Vicki Rubin Kelley

Restoration of tubular epithelial cells during repair of the postischemic kidney occurs independently of bone marrow-derived stem cells

Ischemia causes kidney tubular cell damage and abnormal renal function. The kidney is capable of morphological restoration of tubules and recovery of function. Recently, it has been suggested that cells repopulating the ischemically injured tubule derive from bone marrow stem cells. We studied kidney repair in chimeric mice expressing GFP or bacterial beta-gal or harboring the male Y chromosome exclusively in bone marrow-derived cells. In GFP chimeras, some interstitial cells but not tubular cells expressed GFP after ischemic injury. More than 99% of those GFP interstitial cells were leukocytes. In female mice with male bone marrow, occasional tubular cells (0.06%) appeared to be positive for the Y chromosome, but deconvolution microscopy revealed these to be artifactual. In beta-gal chimeras, some tubular cells also appeared to express beta-gal as assessed by X-gal staining, but following suppression of endogenous (mammalian) beta-gal, no tubular cells could be found that stained with X-gal after ischemic injury. Whereas there was an absence of bone marrow-derived tubular cells, many tubular cells expressed proliferating cell nuclear antigen, which is reflective of a high proliferative rate of endogenous surviving tubular cells. Upon i.v. injection of bone marrow mesenchymal stromal cells, postischemic functional renal impairment was reduced, but there was no evidence of differentiation of these cells into tubular cells of the kidney. Thus, our data indicate that bone marrow-derived cells do not make a significant contribution to the restoration of epithelial integrity after an ischemic insult. It is likely that intrinsic tubular cell proliferation accounts for functionally significant replenishment of the tubular epithelium after ischemia.

Monocyte chemoattractant protein-1 promotes macrophage-mediated tubular injury, but not glomerular injury, in nephrotoxic serum nephritis

Monocyte chemoattractant protein-1 (MCP-1) is upregulated in renal parenchymal cells during kidney disease. To investigate whether MCP-1 promotes tubular and/or glomerular injury, we induced nephrotoxic serum nephritis (NSN) in MCP-1 genetically deficient mice. Mice were analyzed when tubules and glomeruli were severely damaged in the MCP-1-intact strain (day 7). MCP-1 transcripts increased fivefold in MCP-1-intact mice. MCP-1 was predominantly localized within cortical tubules (90%), and most cortical tubules were damaged, whereas few glomerular cells expressed MCP-1 (10%). By comparison, there was a marked reduction (>40%) in tubular injury in MCP-1-deficient mice (histopathology, apoptosis). MCP-1-deficient mice were not protected from glomerular injury (histopathology, proteinuria, macrophage influx). Macrophage accumulation increased adjacent to tubules in MCP-1-intact mice compared with MCP-1-deficient mice (70%, P < 0.005), indicating that macrophages recruited by MCP-1 induce tubular epithelial cell (TEC) damage. Lipopolysaccharide-activated bone marrow macrophages released molecules that induced TEC death that was not dependent on MCP-1 expression by macrophages or TEC. In conclusion, MCP-1 is predominantly expressed by TEC and not glomeruli, promotes TEC and not glomerular damage, and increases activated macrophages adjacent to TEC that damage TEC during NSN. Therefore, we suggest that blockage of TEC MCP-1 expression is a therapeutic strategy for some forms of kidney disease.

IL-12 deficiency in MRL-Fas(lpr) mice delays nephritis and intrarenal IFN-gamma expression, and diminishes systemic pathology.

