch pathway in mouse models of focal segmental glomerulosclerosis ameliorated proteinuria and reduced podocyte loss during the initial phases of glomerular injury, while inducing reduction of progenitor proliferation during the regenerative phases of glomerular injury with worsening of proteinuria and glomerulosclerosis. Taken altogether, these results suggest that the severity of glomerular disorders depends on the Notch-regulated balance between podocyte death and regeneration provided by renal progenitors.

[The role of podocyte damage in the pathogenesis of glomerulosclerosis and possible repair mechanisms]. / Ruolo del danno podocitario nella patogenesi della glomerulosclerosi e possibili meccanismi di riparazione.

Converging evidence suggests that damage to podocytes plays a key role in progression towards glomerulosclerosis, in particular as the primary cause of all forms of focal segmental glomerulosclerosis (FSGS), the most common glomerular disease leading to end-stage renal disease. Any damage occurring to the complex architecture of specialized proteins that constitute the podocyte foot processes, essential to the highly specialized functions of podocytes, leads inevitably to loss of function in the glomerular filtration barrier, and ultimately to proteinuria. Recent studies have also highlighted that a reduction of the podocyte number in a damaged glomerulus is a critical factor for the development of proteinuria and glomerulosclerosis. As long as the podocyte loss is limited, restitution or repair is possible, which shows that the glomerular architecture can be remodeled. However, mature podocytes have limited capacity to divide and display all the phenotypic and functional features of highly specialized, terminally differentiated cells. A potential mechanism for podocyte replacement might be stem-cell-based regeneration, since it has been established that the developmental source of podocytes are resident renal progenitors. Podocyte damage could then be potentially repaired by a stem cell population resident in the kidney.

Renal progenitor cells contribute to hyperplastic lesions of podocytopathies and crescentic glomerulonephritis.

Glomerular injury can involve excessive proliferation of glomerular epithelial cells, resulting in crescent formation and obliteration of Bowman's space. The origin of these hyperplastic epithelial cells in different glomerular disorders is controversial. Renal progenitors localized to the inner surface of Bowman's capsule can regenerate podocytes, but whether dysregulated proliferation of these progenitors contributes to crescent formation is unknown. In this study, we used confocal microscopy, laser capture microdissection, and real-time quantitative reverse transcriptase-PCR to demonstrate that hypercellular lesions of different podocytopathies and crescentic glomerulonephritis consist of three distinct populations: CD133(+)CD24(+)podocalyxin (PDX)(-)nestin(-) renal progenitors, CD133(+)CD24(+)PDX(+)nestin(+) transitional cells, and CD133(-)CD24(-)PDX(+)nestin(+) differentiated podocytes. In addition, TGF-beta induced CD133(+)CD24(+) progenitors to produce extracellular matrix, and these were the only cells to express the proliferation marker Ki67. Taken together, these results suggest that glomerular hyperplastic lesions derive from the proliferation of renal progenitors at different stages of their differentiation toward mature podocytes, providing an explanation for the pathogenesis of hyperplastic lesions in podocytopathies and crescentic glomerulonephritis.

The role of endothelial progenitor cells in acute kidney injury.

Acute kidney injury (AKI) is characterized by a sudden impairment of kidney function, which results in the retention of urea and other nitrogenous waste products and in the perturbation of extracellular fluid volume as well as electrolyte and acid-base homeostasis. The dysfunction and apoptosis of tubular epithelial cells are of key importance for the pathophysiological consequences of AKI. However, a growing body of evidence supports the contribution of altered renal vascular structure and function in potentially initiating and extending the initial tubular injury. Vascular injury and dysfunction result in alterations of renal oxygenation and hemodynamics that may have long-term effects in regards to renal function, predisposing to chronic kidney disease. There is growing evidence that endothelial progenitor cells (EPCs) may improve vascular regeneration in different ischemic organs, and recent data suggest that EPCs are mobilized after acute renal ischemia and recruited in ischemic kidney areas and can ameliorate AKI through both paracrine effects and repair of injured microvasculature. The loss of endothelial cell function may represent an important therapeutic target, in which EPCs may show potential importance in ameliorating the acute and chronic effects of ischemic AKI.

Regeneration of glomerular podocytes by human renal progenitors.

