Recruitment of inflammatory cells, as well as stimulation of resident cells and accumulation of mesangial matrix are key features of various experimental and human glomerular diseases. Mesangial cell proliferation and matrix accumulation may contribute to the development of glomerulosclerosis independently of the underlying primary disease1,2. 1,25(OH)2D3 was shown to affect cell growth and differentiation from a variety of tissues. Thus, apart from its classic role in bone and mineral metabolism, 1,25(OH)2D3 inhibits cell proliferation and promotes cell differentiation3,4. The effects on lymphocyte and macrophage functions are exerted by binding of 1,25(OH)2D3 to vitamin D receptors expressed on monocytes and on activated but not on resting T lymphocytes5,6. Several lines of evidence indicate an immunosuppressive activity of 1,25(OH)2D37,8. It has been recently shown that dendritic cells are the major target of 1,25(OH)2D3-induced immunosuppressive activity since 1,25(OH)2D3 inhibits differentiation, maturation, and survival of these cells, leading to impaired alloreactive T-cell activation9. Moreover, 1,25(OH)2D3 inhibits the production of monocytes-derived cytokines such as interleukin-1
(IL-1), tumor necrosis factor- (TNF-), and IL-6 at the transcriptional level, most likely by reducing the half-life of specific mRNA10. The proliferation of T cells and their release of cytokines such as IL-2 and interferon-
are also suppressed by 1,25(OH)2D311. We recently reported that treatment with 1,25(OH)2D3 inhibits in a dose-dependent manner the production of both IL-1
and TNF- by lymphomonocytes of patients with chronic renal failure12. In a renal cell carcinoma line, Nagakura et al showed a dose-dependent inhibition of proliferation by 1,25(OH)2D313. Moreover, 1,25(OH)2D3 diminished proliferation of cultured mouse14 and human mesangial cells15, and secretion of transforming growth factor- (TGF-) in both human mesangial cells and in a proximal tubular cell line16. In vivo, 1,25(OH)2D3 was shown to reduce proteinuria in active Heymann nephritis17 and renal cell proliferation and glomerular growth, glomerulosclerosis, and albuminuria in a model of subtotal nephrectomy18.
The aim of the present study was to investigate the effects of 1,25(OH)2D3 in a model of proliferative glomerulonephritis induced in the rat by anti-Thy-1.1 antibodies.
METHODS
Experimental design
Animals
Eight-week-old female Wistar rats (Charles River, Calco, Italy) weighing 160 to 200 g were housed in single metabolic cages at constant room temperature (20°C) and humidity (75%) under a controlled light/dark cycle. The rats were fed a standard chow diet and animals had free access to drinking water. Experiments were performed in accordance to the guidelines of our local ethical committee. Rats were randomly allotted to one of the four following groups of treatment Figure 1.
Figure 1.
Rats were randomly allotted to one of the four groups of treatment. (1) The anti-Thy-1.1 group. At day 0, 24 rats received an IV bolus of anti-Thy-1.1 IgG monoclonal antibody (mAb). (2) Anti-Thy-1.1 treated with 1,25(OH)2D3. From day -3 to day 14, 24 rats were given 1,25(OH)2D3 at the dose of 25 ng/100g body wt/day. At day 0, they received anti-Thy-1.1 IgG mAb. (3) Control group. At day 0, 12 rats were injected intravenously with 0.5 mL of vehicle. (4) Control group treated with 1,25(OH)2D3. From day -3 to day 14, 12 rats were given 1,25(OH)2D3 at the dose of 25 ng/100g body wt/day. At day 0, they received 0.5 mL of vehicle. Six rats of each group were sacrificed for the morphological studies where indicated by the arrows.
Full figure and legend (11K)Anti-Thy-1.1 group (group I)
Twenty-four rats at day 0 received 0.5 mL of saline containing 400 
g of ascites-derived ammonium sulfate-precipitated monoclonal antibodies (mAb) anti-Thy-1.1 IgG (Cederlane, Ontario, Canada).
