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 (α, β, γ 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.

In most kidney diseases, leukocytes in the kidney mediate tissue injury. The primary site of infiltration is the renal interstitium. The interaction of leukocytes with resident kidney cells stimulates these parenchymal cells to generate cytokines and incite an amplification cascade resulting in inflammation. Tubular epithelial cells (TECs), which comprise the vast majority of parenchymal cells in the kidney, are a rich source of cytokines and chemokines. The dynamics between the kidney-infiltrating leukocytes and the TECs determine the extent of interstitial inflammation. Activated TECs generate numerous cytokines and chemokines that promote renal injury. IFN-γ produced by T cells is the most potent TEC activator. Since IL-15 is a T cell growth factor (1) that is expressed in IFN-γ–activated human kidney TECs (2), we concluded that IL-15 is a pivotal cytokine in kidney inflammation.

The biologic functions of IL-15 are complex. Although the receptors for IL-15 and IL-2 share two subunits (the IL-2 β and common γ chains), the functions of these molecules during T cell–dependent immune responses are distinct and contrasting (3). Specificity for IL-15 is conferred by the α chain of the IL-15 receptor (IL-15R) (4). While IL-15 and IL-2 have somewhat redundant properties in stimulating T cell proliferation, IL-15, unlike IL-2, does not promote T cell apoptosis. Indeed, IL-15 protects T cells from IL-2–triggered apoptosis (5). Moreover, IL-15 is of particular importance as a growth factor for memory CD8 T cells (6). In fact, mice genetically deficient in IL-15 have a reduction in memory CD8 T cells and NK cells (3).

Of particular importance, IL-15 and IL-2 are not expressed in the same cells. In contrast to the expression of IL-2 in activated T cells, IL-15 is constitutively expressed by a broad array of cell types. Notably, epithelial cells are a principal source of IL-15. In keeping with this data, amplified IL-15 expression at the site of autoimmune disease has been linked to exacerbation of rheumatoid arthritis (7) and inflammatory bowel disease (8). Furthermore, enhanced IL-15 expression is detected in kidneys during human allograft rejection (9). Interestingly, IL-15 blockade with a neutralizing soluble IL-15R or a receptor site antagonist have proven effective in prolonging heart or islet allograft survival (10) and suppressing collagen-induced arthritis (11) and delayed-type hypersensitive reactions (12). Based on these findings, we deduced that preventing signaling via IL-15Rα might be a therapeutic strategy for countering T cell–dependent immune reactions, particularly those involving epithelial cells.

Since T cells mediate immunologic kidney diseases, we hypothesized that IL-15, a potent T cell growth factor produced by kidney TECs, would foster the expansion of activated T cells within the kidney and thus promote T cell–mediated renal injury. To test this hypothesis, we evaluated the role of IL-15 in IL-15–deficient (IL-15^–/–) and IL-15 intact (IL-15^+/+) mice during accelerated nephrotoxic serum nephritis (NSN), a T cell–dependent model of kidney disease (13). Contrary to our hypothesis, renal damage is more severe and macrophages and CD4 T cells are more abundant in IL-15^–/– mice than in IL-15^+/+ mice. Although IL-15 is largely expressed by TECs in nephritic kidneys, it is far more abundant in normal kidneys. Why does a failure to express IL-15 lead to heightened renal injury? We now report that IL-15 functions in an autocrine manner as a survival factor for renal TECs. IL-15 serves to guard against TEC apoptosis. In addition, IL-15 inhibits the expression of the chemokine MCP-1. Perhaps as a consequence, the accumulation of macrophages and CD4 T cells is reduced. Thus, we suggest that IL-15 protects the kidney during certain immunologic assaults.