Depletion of podocytes, common to glomerular diseases in general, plays a role in the pathogenesis of glomerulosclerosis. Whether podocyte injury in adulthood can be repaired has not been established. Here, we demonstrate that in the adult human kidney, CD133+CD24+ cells consist of a hierarchical population of progenitors that are arranged in a precise sequence within Bowman's capsule and exhibit heterogeneous potential for differentiation and regeneration. Cells localized to the urinary pole that expressed CD133 and CD24, but not podocyte markers (CD133+CD24+PDX- cells), could regenerate both tubular cells and podocytes. In contrast, cells localized between the urinary pole and vascular pole that expressed both progenitor and podocytes markers (CD133+CD24+PDX+) could regenerate only podocytes. Finally, cells localized to the vascular pole did not exhibit progenitor markers, but displayed phenotypic features of differentiated podocytes (CD133-CD24-PDX+ cells). Injection of CD133+CD24+PDX- cells, but not CD133+CD24+PDX+ or CD133-CD24- cells, into mice with adriamycin-induced nephropathy reduced proteinuria and improved chronic glomerular damage, suggesting that CD133+CD24+PDX- cells could potentially treat glomerular disorders characterized by podocyte injury, proteinuria, and progressive glomerulosclerosis.

Cannabinoid receptor activation induces apoptosis through tumor necrosis factor alpha-mediated ceramide de novo synthesis in colon cancer cells.

PURPOSE: Cannabinoids have been recently proposed as a new family of potential antitumor agents. The present study was undertaken to investigate the expression of the two cannabinoid receptors, CB1 and CB2, in colorectal cancer and to provide new insight into the molecular pathways underlying the apoptotic activity induced by their activation. EXPERIMENTAL DESIGN: Cannabinoid receptor expression was investigated in both human cancer specimens and in the DLD-1 and HT29 colon cancer cell lines. The effects of the CB1 agonist arachinodyl-2'-chloroethylamide and the CB2 agonist N-cyclopentyl-7-methyl-1-(2-morpholin-4-ylethyl)-1,8-naphthyridin-4(1H)-on-3-carboxamide (CB13) on tumor cell apoptosis and ceramide and tumor necrosis factor (TNF)-alpha production were evaluated. The knockdown of TNF-alpha mRNA was obtained with the use of selective small interfering RNA. RESULTS: We show that the CB1 receptor was mainly expressed in human normal colonic epithelium whereas tumor tissue was strongly positive for the CB2 receptor. The activation of the CB1 and, more efficiently, of the CB2 receptors induced apoptosis and increased ceramide levels in the DLD-1 and HT29 cells. Apoptosis was prevented by the pharmacologic inhibition of ceramide de novo synthesis. The CB2 agonist CB13 also reduced the growth of DLD-1 cells in a mouse model of colon cancer. The knockdown of TNF-alpha mRNA abrogated the ceramide increase and, therefore, the apoptotic effect induced by cannabinoid receptor activation. CONCLUSIONS: The present study shows that either CB1 or CB2 receptor activation induces apoptosis through ceramide de novo synthesis in colon cancer cells. Our data unveiled, for the first time, that TNF-alpha acts as a link between cannabinoid receptor activation and ceramide production.

Stem-cell approaches for kidney repair: choosing the right cells.

With the increasing rate of end-stage renal failure and limited alternatives for its treatment, stem cell (SC) therapy for kidney injury is urgently needed. Choosing the right SC type is the critical step in realizing the potential of this therapeutic approach. Four possible sources of SCs are envisioned for the development of this type of treatment: (i) bone-marrow-derived SCs (BMSCs), (ii) renal adult SCs, (iii) embryonic SCs and (iv) fetal renal SCs. We suggest that resident SCs recently identified in the Bowman's capsule of adult human kidneys might prospectively be the ideal cell type for treatment of both acute and chronic renal injury because they display the potential to differentiate into multiple types of renal cells. However, BMSCs also represent an attractive alternative, especially for the treatment of patients affected by acute renal failure.

Human immature myeloid dendritic cells trigger a TH2-polarizing program via Jagged-1/Notch interaction.