Anti-Thy-1.1 treated with 1,25(OH)2D3 group (group II)
From day -3 to day 14, 24 rats were given 1,25(OH)2D3 (Calcijex; Abbott, North Chicago, IL, USA) at the dose of 25 ng/100 g body wt/day. At day 0, rats received 0.5 mL of saline containing 400 g of ascites-derived ammonium sulfate-precipitated mAb anti-Thy-1.1 IgG.
Control group (group III)
At day 0, 12 rats were injected intravenously with 0.5 mL of vehicle.
Control group treated with 1,25(OH)2D3 (group IV)
From day -3 to day 14, 12 rats were given 1,25(OH)2D3 at the dose of 25 ng/100 g body wt/day. At day 0, they received 0.5 mL of vehicle.
Anti-Thy-1 antibody and vehicle were given by intrafemoral venous injection, whereas 1,25(OH)2D3 was administered subcutaneously by osmotic minipump (Charles River).
Measurements
Twenty-four–hour urine samples were collected with the aid of metabolic cages.
Proteinuria was measured daily by using the Bio-Rad Protein Assay (Bio-Rad Laboratories GmbH, München, Germany) and bovine serum albumin (Sigma Chemical Co., St. Louis, MO, USA) as a standard. Blood samples for urea, creatinine, calcium, phosphate were drawn from conscious rat via the caudal vein at days -3, 0, 4, 7, and 14 using ethylenediaminetetraacetic acid (EDTA) as the anticoagulant, and the plasma was stored below -20°C. Urinary calcium was determined daily. Blood and urinary samples were analyzed using an autoanalyzer (Beckman Instruments GmbH, Munchen, Germany).
Body weight was measured at regular intervals.
IL-6 urinary activity
Interleukin-6 activity was examined daily in the urine using a highly sensitive specific anti-rat enzyme-linked immunosorbent assay (ELISA; Biosource, Los Angeles, CA, USA) with an intra-assay and interassay variability of lower than 5%. The lower detection limit of the assay was 20 pg/mL. No cross-reactivity with recombinant TNF-, TNF-, IL-1 and IL-1, IL-2, IL-3, and IL-4 was observed when these cytokines were added to blank samples in concentrations of up to 50 ng/mL.
Morphologic studies
At the end of the experiment, the kidneys were removed: One half was snap-frozen in liquid nitrogen-cooled isopentane and stored at -70°C, and the other half was fixed in 10% neutral-buffered formalin and embedded in paraffin.
To investigate glomerular morphology, sections of 3 m of thickness were stained with periodic acid-Schiff (PAS) reagent and hematoxylin and eosin (HE). For each rat, all glomerular cross-sections present in a specimen were evaluated. Glomerular diameters of each kidney in a cross-section were measured by a graduated lens (
250).
Three micrometer thick frozen sections were cut and fixed in acetone, washed with phosphate-buffered saline (PBS), incubated with primary antibodies against anti-Thy-1.1 antibody, anti-rat C3 (ED11 antigen, MCA733 Serotec Ltd., UK) and anti-rat granulocytes His48 antigen (MCA967 Serotec Ltd.). Then the slides were incubated with FITC-conjugated antibodies (Sigma, MA, USA) specific for each primary antibody.
The slides were examined by a fluorescence microscope (Lietz, Wetzlar, Germany).