Renal tubular pathology is markedly increased, while glomerular pathology is modestly worse and BUN is elevated in IL-15^–/– mice during NSN. To investigate the importance of IL-15 in kidneys undergoing immune injury, we induced NSN in IL-15^–/– and IL-15^+/+ mice. Unexpectedly, we detected a striking increase in renal pathology in the IL-15^–/– mice compared with IL-15^+/+ mice; this was most notable in the tubules on day 11 (Figure 1, a–c). Tubular damage (which included one or more of the following: dilation with flattened or degraded epithelium, atrophy, thickening, and rupture of basement membranes)was increased in IL-15^–/– mice on days 11 and 14 (Figure 1, b and c) compared with IL-15^+/+ mice (Figure 1, a and c). In addition, peritubular leukocytic infiltrates and casts in tubule lumens were associated with damaged tubules (Figure 1, a and b). The change in glomerular pathology was subtler. We noted an increase in cell number of 30–40% within glomeruli in IL-15^–/– mice compared with glomeruli in wild-type mice at 6, 11, and 14 days (Figure 2). On the other hand, we did not detect a difference in the number of glomerular crescents.

Figure 1

Tubular pathology and apoptosis are markedly increased in IL-15^–/– kidneys during NSN. Tubular damage is increased in IL-15^–/– kidneys (a) compared with IL-15^+/+ kidneys (b) with NSN measured on day 11 after induction (inset; arrow indicates damaged tubule). Tubular damage (c), and apoptosis (d) (measured on day 11 by TUNEL assay) are increased during NSN in IL-15^–/– kidneys. Data were analyzed by Student t test and are presented as mean ± SEM. n = 8 per group. *P < 0.05. Magnification: a and b, ×100; insets, ×400.

Figure 2

The number of cells in glomeruli is modestly increased in IL-15^–/– kidneys. We detected an increase in cell/glomerulus at days 6, 11, and 14 in IL-15^–/– glomeruli (a and c) compared with IL-15^+/+ glomeruli (b and c) after induction of NSN. Magnification: a and b, ×100. Cells in glomeruli were counted in sections stained with periodic acid–Schiff reagent (50 glomeruli/kidney, ×400). Data were analyzed by Student t test and are presented as mean ± SEM. n = 8 per group. *P < 0.05.

Accompanying the increased renal pathology in the IL-15^–/– mice was a greater loss of renal function. BUN levels measured on days 11 and 14 after initiation of NSN were elevated in the IL-15^–/– mice compared with wild-type mice (Figure 3). Urine protein levels in IL-15^+/+ and IL-15^–/– mice were increased to a similar degree on days 5 and 13 after NSN induction (data not shown). However, urinary protein is not a suitable functional marker of tubulointerstitial injury in this model, because nephrotoxic serum reacts with several podocyte surface antigens and causes proteinuria by direct podocyte injury in the absence of an inflammatory response (19).

Figure 3

BUN is higher in IL-15^–/– mice with NSN than in wild-type mice with NSN. Measurements were taken on day 11 and day 14 after induction of NSN and results were analyzed by ANOVA with replication. Data are presented as mean ± SEM. The BUN of these mice prior to receiving nephrotoxic serum was 33.7 ± 1.5 mg/dl. n = 4 per group. *P = 0.014.

Apoptotic cells are increased in IL-15^–/– mice during NSN. The increase in tubular injury in IL-15^–/– compared with IL-15^+/+ mice during NSN indicated that IL-15 might be an epithelial cell survival factor. To address this issue, we enumerated the number of apoptotic cells in the kidney. Apoptotic cells in the kidney were almost exclusively within cortical tubules; IL-15^–/– kidneys had twice as many apoptotic cells as did IL-15^+/+ kidneys (Figure 1d).

IL-15 expression in the kidney is decreased after the induction of NSN. The increased renal pathology in IL-15^–/– mice prompted us to probe for the intrarenal expression of IL-15 in wild-type mice with NSN. As opposed to most cytokines and chemokines, which are not constitutively expressed, we detected an abundant expression of IL-15 in kidneys prior to NSN.This IL-15 expression decreased dramatically (threefold) on day 14 of NSN (Figure 4). Intrarenal IL-15 was detected by immunostaining. In agreement with the IL-15 transcripts, IL-15 protein was prominent in untreated wild-type kidneys without NSN. We detected IL-15 mainly in tubules (Figure 5a), with lesser amounts in glomeruli (Figure 5b) and vascular endothelium in the normal kidneys. However, during NSN, IL-15 measured on day 14 was markedly reduced in the tubules (Figure 5c) and glomeruli (Figure 5d) of IL-15^+/+ mice. Sections stained for IL-15 with IL-15 antibody that was preabsorbed with an excess amount of mouse rIL-15 were negative, confirming specificity (not shown). Based on the abundant expression of IL-15 in normal TECs that decreases during NSN, and the enhanced TEC pathology and apoptosis in IL-15^–/– kidneys during NSN, we hypothesized that IL-15 is a survival factor for epithelial cells.

