Maria Lucia Angelotti

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 Bowmans 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.

Toll-Like Receptors 3 and 4 Are Expressed by Human Bone Marrow-Derived Mesenchymal Stem Cells and Can Inhibit Their T-Cell Modulatory Activity by Impairing Notch Signaling

Bone marrow (BM)‐derived mesenchymal stem cells (MSCs) are multipotent, nonhemopoietic progenitors that also possess regulatory activity on immune effector cells through different mechanisms. We demonstrate that human BM‐derived MSCs expressed high levels of Toll‐like receptors (TLRs) 3 and 4, which are both functional, as shown by the ability of their ligands to induce nuclear factor κB (NF‐κB) activity, as well as the production of interleukin (IL)‐6, IL‐8, and CXCL10. Of note, ligation of TLR3 and TLR4 on MSCs also inhibited the ability of these cells to suppress the proliferation of T cells, without influencing their immunophenotype or differentiation potential. The TLR triggering effects appeared to be related to the impairment of MSC signaling to Notch receptors in T cells. Indeed, MSCs expressed the Notch ligand Jagged‐1, and TLR3 or TLR4 ligation resulted in its strong downregulation. Moreover, anti‐Jagged‐1 neutralizing antibody and N[N‐(3,5‐difluorophenacetyl‐l‐alanyl)]‐S‐phenylglycine t‐butyl ester (DAPT), an inhibitor of Notch signaling, hampered the suppressive activity of MSCs on T‐cell proliferation. These data suggest that TLR3 and TLR4 expression on MSCs may provide an effective mechanism to block the immunosuppressive activity of MSCs and therefore to restore an efficient T‐cell response in the course of dangerous infections, such as those sustained by double‐stranded RNA viruses or Gram‐negative bacteria, respectively.

Essential but differential role for CXCR4 and CXCR7 in the therapeutic homingof 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.

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 Bowmans 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. STEM CELLS2012;30:1714–1725

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 Bowmans 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.

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 Bowmans space. The origin of these hyperplastic epithelial cells in different glomerular disorders is controversial. Renal progenitors localized to the inner surface of Bowmans 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.

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 Bowmans 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 G2/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. STEM CELLS 2010; 28:1674–1685.

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.

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

Summary 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.

The Antiviral Cytokines IFN-α and IFN-β Modulate Parietal Epithelial Cells and Promote Podocyte Loss: Implications for IFN Toxicity, Viral Glomerulonephritis, and Glomerular Regeneration

Interferon (IFN)-α and IFN-β are the central regulators of antiviral immunity but little is known about their roles in viral glomerulonephritis (eg, HIV nephropathy). We hypothesized that IFN-α and IFN-β would trigger local inflammation and podocyte loss. We found that both IFNs consistently activated human and mouse podocytes and parietal epithelial cells to express numerous IFN-stimulated genes. However, only IFN-β significantly induced podocyte death and increased the permeability of podocyte monolayers. In contrast, only IFN-α caused cell-cycle arrest and inhibited the migration of parietal epithelial cells. Both IFNs suppressed renal progenitor differentiation into mature podocytes. In Adriamycin nephropathy, injections with either IFN-α or IFN-β aggravated proteinuria, macrophage influx, and glomerulosclerosis. A detailed analysis showed that only IFN-β induced podocyte mitosis. This did not, however, lead to proliferation, but was associated with podocyte loss via podocyte detachment and/or mitotic podocyte death (mitotic catastrophe). We did not detect TUNEL-positive podocytes. Thus, IFN-α and IFN-β have both common and differential effects on podocytes and parietal epithelial cells, which together promote glomerulosclerosis by enhancing podocyte loss while suppressing podocyte regeneration from local progenitors.