BACKGROUND: The mechanisms by which human dendritic cells (DCs) activate a TH1-polarizing or TH2-polarizing program are still partially unclear. OBJECTIVE: Study of the mechanisms responsible for the TH1/TH2-polarizing activity of human circulating myeloid DCs before and after ligation of their Toll-like receptors (TLRs). METHODS: IL-4 and IFN-gamma production by CD4+ T cells was assessed in cocultures with myeloid DCs before or after TLR triggering. Expression of Jagged-1 and Delta-4 Notch ligands and of GATA-3 and T-box expressed in T cells transcription factors was evaluated by real-time quantitative PCR. Signal transducer and activator of transcription 4 and 6 phosphorylation was assessed by flow cytometry. Knockdown of Jagged-1 or Delta-4 was performed by transfection of DCs with appropriate silencing mRNAs. RESULTS: Myeloid immature DCs constitutively expressed Jagged-1, which induces in CD4+ T cells a TH2 polarization, as shown by Jagged-1 gene silencing. The TH2 polarization associated with high GATA-3/T-box expressed in T cells ratio and was at least partially dependent on the early induction of IL-4. Maturation of DCs by TLR ligation resulted in the reduction of Jagged-1 and upregulation of Delta-4, which was at least in part responsible for the polarization of CD4+ T cells to the TH1 phenotype. CONCLUSION: CD4+ T-cell responses are usually characterized by a prevalent TH2 phenotype unless TLRs are triggered on DCs by microbial components.

Essential but differential role for CXCR4 and CXCR7 in the therapeutic homing of human renal progenitor cells.

Recently, we have identified a population of renal progenitor cells in human kidneys showing regenerative potential for injured renal tissue of SCID mice. We demonstrate here that among all known chemokine receptors, human renal progenitor cells exhibit high expression of both stromal-derived factor-1 (SDF-1) receptors, CXCR4 and CXCR7. In SCID mice with acute renal failure (ARF), SDF-1 was strongly up-regulated in resident cells surrounding necrotic areas. In the same mice, intravenously injected renal stem/progenitor cells engrafted into injured renal tissue decreased the severity of ARF and prevented renal fibrosis. These beneficial effects were abolished by blocking either CXCR4 or CXCR7, which dramatically reduced the number of engrafting renal progenitor cells. However, although SDF-1-induced migration of renal progenitor cells was only abolished by an anti-CXCR4 antibody, transendothelial migration required the activity of both CXCR4 and CXCR7, with CXCR7 being essential for renal progenitor cell adhesion to endothelial cells. Moreover, CXCR7 but not CXCR4 was responsible for the SDF-1-induced renal progenitor cell survival. Collectively, these findings suggest that CXCR4 and CXCR7 play an essential, but differential, role in the therapeutic homing of human renal progenitor cells in ARF, with important implications for the development of stem cell-based therapies.

Pretransplant serum FT3 levels in kidney graft recipients are useful for identifying patients with higher risk for graft failure.

OBJECTIVE: End-stage renal disease (ESRD) is a condition associated with thyroid disturbances both in function and morphology. Recent studies demonstrated that serum free triiodothyronine 3 (FT3) levels are negatively correlated with serum markers of inflammation and endothelial activation in patients with ESRD. However, no previous research evaluated serum thyroid function parameters in relation to kidney graft outcome, as we aim to do so in this study. DESIGN: Serum FT3, free thyroxine 4 (FT4) and TSH levels were measured before transplantation in 196 kidney graft recipients. RESULTS: The graft survival rate at 5 years for all patients was 92.3%. Kidney graft recipients with normally functioning grafts showed serum pretransplant thyroid parameters similar to patients who experienced graft failure. Life-time analysis was performed after stratification of patients according to pretransplant serum FT3 levels < 3.1 pmol/l or > 3.1 pmol/l. A significantly different 5-year death-censored graft survival rate (93.9%vs. 76.5% for patients with normal or low FT3 levels, respectively; P < 0.01) and similar survival rate (death of patients with functioning grafts) (21.1%vs. 5.9%; P = 0.288) were observed. No similar feature was found for FT4 or TSH, suggesting that the effect is not related to hypothyroidism but rather dependent upon inappropriately low FT3 levels. Pretransplant serum FT3 levels were similar in patients who experienced early acute rejections as compared with nonrejector patients. CONCLUSIONS: The results of this study demonstrate that among patients with ESRD undergoing kidney transplantation, those displaying lower pretransplant serum FT3 levels are at higher risk for subsequent graft failure. The demonstration of a predictive value of serum FT3 levels for graft survival suggests that measurement of pretransplant serum FT3 levels might represent a clinically useful parameter to identify patients with increased risk for graft failure.