Immunohistochemical staining for proliferating glomerular cells with a biotinylated mouse antiproliferating cell nuclear antigen (PCNA; Zymed, South San Francisco, CA, USA) and for infiltrating monocytes with a mouse anti-rat ED1-mAB (MCA341R; Serotec). Briefly, 3 m renal section were microwaved in 0.01 mol/L citrate buffer, pH 6.0, for two 10-minute periods at 600 W to retrieve the antigens. The sections were then treated with 3% hydrogen peroxide in methanol for 10 minutes at room temperature to block endogenous peroxidase. Sections were subsequently blocked with 10% normal goat antiserum for 30 minutes at room temperature, and the primary antibodies were added followed by incubation at 37°C for two hours. Then sections were washed three times in PBS for 10 minutes. Anti-PCNA–treated section were incubated with streptavidin-peroxidase for 10 minutes, and then the reaction was detected with peroxidase substrate containing diaminobenzidine chromogen (DAB). Anti-ED1–treated sections were incubated with a biotinylated anti-mouse secondary antibody (100 L for 20 min at room temperature); then a substrate-chromogen mixture containing 3-amino-9-ethylcarbazole (AEC) was added to each section (10 min at room temperature).
Finally, all sections were counterstained with 100 L hematoxylin. For the negative controls, the specific antibodies were omitted.
To measure DNA nicking, a marker of apoptosis, the TUNEL assay (In situ cell death detection kit-POD; Roche Diagnostics S.p.A., Italy) was performed on formalin-fixed tissue. Terminal deoxynucleotidyl transferase (TdT) was omitted from the nucleotide mixture as a negative control. Tissue sections treated with DNAse to introduce DNA breaks in all nuclei were used as positive controls.
Calculation of cell number was performed by the investigators in a blinded fashion using coded samples until the analysis was complete. Results were expressed as the number of positive cells per glomerular cross section; the percentage of positive glomeruli for each kidney cross section was also calculated.
Statistical analysis
Analysis of variance and the Student–Neumann–Keul's t test were used for statistical analysis. Values are expressed as mean 
SD; the null hypothesis was rejected when P < 0.05.
RESULTS
Effects of 1,25(OH)2D3 pretreatment on proteinuria
During the course of the study, no animals showed clinically adverse effects. There was no difference in body weight, fluid intake, or urinary volume among the four groups of rats.
As shown in Figure 2a, the four groups had similar levels of protein excretion before the injection of anti-Thy-1.1 antibodies. Compared with control rats, anti-Thy-1.1 nephritic rats (anti-Thy-1.1 group) demonstrated frank proteinuria from day 2 to day 14. In nephritic rats in which 1,25(OH)2D3 was administered [anti-Thy-1.1 treated with 1,25(OH)2D3 group], a significant reduction of proteinuria was detected.
Figure 2.
Protein excretion in the four groups of rats before the injection of anti-Thy-1.1 antibodies. Symbols are: (
) anti-Thy-1.1; (
) anti-Thy-1.1 + vitamin D3; (
) controls; (
) controls + vitamin D3. (A) Compared with control rats, anti-Thy-1.1 nephritic rats (anti-Thy-1.1) demonstrated frank proteinuria from days 2 to 14 [ANOVA with Student-Neumann-Keul's multicomparison t test was performed: *P < 0.05 vs. control group; §P < 0.05 vs. anti-Thy-1.1 + 1,25(OH)2D3]. In nephritic rats in which 1,25(OH)2D3 was administered [anti-Thy-1.1 + 1,25(OH)2D3], a significant reduction of proteinuria was detected. The enhanced IL-6 urinary excretion (B) observed in nephritic rats at days 2, 3, 4, and 7 was significantly inhibited by 1,25(OH)2D3 administration.
IL-6 urinary excretion
The results regarding IL-6 urinary excretion in the four groups of rats are depicted in Figure 2b. IL-6 urinary excretion was significantly increased in nephritic rats at days 2, 3, 4, and 7 versus all groups.
1,25(OH)2D3 treatment significantly inhibited the enhanced IL-6 urinary excretion observed in nephritic rats at days 2, 3, 4, and 7 Figure 2b.