Figure 4

IL-15 transcription is downregulated in the kidneys of mice with NSN. There is a decrease in IL-15 transcript in the kidneys of IL-15^+/+ mice injected with nephrotoxic serum (measured on day 14 after induction of NSN) compared with transcript in kidneys of uninjected IL-15^+/+ mice, as measured by RT-PCR. Data were analyzed by Bonferroni-Dunn test and are presented as mean ± SEM. n = 3 per group. *P < 0.05.

Figure 5

IL-15 protein is decreased in tubules and glomeruli during NSN in wild-type mice. IL-15 was strongly expressed in the tubules (arrows) and glomeruli (arrowheads) in normal kidneys (a and b). IL-15 expression was markedly decreased within tubules and glomeruli during NSN (day 14) (c and d). Magnifications: a and c, ×100; b and d, ×400.

IL-15 protects TECs from AD-induced death and apoptosis. To test the hypothesis that IL-15 is a survival factor for TECs, we determined whether AD-induced cell death was enhanced in IL-15^–/– TECs. We incubated primary cultured TECs derived from IL-15^+/+ and IL-15^–/– mice with AD (0.05 μg/ml and 0.1 μg/ml) for 24 hours. IL-15^–/– TECs were more vulnerable to AD-induced cell death than IL-15^+/+ TECs were (Figure 6a). We incubated the IL-15^+/+ TECs with soluble IL-15Rα (sIL-15Rα; 200 ng/ml), a molecule that competitively blocks the binding of IL-15 to IL-15Rα, to determine whether enhanced TEC survival was specific to IL-15. The soluble mutant IL-15R, smIL-15Rα (200 ng/ml), which does not block the binding of IL-15 to IL-15Rα, served as a control. The sIL-15Rα increased cell death in IL-15^+/+ TECs, while the smIL-15Rα did not alter IL-15^+/+ TEC survival (Figure 6b).

Figure 6

Cultured IL-15^–/– TECs are more susceptible to AD-induced cell death and apoptosis. AD causes a concentration-dependent increase in the number of dead cells that is more severe in IL-15^–/– TECs (a). Blocking IL-15 signaling with soluble receptor (sIL-15Rα) in IL-15^+/+ TECs increased AD-induced cell death (b). Blocking IL-15 with sIL-15Rα increased AD-induced apoptosis in IL-15^+/+ TECs, and exogenous rIL-15 protected IL-15^–/– TECs from AD-induced apoptosis (c). In comparison, adding smIL-15R did not alter findings (b and c). Data (presented as mean ± SEM) were analyzed by the Fisher t test (a and b) and the Bonferroni-Dunn test (c) and are representative of three experiments using TECs isolated from different mice (n = 5). *P < 0.05.

To determine whether IL-15 is anti-apoptotic, we exposed IL-15^–/– and IL-15^+/+ TECs to AD as above and evaluated apoptosis by the TUNEL assay. IL-15^–/– TECs had an increase (56%) in apoptosis compared with IL-15^+/+ TECs (Figure 6c). Blocking the IL-15R on IL-15^+/+ TECs exposed to AD with sIL-15Rα increased apoptosis compared with IL-15^+/+ TECs exposed to smIL-15Rα, the control molecule (Figure 6c). Blockade of IL-15R on IL-15^+/+ TECs resulted in an amount of AD-induced apoptosis that was similar to that in IL-15^–/– TECs (Figure 6c). Furthermore, adding rIL-15 (50 ng/ml and 200 ng/ml) dramatically reduced AD-induced apoptosis of IL-15^–/– TECs. Taken together, these results indicate that IL-15 protects TECs from AD-induced apoptosis.