Regenerative potential of embryonic renal multipotent progenitors in acute renal failure.

Bone marrow-and adult kidney-derived stem/progenitor cells hold promise in the development of therapies for renal failure. Here is reported the identification and characterization of renal multipotent progenitors in human embryonic kidneys that share CD24 and CD133 surface expression with adult renal progenitors and have the capacity for self-renewal and multilineage differentiation. It was found that these CD24+CD133+ cells constitute the early primordial nephron but progressively disappear during nephron development until they become selectively localized to the urinary pole of Bowman's capsule. When isolated and injected into SCID mice with acute renal failure from glycerol-induced rhabdomyolysis, these cells regenerated different portions of the nephron, reduced tissue necrosis and fibrosis, and significantly improved renal function. No tumorigenic potential was observed. It is concluded that CD24+CD133+ cells represent a subset of multipotent embryonic progenitors that persist in human kidneys from early stages of nephrogenesis. The ability of these cells to repair renal damage, together with their apparent lack of tumorigenicity, suggests their potential in the treatment of renal failure.

PF-4/CXCL4 and CXCL4L1 exhibit distinct subcellular localization and a differentially regulated mechanism of secretion.

PF-4/CXCL4 is a member of the CXC chemokine family, which is mainly produced by platelets and known for its pleiotropic biological functions. Recently, the proteic product of a nonallelic variant gene of CXCL4 was isolated from human platelets and named as CXCL4L1. CXCL4L1 shows only 4.3% amino acid divergence in the mature protein, but exhibits a 38% amino acid divergence in the signal peptide region. We hypothesized that this may imply a difference in the cell type in which CXCL4L1 is expressed or a difference in its mode of secretion. In different types of transfected cells, CXCL4 and CXCL4L1 exhibited a distinct subcellular localization and a differential regulation of secretion, CXCL4 being stored in secretory granules and released in response to protein kinase C activation, whereas CXCL4L1 was continuously synthesized and secreted through a constitutive pathway. A protein kinase C-regulated CXCL4 secretion was observed also in lymphocytes, a cell type expressing mainly CXCL4 mRNA, whereas smooth muscle cells, which preferentially expressed CXCL4L1, exhibited a constitutive pathway of secretion. These results demonstrate that CXCL4 and CXCL4L1 exhibit a distinct subcellular localization and are secreted in a differentially regulated manner, suggesting distinct roles in inflammatory or homeostatic processes.

Isolation and characterization of multipotent progenitor cells from the Bowman's capsule of adult human kidneys.

Regenerative medicine represents a critical clinical goal for patients with ESRD, but the identification of renal adult multipotent progenitor cells has remained elusive. It is demonstrated that in human adult kidneys, a subset of parietal epithelial cells (PEC) in the Bowman's capsule exhibit coexpression of the stem cell markers CD24 and CD133 and of the stem cell-specific transcription factors Oct-4 and BmI-1, in the absence of lineage-specific markers. This CD24+CD133+ PEC population, which could be purified from cultured capsulated glomeruli, revealed self-renewal potential and a high cloning efficiency. Under appropriate culture conditions, individual clones of CD24+CD133+ PEC could be induced to generate mature, functional, tubular cells with phenotypic features of proximal and/or distal tubules, osteogenic cells, adipocytes, and cells that exhibited phenotypic and functional features of neuronal cells. The injection of CD24+CD133+ PEC but not of CD24-CD133- renal cells into SCID mice that had acute renal failure resulted in the regeneration of tubular structures of different portions of the nephron. More important, treatment of acute renal failure with CD24+CD133+ PEC significantly ameliorated the morphologic and functional kidney damage. This study demonstrates the existence and provides the characterization of a population of resident multipotent progenitor cells in adult human glomeruli, potentially opening new avenues for the development of regenerative medicine in patients who have renal diseases.