Renal function, serum calcium and phosphate, urinary calcium
Data regarding renal function at days 0, 4, 7, and 14 in all groups of rats are shown in Table 1. No significant variations in serum calcium and phosphate were observed at day 14 in 1,25(OH)2D3-treated rats. Urinary calcium (expressed as mg calcium/mg creatininuria) at day 14 showed no significant variation in all groups of rats: The mean values SD were, respectively, 0.47 0.20 in the control group, 0.54 0.29 in the control + 1,25(OH)2D3 group, 0.45 0.26 in the anti-Thy-1.1 group, and 0.60 0.28 in the anti-Thy-1.1 treated with 1,25(OH)2D3 group. A significant increase of plasma blood urea nitrogen and creatinine was observed in nephritic rats (anti-Thy-1.1 group) at day 4 (P < 0.01 vs. controls). In contrast, no variations in renal function were observed in nephritic rats treated with 1,25(OH)2D3 [anti-Thy-1.1 treated with 1,25(OH)2D3 group].
Morphological studies
Immunofluorescence studies
No significant difference in immunofluorescence staining for anti-Thy-1.1 antibodies and complement was detected in glomeruli of rats injected with anti-Thy-1.1 antibodies and treated [anti-Thy-1.1 treated with 1,25(OH)2D3 group] or untreated with 1,25(OH)2D3 (anti-Thy-1.1 group; Figure 3).
Figure 3.
No significant difference in immunofluorescence staining for anti-rat C3 antibody was detected in glomeruli of rats injected with anti-Thy-1.1 antibodies (anti-Thy-1.1;A) and of rats injected with anti-Thy-1.1 antibodies treated with 1,25(OH)2D3 (anti-Thy-1.1 + vitamin D3;B).
Full figure and legend (48K)Glomerular size and morphological findings
The size of glomeruli was evaluated by measuring the cross-sectional diameter. The mean glomerular diameter of rats receiving anti-Thy-1.1 antibody (anti-Thy-1.1 group) but not of rats treated with 1,25(OH)2D3 [anti-Thy-1.1 treated with 1,25(OH)2D3 group] was significantly increased in respect to the controls at days 1, 4, 7, and 14 Table 2. Moreover, the mean glomerular diameter of rats treated with 1,25(OH)2D3 [anti-Thy-1.1 treated with 1,25(OH)2D3 group] was significantly lower then that of rats injected with anti-Thy-1.1 antibody (anti-Thy-1.1 group). In untreated nephritic rats (anti-Thy-1.1 group) at day 4, most glomeruli showed mesangial cells destruction and aneurysms of the capillary loops.
Infiltration of inflammatory cells
A peak of neutrophil accumulation was detectable at day 4 by morphometric analysis of sections stained with His48 Figure 4. The number of neutrophils decreased in the following days.
Figure 4.
A peak of neutrophil accumulation was detectable at day 4 in rats receiving anti-Thy-1.1 antibody (anti-Thy-1.1) by morphometric analysis of sections stained with His48 (20 12 His48-positive cells/glomerulus; * P< 0.001 vs. control groups). Symbols are: () anti-Thy-1.1; () anti-Thy-1.1 + vitamin D3; () controls; () controls + vitamin D3. The number of neutrophils decreased in the following days (12 2 His48-positive cells/glomerulus at day 7 and 10 1 His48-positive cells/glomerulus at day 14; *P < 0.001 vs. controls groups). Treatment with 1,25(OH)2D3 (anti-Thy-1.1 + vitamin D3) completely abrogated the glomerular accumulation of His48-positive cells (§P < 0.001 nephritic rats vs. anti-Thy-1.1 + vitamin D3).
ED1-positive monocytes were significantly increased one day after injection of anti-Thy-1.1 antibodies, and after a transient reduction, their number peaked at day 7 and decreased thereafter Figure 5c. Treatment with 1,25(OH)2D3 [anti-Thy-1.1 treated with 1,25(OH)2D3 group] completely abrogated the glomerular accumulation of His48-positive polymorphonuclear cells. Whereas 1,25(OH)2D3 did not significantly reduce the early accumulation of ED1-positive monocytes observed at day 1, the number of glomerular monocytes was significantly reduced at day 7 Figure 5d.