IL-15 protects TECs from Fas-induced cell death and apoptosis. To eliminate the possibility that IL-15 is a survival factor unique to AD-induced TEC death, we explored the importance of IL-15 in Fas-mediated TEC death. We primed TECs with LPS and IFN-γ to induce Fas expression on TECs, and then incubated the TECs with anti-Fas antibody and protein G to initiate Fas-induced cell death. Fas-induced TEC death was more pronounced (twofold) in IL-15^–/– TECs than in IL-15^+/+ TECs and was diminished by the addition of rIL-15 (Figure 7a). As in AD-induced cell death, sIL-15Rα (200 ng/ml), but not smIL-15Rα, increased death of IL-15^+/+ TECs to the level of IL-15^–/– TEC death (Figure 7b). In addition, we determined that IL-15^–/– TECs had an increase in Fas-induced apoptosis that was more than IL-15^+/+ TECs (Figure 7c). Moreover, adding rIL-15 (200 ng/ml) reduced TEC apoptosis in IL-15^–/– TECs to the level detected in IL-15^–/– TECs (Figure 7c). Thus, IL-15 is a survival factor for Fas- and AD-induced TEC apoptosis.

Figure 7

IL-15 protects TECs from Fas-induced cell death and apoptosis. (a) Adding rIL-15 reduced TEC death in IL-15^–/– TECs but not in IL-15^+/+ TECs (n = 6 per group). (b) Blocking IL-15 with sIL-15Rα induced an increase in cell death in IL-15^+/+ TECs, while the mutant sIL-15Rα molecule had no effect (n = 3 per group). (c) Fas-mediated apoptosis (measured by TUNEL assay) was increased in IL-15^–/– TECs compared with IL-15^+/+ TECs. Adding rIL-15 reduced IL-15^–/– TEC apoptosis to IL-15^+/+ TEC levels (n = 5 per group). Data (presented as mean ± SEM) were analyzed by the Bonferroni-Dunn test and are representative of more than three experiments using TECs isolated from different mice. *P < 0.05.

IL-15 and IL-15R are expressed by normal TECs. To determine whether IL-15 could act as an autocrine molecule, we probed kidneys and primary TECs from normal C57BL/6 mice for IL-15 and IL-15R expression. IL-15 transcripts were detected in the normal kidneys and TECs using RT-PCR (Figure 8a). The transcripts of the IL-15R, comprised of the IL-15Rα, IL-2Rβ, and common γ chains, were identified in TECs (Figure 8b). Therefore, normal TECs express both IL-15 and all three chains of IL-15R.

Figure 8

IL-15 and IL-15R mRNA is expressed in normal TECs. (a) Normal kidney and TECs express IL-15. (b) Normal TECs express the IL-15R α, β, and common γ chains. IL-15R transcripts were detected in TECs derived from three different mice. The data are representative of more than ten experiments.

Renal interstitial leukocytic infiltrates are more prominent in IL-15^–/– mice during NSN. Leukocytic infiltrates in the renal interstitium are prominent in NSN (see Figure 9). In the IL-15^–/– kidneys during NSN, we detected an initial (on day 6 and day 11) enhanced increase in macrophages, and a more enduring increase in CD4 T cells measured on days 6, 11, and 14 in the interstitium versus wild-type kidneys (Figure 9, a and b) In contrast, CD8 T cells increased in IL-15^+/+ kidneys but not in IL-15^–/– kidneys compared with pre-NSN levels (measured on day 6, day 11, and day 14; Figure 9c).

Figure 9

Interstitial CD4 T cells and macrophages are increased, while CD8 T cells are decreased in IL-15^–/– mice as compared with IL-15^+/+ mice with NSN. Macrophages and CD4 T cells in the interstitium were markedly increased, while CD8 T cells were decreased in IL-15^–/– kidneys. Data (presented as mean ± SEM) were analyzed by Bonferroni-Dunn test. n = 5 per group. *P < 0.05.