Autoimmune disease in MRL-Faslpr mice is characterized by fatal nephritis, systemic pathology, and autoantibodies, mimicking human lupus. We previously reported that 1) intrarenal IL-12 elicits nephritis by fostering the accumulation of intrarenal IFN-γ-secreting T cells, and 2) MRL-Faslpr mice deficient in the IFN-γ receptor were spared from nephritis. Therefore, we hypothesized that eliminating IL-12 in MRL-Faslpr mice reduces IFN-γ-secreting cells and thereby prevents systemic pathology. For this purpose, we constructed an IL-12p40-deficient MRL-Faslpr(IL-12−/−) strain. We determined that glomerular and interstitial, but not perivascular, renal pathology were decreased in IL-12−/− mice vs the wild-type (WT) strain (5 mo of age). Similarly, systemic pathology (lung, lacrimal and salivary glands, skin, and lymphadenopathy) was diminished. The intrarenal accumulation of T cells (CD4+, CD8+, CD4−CD8−B220+) and macrophages was dramatically reduced in IL-12−/− MRL-Faslpr kidneys. We determined that there were fewer IFN-γ transcripts (>70%) in the IL-12−/− protected kidneys compared with the WT kidneys. Similarly, cells propagated from IL-12−/− MRL-Faslpr kidneys generated substantially less IFN-γ when stimulated with IL-12 and IL-18 compared with those from WT kidneys, and we detected fewer CD8 and B220 T cells producing IFN-γ in these IL-12−/− MRL-Faslpr kidneys. Of note, survival was modestly extended in the IL-12−/− MRL-Faslpr mice. While lung and lacrimal and salivary gland pathology remained reduced in moribund IL-12−/− MRL-Faslpr mice, renal pathology and IFN-γ expression were equivalent to those in the WT strain. Thus, we suggest that IL-12 is a therapeutic target for multiple tissues in lupus; however blocking IL-12 alone is not sufficient to confer enduring protection from lupus nephritis.

CSF-1 signals directly to renal tubular epithelial cells to mediate repair in mice

Tubular damage following ischemic renal injury is often reversible, and tubular epithelial cell (TEC) proliferation is a hallmark of tubular repair. Macrophages have been implicated in tissue repair, and CSF-1, the principal macrophage growth factor, is expressed by TECs. We therefore tested the hypothesis that CSF-1 is central to tubular repair using an acute renal injury and repair model, ischemia/reperfusion (I/R). Mice injected with CSF-1 following I/R exhibited hastened healing, as evidenced by decreased tubular pathology, reduced fibrosis, and improved renal function. Notably, CSF-1 treatment increased TEC proliferation and reduced TEC apoptosis. Moreover, administration of a CSF-1 receptor-specific (CSF-1R-specific) antibody after I/R increased tubular pathology and fibrosis, suppressed TEC proliferation, and heightened TEC apoptosis. To determine the contribution of macrophages to CSF-1-dependent renal repair, we assessed the effect of CSF-1 on I/R in mice in which CD11b+ cells were genetically ablated and determined that macrophages only partially accounted for CSF-1-dependent tubular repair. We found that TECs expressed the CSF-1R and that this receptor was upregulated and coexpressed with CSF-1 in TECs following renal injury in mice and humans. Furthermore, signaling via the CSF-1R stimulated proliferation and reduced apoptosis in human and mouse TECs. Taken together, these data suggest that CSF-1 mediates renal repair by both a macrophage-dependent mechanism and direct autocrine/paracrine action on TECs.

Reduced macrophage recruitment, proliferation, and activation in colony-stimulating factor-1-deficient mice results in decreased tubular apoptosis during renal inflammation.

Kidney tubular epithelial cell (TEC) death may be dependent on the number and activation state of macrophages (Mφ) during inflammation. Our prior studies indicate that activated Mφ release soluble mediators that incite TEC death, and reducing intrarenal Mφ during kidney disease diminishes TEC apoptosis. CSF-1 is required for Mφ proliferation and survival. We hypothesized that in the absence of CSF-1, Mφ-mediated TEC apoptosis would be prevented during renal inflammation. To test this hypothesis, we evaluated renal inflammation during unilateral ureter obstruction in CSF-1-deficient (Csf1op/Csf1op) mice. We detected fewer Mφ and T cells and less apoptotic TEC in the obstructed kidneys of Csf1op/Csf1op mice compared with wild-type (WT) mice. The decrease in intrarenal Mφ resulted from diminished recruitment and proliferation, not enhanced apoptosis. CSF-1 enhanced Mφ activation. There were far fewer activated (CD69, CD23, Ia, surface expression) Mφ in obstructed CSF-1-deficient compared with WT obstructed kidneys. Similarly, bone marrow Mφ preincubated with anti-CSF-1 receptor Ab or anti-CSF-1 neutralizing Ab were resistant to LPS- and IFN-γ-induced activation. We detected fewer apoptotic-inducing molecules (reactive oxygen species, TNF-α, inducible NO synthase) in 1) Mφ propagated from obstructed Csf1op/Csf1op compared with WT kidneys, and 2) WT bone marrow Mφ blocked with anti-CSF-1 receptor or anti-CSF-1 Ab compared with the isotype control. Furthermore, blocking CSF-1 or the CSF-1 receptor induced less TEC apoptosis than the isotype control. We suggest that during renal inflammation, CSF-1 mediates Mφ recruitment, proliferation, activation, and, in turn, TEC apoptosis.