Figure 5.
Immunohistochemical detection of ED1-positive monocytes in a kidney section of anti-Thy-1.1 nephritic rats (anti-Thy-1.1;A andC) and anti-Thy-1.1 nephritic rats treated with 1,25(OH)2D3 (anti-Thy-1.1 + vitamin D3;B andD) four days (A and B) and seven days (C and D) after antibody injection. ED1-positive nuclei are stained in black. The number of glomerular monocytes was significantly reduced at days 4 and 7 in the anti-Thy-1.1 + vitamin D3 group.
Full figure and legend (177K)Apoptosis and mesangial cell proliferation
By TUNEL, the first peak of apoptotic cells was detected at day 4, concomitantly with morphological aspects of mesangiolysis Figure 6. Figure 7 shows that the number of apoptotic cells was reduced in rats treated with 1,25(OH)2D3 [anti-Thy-1.1 treated with 1,25(OH)2D3 group] in respect to nephritic rats (anti-Thy-1.1 group). Apoptosis was reduced at day 7, and a second peak was detected at day 14 Figure 6 concomitantly with the reduction of mesangial cell proliferation detected by PCNA Figures 8 and 9a, c. Mesangial proliferation peaked at day 7 and then decreased thereafter. The early apoptotic response detected at day 4 was completely abrogated in 1,25(OH)2D3-treated rats (anti-Thy-1.1 treated with 1,25(OH)2D3 group; Figures 6 and 7b). The extent of proliferation was reduced overall in the treated group Figures 5b and 9b, d). However, at day 14, the extent of apoptosis was not different in treated [anti-Thy-1.1 treated with 1,25(OH)2D3 group] or untreated nephritic rats (anti-Thy-1.1 group).
Figure 6.
As shown by TUNEL, a first peak of apoptotic cells was detected at day 4 in the anti-Thy-1.1 group (* P < 0.01 nephritic rats vs. control groups). Symbols are: () anti-Thy-1.1; () anti-Thy-1.1 + vitamin D3; () controls; () controls + vitamin D3. Apoptosis was reduced at day 7, and a second peak was detected at day 14 concomitantly with the reduction of mesangial cell proliferation detected by PCNA (*P < 0.01 nephritic rats vs. control groups). The early apoptotic response detected at day 4 was completely abrogated in 1,25(OH)2D3-treated rats (anti-Thy-1.1 + vitamin D3, §P < 0.01 nephritic rats vs. anti-Thy-1.1 + vitamin D3). However, at 14, the extent of apoptosis was not different in treated (anti-Thy-1.1 + vitamin D3) or untreated nephritic rats (anti-Thy-1.1), suggesting that the apoptosis involved in the regression of glomerular proliferation was not affected by treatment with 1,25(OH)2D3.
Figure 7.
Detection of apoptotic cells by TUNEL in kidney sections of anti-Thy-1.1 nephritic rats (anti-Thy-1.1;A) and anti-Thy-1.1 nephritic rats treated with 1,25(OH)2D3 (anti-Thy-1.1 + vitamin D3;B) four days after antibody injection. Positive nuclei are stained in black. An important reduction of apoptotic cells was detected in the anti-Thy-1.1 + vitamin D3-treated rat.
Full figure and legend (55K)Figure 8.
An important reduction of mesangial cell proliferation assessed by proliferating cell nuclear antigen (PCNA) was detected in anti-Thy-1.1 + vitamin D3-treated rats versus nephritic rats after 4, 7, and 14 days from antibody injection. Symbols are: () anti-Thy-1.1; () anti-Thy-1.1 + vitamin D3; () controls; () controls + vitamin D3. Mesangial proliferation peaked at day 7 and decreased to day 14 (*P < 0.01 vs. control groups; §P < 0.05 vs. anti-Thy-1.1 + 1,25(OH)2D3).