Glomerular leukocytic infiltrates are increased in IL-15^–/– kidneys. To determine whether leukocytes were responsible for increasing the numbers of cells in glomeruli, we stained for the presence of macrophages, CD4 T cells, and CD8 T cells. Initially, we detected more macrophages (day 6) and CD4 T cells (days 6 and 11) in IL-15^–/– glomeruli than in IL-15^+/+ glomeruli (Figure 10, a and b). In contrast, as in the renal interstitium, the numbers of CD8 T cells in IL-15^–/– glomeruli remained normal, while there was an increase in CD8 T cells in IL-15^+/+ glomeruli (days 6, 11, and 14; Figure 10c). Taken together, this data indicates that macrophages and CD4 T cells are responsible, in part, for the increase in glomerular cells in IL-15^–/– during NSN.

Figure 10

Glomerular macrophages and CD4 T cells are initially increased in IL-15^–/– mice with NSN. In contrast, CD8 T cells were substantially lower in glomeruli of IL-15^–/– mice than in glomeruli of IL-15^+/+ mice. Data were evaluated by Bonferroni-Dunn test and are presented as mean ± SEM. n = 8 per group. *P < 0.01.

IL-15 inhibits intrarenal MCP-1 expression during NSN. We have previously reported that MCP-1, CSF-1, and RANTES recruit macrophages and T cells into the kidney (15, 18, 20, 21). In addition, we established that IFN-γ and IL-12 are nephritogenic (16, 22). During NSN, MCP-1, CSF-1 and RANTES transcripts were increased in the renal cortex in IL-15^–/– and IL-15^+/+ mice compared with normal mice (day 11; Figure 11). However, only MCP-1 was greater in the IL-15^–/– kidneys compared with the IL-15^+/+ kidneys. Thus, IL-15 inhibits intrarenal MCP-1, and in turn may be responsible for dampening the influx of macrophages and T cells during NSN.

Figure 11

IL-15 inhibits MCP-1 expression during NSN. MCP-1, CSF-1, and RANTES transcripts, but not IL-12 (p40) or IFN-γ, are upregulated in both IL-15^–/– and IL-15^+/+ mice in the renal cortex during NSN. MCP-1 transcripts are further increased in IL-15^–/– kidneys compared with IL-15^+/+ kidneys during NSN (day 11). Thus, IL-15 inhibits MCP-1 expression during NSN. Analysis was done by real-time PCR normalized to GAPDH expression. Data were analyzed by Bonferroni-Dunn test and are presented as mean ± SEM. n = 3 per group. *P < 0.05 versus normal; ^#P < 0.05 versus IL-15^+/+. Normal, wild-type mice without induced NSN.

IL-15^–/– mice do not have an increase in circulating anti–sheep Ig isotypes, or IgG in the kidney during NSN. We evaluated serum Ig isotypes in IL-15^–/– and wild-type mice during NSN on day 11 and day 14. Although there is a consistent trend to lower serum isotype titres on day 14 in IL-15^–/– mice, these values were not statistically significant. The OD₄₅₀ readings were: IgG, 1.0 ± 0.1 and 0.8 ± 0.1; IgG1, 1.1 ± 0.1 and 0.7 ± 0.1; IgG2a, 0.4 ± 0.1 and 0.2 ± 0.1, for IL-15^–/– (n = 5) and wild type (n = 6), respectively. The serum isotype findings in IL-15^–/– and wild-type mice at day 11 were similar to those of day 14 with the exception that total IgG levels were statistically decreased in the IL-15^–/– mice (P < 0.03). Taken together, this data suggests that the increased renal disease in IL-15^–/– mice was not a result of an increase in serum Ig’s or a result of immune deviation toward a Th1 phenotype. In fact, the tendency for the serum Ig isotypes to be decreased in IL-15^–/– mice would suggest that renal disease should have been less, and not greater, in IL-15^–/– mice during NSN.

To establish whether the deposition of IgG differed between IL-15^–/– and wild-type mice, we quantitatively evaluated the deposition of host (mouse) IgG in the IL-15^–/– kidneys on day 14 after the induction of NSN. The results revealed similar amounts of IgG in the IL-15^–/– and wild-type kidneys (12.09 ± 1.18 and 11.39 ± 0.74 luminosity units, respectively; n = 5 per group; P < 0.6). Mouse IgG is located in the capillary walls of the glomeruli, and the distribution is similar in both groups. This indicates that the total amount of mouse anti–sheep IgG deposited in the glomeruli is not different in the two groups, and that the amount of antibody deposited in the kidney in IL-15^–/– mice was not influenced by the lack of IL-15.