Negative Role of Colony-Stimulating Factor-1 in Macrophage, T Cell, and B Cell Mediated Autoimmune Disease in MRL-Faslpr Mice

Inflammation in the kidney and other tissues (lung, and salivary and lacrimal glands) is characteristic of MRL-Faslpr mice with features of lupus. Macrophages (Mφ) are prominent in these tissues. Given that 1) Mφ survival, recruitment, proliferation, and activation during inflammation is dependent on CSF-1, 2) Mφ mediate renal resident cell apoptosis, and 3) CSF-1 is up-regulated in MRL-Faslpr mice before, and during nephritis, we hypothesized that CSF-1-deficient MRL-Faslpr mice would be protected from Mφ-mediated nephritis, and the systemic illness. To test this hypothesis, we compared CSF-1-deficient MRL-Faslpr with wild-type strains. Renal pathology is suppressed and function improved in CSF-1-deficient MRL-Faslpr mice. There are far fewer intrarenal Mφ and T cells in CSF-1-deficient MRL-Faslpr vs wild-type kidneys. This leukocytic reduction results from suppressed infiltration, and intrarenal proliferation, but not enhanced apoptosis. The CSF-1-deficient MRL-Faslpr kidneys remain preserved as indicated by greatly reduced indices of injury (nephritogenic cytokines, tubular apoptosis, and proliferation). The renal protective mechanism in CSF-1-deficient mice is not limited to reduced intrarenal leukocytes; circulating Igs and autoantibodies, and renal Ig deposits are decreased. This may result from enhanced B cell apoptosis and fewer B cells in CSF-1-deficient MRL-Faslpr mice. Furthermore, the systemic illness including, skin, lung, and lacrimal and salivary glands pathology, lymphadenopathy, and splenomegaly are dramatically suppressed in CSF-1-deficient MRL-Faslpr as compared with wild-type mice. These results indicate that CSF-1 is an attractive therapeutic target to combat Mφ-, T cell-, and B cell-mediated autoimmune lupus.

Costimulation by B7-1 and B7-2 Is Required for Autoimmune Disease in MRL-Faslpr Mice

Autoimmune lupus nephritis is dependent on infiltrating autoreactive leukocytes and Igs. B7 costimulatory molecules (B7-1 and B7-2) provide signals essential for T cell activation and Ig class switching. In MRL-Faslpr mice, a model of human lupus, although multiple tissues are targeted for autoimmune injury, nephritis is fatal. We identified intrarenal B7-1 and B7-2 expression, restricted to kidney-infiltrating leukocytes, before and increasing with progressive nephritis in MRL-Faslpr mice. Thus, we hypothesized that the B7 pathway is required for autoimmune disease in MRL-Faslpr mice. To investigate the role of B7 costimulatory molecules in this autoimmune disease, we generated a MRL-Faslpr strain deficient in B7-1 and B7-2. Strikingly, MRL-Faslpr mice lacking both B7 costimulators do not develop kidney (glomerular, tubular, interstitial, vascular) pathology, or proteinuria, and survive far longer. Intrarenal downstream effector transcripts (IFN-γ, IL-12, monocyte chemoattractant protein-1, CSF-1) linked to nephritis remained at normal levels compared with wild-type mice. Skin lesions and lymphoid enlargement characteristic of MRL-Faslpr mice were diminished in B7-1/B7-2-deficient MRL-Faslpr mice. B7-1/B7-2-deficient MRL-Faslpr mice did not develop leukocytic infiltrates, elevated serum IgG and isotypes (G1,G2b,G3), autoantibodies, and intrarenal IgG deposits. Our findings demonstrate that B7-1 and B7-2 costimulatory pathways are critical to the pathogenesis of autoimmune lupus.