Figure 9.
Immunohistochemical detection of the proliferation marker, PCNA, in kidney sections of anti-Thy-1.1 nephritic rats (anti-Thy-1.1) and anti-Thy-1.1 nephritic rats treated with 1,25(OH)2D3 (anti-Thy-1.1 + vitamin D3) 7 (A andB) and 14 (C andD) days after antibody injection. PCNA-positive nuclei are stained in black. An important reduction of mesangial cell proliferation assessed by PCNA was detected in anti-Thy-1.1 + vitamin D3-treated rats after seven and 14 days (B and D).
Full figure and legend (239K)DISCUSSION
Rat mesangial cells express the thymocyte anti-Thy-1.1. The administration of anti-Thy-1.1 antibodies induces a mesangial proliferative glomerulonephritis19,20. The early pathological events are characterized by mesangiolysis and accumulation within hours after antibody injection of polymorphonuclear neutrophils and monocytes into the glomeruli21,22. Early mesangiolysis is followed by mesangial proliferation mediated by a complex interplay of several proinflammatory mediators and growth factors. The mesangial alterations are followed by a recovery phase that is characterized by capillary angiogenesis, matrix remodeling, and the restoration of normal glomerular cellularity by apoptosis of inflammatory and excess glomerular mesangial cells23,24.
This study provides evidence that 1,25(OH)2D3 can attenuate proteinuria, inflammation, and glomerular hypercellularity in anti-Thy-1.1 nephritis.
Few studies have investigated the effects of 1,25(OH)2D3 on the development of glomerular pathology. Branisteanu et al induced active Heymann nephritis in Lewis rats treated with 1,25(OH)2D317. In their model, 1,25(OH)2D3 (0.5 g/kg body wt) was administered on alternate days during the first 13 days after active immunization significantly reduced proteinuria. This reduction of proteinuria was comparable to our observation in rats treated with CsA (20 mg/kg) on alternate days. These results were interpreted as an immunosuppressive effect of 1,25(OH)2D3, which is supported by several studies indicating an immunosuppressive activity of 1,25(OH)2D33,4,5,6,7. The inhibition of dendritic cell differentiation and maturation, as well as the modulation of their activation and survival leading to T-cell hyporesponsiveness may explain the immunosuppressive activity of 1,25(OH)2D39.
Moreover, in a rat model of subtotal nephrectomy, Schwarz et al demonstrated that 1,25(OH)2D3 possesses an antiproliferative action during the compensatory growth of nephrons in response to subtotal nephrectomy18. In their model, 1,25(OH)2D3 reduces renal cell proliferation and glomerular growth as well as glomerulosclerosis and albuminuria as indicators of progressive glomerular damage. They concluded that 1,25(OH)2D3 has antiproliferative actions during the compensatory growth of nephrons in response to subtotal nephrectomy, and that this effect is independent of parathyroid hormone (PTH).
The results of our study provide evidence that 1,25(OH)2D3 also may display an anti-inflammatory action that protects the model of anti-Thy-1.1–induced nephritis against immune-mediated glomerular injury, by using a passive administration of an antibody that is reactive with mesangial cells.
In this model, an antibody and complement-dependent early mesangiolysis occurs22. We also observed an increased apoptosis in concomitance with mesangiolysis. These events were associated with an early recruitment of inflammatory cells, which include a transient accumulation of neutrophils and a more persistent infiltration of monocytes.
The role of neutrophils in the process of glomerular damage in this model is considered to be of minor relevance, as shown by neutrophil depletion studies25. In contrast, macrophages are considered instrumental in the development of glomerular alterations, since they produce both nitric oxide (NO) and reactive oxygen species that may induce apoptosis and mediate mesangiolysis26. Moreover, secretion from macrophages of growth factors and cytokines may contribute to mesangial cell proliferation and accumulation of mesangial matrix that occurs in the late stage of anti-Thy-1.1–induced glomerular injury.