We now report that IL-15 counteracts renal injury in a T cell–dependent disease akin to human kidney illnesses. This is the first report that: (a) IL-15 is a survival factor for kidney epithelial cells; (b) IL-15 is protective in a T cell–dependent disease; (c) IL-15 inhibits the induction of MCP-1 in vivo; and (d) IL-15 protects the kidney during nephritis. In addition, this data is particularly compelling since it was generated using a strain genetically deficient in IL-15.

Interstitial inflammation is a major determinant of the outcome of human renal disease (23, 24). Most cytokines are not expressed by normal kidneys, but are induced during inflammation (18, 25) and are largely expressed in TECs (15, 18, 26, 27). The interplay between the kidney-infiltrating leukocytes and the TECs determines the extent of interstitial inflammation (28, 29). An amplification cascade drives kidney disease: interstitial T cells release IFN-γ which activates adjacent TECs (15, 18, 30, 31), which in turn release chemokines (MCP-1) and cytokines (CSF-1) that are responsible for recruiting macrophages and T cells (32). Subsequently, upon activation, macrophages release molecules that induce apoptosis in TECs (15). Since TEC apoptosis correlates with tubulointerstitial fibrosis, protecting the TECs from injury is critical in combating kidney disease (33).

We now report that intrarenal IL-15 inhibits TEC apoptosis. Typically, cytokines are not constitutively expressed in TECs, but rather are upregulated during renal disease (30, 31). IL-15 is atypical. It is constitutively and abundantly expressed in normal TECs and is reduced during renal injury, as shown in NSN and in spontaneous lupus nephritis in MRL-Fas^lpr mice (data not shown). Thus, IL-15 in normal TECs is well positioned to guard against unwanted immunologic insults.

Initially, IL-15 was identified as a survival factor in T cells. It is now clear that IL-15 is a survival factor for a broader array of cell types. IL-15 promotes cell survival in hematopoietic cells, including T cells (34), B cells, mast cells (35), and neutrophils (36). The evidence that IL-15 is a survival factor for nonhematopoietic cells is far more limited. In vitro data supports the concept that IL-15 is anti-apoptotic in fibroblasts (37) and keratinocytes (38). Moreover, in vivo data is limited to suppression of apoptosis in hepatocytes using a long-acting IL-15 (IL-15–IgGb fusion protein) to inhibit anti-Fas–induced rapid hepatic failure (39). Thus, it is plausible that IL-15 is a general survival factor for epithelial cells.

The mechanism responsible for IL-15 counteracting TEC apoptosis is not known. We have established that TECs express all three chains of the IL-15R (α, β, and γ). In a murine fibroblast cell line (L929), the binding of IL-15 to IL-15Rα competes with the TNFR1 complex for TRAF2 binding, impeding the assembly of key adapter proteins in the TNFR1 complex and in turn inducing IκBα phosphorylation (37). Thus, it is possible that IL-15Rα signaling of TECs may induce similar anti-apoptotic mechanisms. In addition, members of the Bcl2 protein family have been implicated as regulators of TEC cell death during acute renal failure (40). Since IL-15 induces Bcl-x_L expression and prevents apoptosis in a mouse mast cell line, it is possible that IL-15 induces Bcl-x_L in TECs (41). We are investigating the precise IL-15–mediated anti-apoptotic pathways in TECs.

Although there is a substantial increase in TEC apoptosis in IL-15^–/– mice, many cells survive. Thus, IL-15 is not the only TEC survival factor. Osteopontin, a secreted phosphoprotein expressed by TECs, protects TECs from apoptosis (42). However, unlike IL-15, osteopontin is a macrophage chemoattractant, and therefore contributes to nephritis. We are probing for the other TEC survival factors that combat renal disease.