IL-15, a survival factor for kidney epithelial cells, counteracts apoptosis and inflammation during nephritis

IL-15, a T cell growth factor, has been linked to exacerbating autoimmune diseases and allograft rejection. To test the hypothesis that IL-15-deficient (IL-15-/-) mice would be protected from T cell-dependent nephritis, we induced nephrotoxic serum nephritis (NSN) in IL-15-/- and wild-type (IL-15+/+) C57BL/6 mice. Contrary to our expectations, IL-15 protects the kidney during this T cell-dependent immunologic insult. Tubular, interstitial, and glomerular pathology and renal function are worse in IL-15-/- mice during NSN. We detected a substantial increase in tubular apoptosis in IL-15-/- kidneys. Moreover, macrophages and CD4 T cells are more abundant in the interstitia and glomeruli in IL-15-/- mice. This led us to identify several mechanisms responsible for heightened renal injury in the absence of IL-15. We now report that IL-15 and the IL-15 receptor (alpha, beta, gamma chains) are constitutively expressed in normal tubular epithelial cells (TECs). IL-15 is an autocrine survival factor for TECs. TEC apoptosis induced with anti-Fas or actinomycin D is substantially greater in IL-15-/- than in wild-type TECs. Moreover, IL-15 decreases the induction of a nephritogenic chemokine, MCP-1, that attracts leukocytes into the kidney during NSN. Taken together, we suggest that IL-15 is a therapeutic for tubulointerstitial and glomerular kidney diseases.

Interferon-β: A Therapeutic for Autoimmune Lupus in MRL-Faslpr Mice

Type I interferons are associated with lupus. Genes that are regulated by IFN-alpha are upregulated in pediatric lupus patients. Gene deletion of the IFN-alpha/beta receptor in experimental lupus-like NZB mice results in reduced disease activity. Conversely, IFN-beta is a well-established treatment in multiple sclerosis, another autoimmune disease. For determining whether IFN-beta treatment is harmful or beneficial in lupus, MRL-Fas(lpr) mice were injected with this type I IFN. Treatment was initiated in MRL-Fas(lpr) mice with mild and advanced disease. IFN-beta was highly effective in prolonging survival and ameliorating the clinical (renal function, proteinuria, splenomegaly, and skin lesions), serologic (autoantibodies and cytokines), and histologic parameters of the lupus-like disease in mice that had mild and advanced disease. Several underlying mechanisms of IFN-beta therapy involving cellular (decreased T cell proliferation and infiltration of leukocytes into the kidney) and humoral (decrease in IgG3 isotypes) immune responses and a reduction in nephrogenic cytokines were identified. In conclusion, IFN-beta treatment of lupus nephritis in MRL-Fas(lpr) mice is remarkably beneficial and suggests that IFN-beta may be an appealing therapeutic candidate for subtypes of human lupus.

CXCL9, but not CXCL10, Promotes CXCR3-Dependent Immune-Mediated Kidney Disease

Chemokines are instrumental in macrophage- and T cell-dependent diseases. The chemokine CCL2 promotes kidney disease in two models of immune-mediated nephritis (MRL-Fas(lpr) mice and the nephrotoxic serum nephritis model), but evidence suggests that multiple chemokines are involved. For identification of additional therapeutic targets for immune-mediated nephritis, chemokine ligands and receptors in CCL2-/- and wild-type (WT) MRL-Fas(lpr) kidneys were profiled. The focus was on intrarenal chemokine ligand/receptor pairs that were highly upregulated downstream of CCL2; the chemokine CXCL10 and its cognate receptor, CXCR3, stood out as potential therapeutic targets. However, renal disease was not suppressed in CXCL10-/- MRL-Fas(lpr) mice, and CXCL10-/- C57BL/6 mice were not protected from nephrotoxic serum nephritis compared with WT mice. Because CXCR3 engages with the ligand CXCL9, CXCR3-/- , CXCL9-/- , and CXCL10-/- B6 mice were compared with WT mice with nephrotoxic serum nephritis. Kidney disease, measured by loss of renal function and histopathology, was suppressed in both CXCR3-/- and CXCL9-/- mice but not in CXCL10-/- mice. With nephrotoxic serum nephritis, CXCR3-/- and CXCL9-/- mice had fewer intrarenal activated T cells and activated macrophages. Both IgG glomerular deposits and antigen-specific IgG in serum were reduced in these mice, suggesting that although CXCR3 and CXCL9 initiate nephritis through cell-mediated events, renal inflammation may be sustained by their regulation of IgG. It is concluded that specific blockade of CXCL9