The results of the present study demonstrate that the antiproteinuric effect of 1,25(OH)2D3 is associated with a reduction in mesangiolysis and early apoptosis, and in the glomerular inflammatory process. Indeed, apoptotic cells detected by TUNEL were significantly inhibited at day 4 in rats treated with 1,25(OH)2D3. Moreover, the inhibition of urinary excretion of IL-6 and the accumulation of neutrophils and monocytes within the glomeruli suggest that 1,25(OH)2D3 affects glomerular inflammation in Thy-1.1-induced injury. In particular, 1,25(OH)2D3 prevents the early accumulation of neutrophils, suggesting an action at the step of recruitment of these cells. In contrast, 1,25(OH)2D3 does not abrogate the early accumulation of monocytes but favors their disappearance from glomeruli, suggesting that its action is that of cell activation rather than recruitment. A decreased production of pro-inflammatory cytokine, which is suggested by the early reduction of IL-6, may reflect an inhibitory action of 1,25(OH)2D3 on monocyte activation, such as that previously seen for dendritic cells9. These observations should stimulate further in vitro studies to investigate the differential effects of 1,25(OH)2D3 on different populations of inflammatory cells.
In agreement with the results obtained by Schwarz et al in a model of subtotal nephrectomy, a significant inhibition of mesangial proliferation in rats treated with 1,25(OH)2D3 was observed18. Indeed, as detected by PCNA staining, glomerular proliferation was reduced in Thy-1.1 nephritic rats treated with 1,25(OH)2D3. These antiproliferative effects may depend either on a direct action of 1,25(OH)2D3 on mesangial cells (abstract; Ooi et al, J Am Soc Nephrol 102:2107, 1998)27 or on the inhibitory effects on monocyte accumulation and/or activation. Several studies suggest that inhibition of monocyte accumulation prevents mesangial cells proliferation and matrix expansion in anti-Thy-1.1 glomerular injury28.
The anti-Thy-1.1 model is transient, and glomeruli can recover from the massive cell proliferation and mesangial matrix expansion. Shimizu et al showed that in anti-Thy-1.1 glomerulonephritis, apoptosis has a critical role in the regression of glomerular injury leading to the disappearance of the inflammatory cells and reduction in number of proliferating mesangial cells24. In recent years, apoptosis has been implicated in the resolution of the inflammatory response. Darby, Skalli, and Gabbiani reported that myofibroblasts can undergo apoptosis during experimental wound healing29. Therefore, the observation of Shimitzu et al in anti-Thy-1.1 glomerulonephritis supports the contention of previous authors30,31,32 that apoptosis may favor recovery from glomerular hypercellularity24.
Using the TUNEL technique we observed that apoptosis was significantly increased in anti-Thy-1.1 injected rats at day 14, which was concomitant with the reduction of glomerular hypercellularity. In rats treated with 1,25(OH)2D3, this late increase in apoptosis remained intact, demonstrating that the effect of 1,25(OH)2D3 observed in the early apoptotic changes (day 4) was not dependent on an anti-apoptotic action, but rather on the anti-inflammatory action of the drug.
In conclusion, our study suggests that 1,25(OH)2D3 attenuates proteinuria as well as morphological and functional alterations occurring in the anti-Thy-1.1–induced GN as a result of an anti-inflammatory action. Future studies are needed to evaluate the mechanisms by which 1,25(OH)2D3 is able to reduce inflammatory cell infiltration and glomerular proliferation, and accelerate the resolution of glomerular injury.
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Acknowledgments
This work was supported by the National Research Council (CNR), Targeted Project Biotechnology to G.C. We thank Mr. Pierluigi Frangioni (immunohistochemestry and light microscopy) and Mr. Leonardo Casarosa (immunofluorescence and proteinuria determination) for their skilled technical assistance.