It is intriguing that despite the absence of the T cell growth factor IL-15, there is a marked increase in CD4 T cells and macrophages in the renal interstitium, and a subtler increase in these leukocytes in glomeruli during NSN. This suggests that the action of IL-15 as a survival factor for epithelial cells and an inhibitor of MCP-1 has a greater impact on the pathogenesis of renal disease than does IL-15’s role in promoting the expansion of T cells. Moreover, this data indicates that IL-15 is not required to drive the expansion of CD4 T cells in kidney diseases, and suggests that the T cell growth factor IL-2 is sufficient for this process. IL-15–deficient mice have a pronounced reduction in CD8 T cells and NK cells (3). During NSN, few CD8 T cells accumulate in the kidney or in the spleen (data not shown) in the IL-15–deficient strain. By extension, we suggest that CD8 T cells are not instrumental in promoting some T cell–dependent renal diseases.

It is noteworthy that tubular injury and TEC apoptosis were more severe in IL15^–/– mice than in wild-type mice in our study, despite a similar amount of proteinuria. This has several implications. First, there is a growing consensus that proteinuria is an important factor in the development of tubular injury and consequent interstitial fibrosis (43). Although cytokines, chemokines, and growth factors have been implicated in the interstitial inflammation and postinflammatory fibrogenic reaction associated with proteinuria, less is known about the mechanisms by which proteinuria causes TEC damage. Complement, modified albumin, and other molecules have been implicated (43–45). Regardless of the mechanism, our results demonstrate that IL-15–deficient TECs are more susceptible to injury in NSN, and are more likely than are wild-type TECs to undergo apoptosis, despite equivalent amounts of proteinuria. In addition, since proteinuria is similar in the two groups, this would suggest that there is equivalent glomerular injury. However, this is at variance with the pathology and elevated BUN levels in the IL15^–/– mice. To understand this apparent discrepancy, it is important to recognize the limitations of proteinuria as a marker of renal injury in this model. Nephrotoxic serum is capable of inducing proteinuria by binding to one or more podocyte antigens (19) or by recruiting inflammatory mediators. These include the proinflammatory and direct effects of complement (46, 47), leukocyte recruitment by Fc receptor binding (48), and, in animals presensitized to the heterologous Ig, a cell-mediated macrophage-dependent reaction (13). This dissociation between glomerular injury and proteinuria was further illustrated in P-selectin–deficient mice in which proteinuria was unaffected despite differences in glomerular leukocyte infiltration during the heterologous phase of NTN (49). Finally, tubular injury itself is able to reduce the glomerular filtration rate and raise BUN by altering tubular-glomerular feedback and thereby decreasing the number of functioning nephrons. Thus, we conclude that IL-15–deficient mice suffer from a greater loss of renal function than wild-type mice do because they are more susceptible to tubular damage despite similar levels of proteinuria.

Deleting a gene in advance of disease, and blockade of a molecule are often but not always similar. For example, a P-selectin deficiency exacerbates NSN (50), while blocking antibodies to P-selectin diminishes the acute phase of this disease (51). Since blockade of IL-15 protects against murine collagen arthritis, the generalized Shwartzman reaction, and delayed-type hypersensitivity, we plan to determine whether blocking IL-15 during NSN enhances or protects against renal injury (10–12, 52).

There are several mechanisms that may be responsible for enhanced apoptosis in IL-15^–/– TECs during NSN. MCP-1 plays a major pathogenic role in this model (15, 53). MCP-1 recruits macrophages into the interstitium, and activated macrophages induce TEC apoptosis (15). During NSN, MCP-1 is increased in IL-15^–/– kidneys compared with IL-15^+/+ kidneys. This finding is consistent with IL-15 downregulating MCP-1 in a colonic epithelial cell line (54). Therefore, it is possible that an IL-15 deficiency leads to exaggerated TEC apoptosis for two reasons: anti-apoptotic molecules are not induced, and MCP-1 expression (and in turn, the increased interstitial macrophage infiltration) is not inhibited.

In conclusion, we suggest that provision of IL-15 in some human kidney diseases may be therapeutic.

This work was supported in part by a National Kidney Foundation grant to J. Hirahashi, and by NIH grants to V.R. Kelley (DK-36149, DK-56848, and DK-52369) and D.J. Salant (DK-30932 and HL-63894). We wish to acknowledge Surya M. Nauli for his assistance in preparation of this manuscript.

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