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【摘要】 1,25(OH) 2 D 3 has antiproliferative effects and promotes cell differentiation. This consideration has provided the rationale for studies in subtotally nephrectomized rats showing that 1,25(OH) 2 D 3 interferred with glomerulosclerosis. The cellular mechanisms involved have remained obscure, however. It was the purpose of the present study to assess glomerular structure and cellular composition in subtotally nephrectomized (SNX) rats treated with nonpharmacological doses of 1,25(OH) 2 D 3. Male Sprague-Dawley rats were sham operated (sham) or underwent SNX under general anesthesia and received either solvent or 1,25(OH) 2 D 3 (3 ng·100 g body wt -1 ·day -1 sc). Blood pressure (BP) and albuminuria were measured. After 16 wk, the remnant renal tissue was perfusion fixed and morphometric and stereological measurements were carried out. The expression of proliferating cellular antigen (PCNA), cyclin-dependent kinase inhibitor p27, Wilms tumor gene (WT1), and desmin, a marker of early podocyte damage, was investigated by immunohistology. BP, serum creatinine, and urinary albumin excretion were significantly higher in SNX than in sham rats. Albuminuria was significantly lower in SNX+1,25(OH) 2 D 3 compared with SNX+solvent rats. Mean glomerular tuft volume was significantly higher in SNX+solvent (2.69 ± 0.21 g x 10 6 µm 3 ) than in sham rats (1.44 ± 0.17 and 1.28 ± 0.14 x 10 6 µm 3 ); it was significantly ( P < 0.05) lower in SNX+1,25(OH) 2 D 3 rats (1.81 ± 0.16 x 10 6 µm 3 ). The main finding was a significantly higher number of podocytes in SNX+1,25(OH) 2 D 3 (88 ± 9) and sham (98 ± 17) compared with SNX+solvent rats (81 ± 8.7). In parallel, the increase in podocyte volume in SNX+solvent rats was abrogated by treatment with 1,25(OH) 2 D 3, and immunohistochemistry revealed less expression of desmin, PCNA, and p27, suggesting less podocyte injury and activation of the cyclin cascade. This study identifies the podocyte as an important target cell for the renoprotective action of 1,25(OH) 2 D 3. This notion is suggested by less evidence of podocyte injury, decreased podocytes loss, and abrogation of podocyte hypertrophy, findings that may also explain less pronounced albuminuria and glomerulosclerosis.
【关键词】 visceral glomerular epithelial cell mesangial cell glomerular endothelial cell progression
THE VITAMIN D ENDOCRINE SYSTEM exerts numerous pleiotropic effects unrelated to its traditionally recognized function of regulating plasma calcium levels ( 7, 31, 53 ). Previous studies in this laboratory documented that 1,25(OH) 2 D 3 treatment attenuated the development of glomerulosclerosis and progression of albuminuria in subtotally nephrectomized (SNX) rats ( 45 ). This observation is in line with several studies assessing the role of active vitamin D in nonimmune ( 14 ) and immune-mediated models of injury ( 2, 24, 33, 35 ).
These studies found reduced mesangial cell proliferation, glomerulomegaly, and development of glomerulosclerosis by administration of 1,25(OH) 2 D 3. Consequently, the action of 1,25(OH) 2 D 3 was attributed to prevention of mesangial cell proliferation ( 14, 45 ).
There has been increasing recognition of an important role for podocytes in the progression of renal disease ( 27 ). It has recently become clear that initial glomerular injury affects the podocyte as an important target cell for progression ( 21 ), e.g., in diabetic nephropathy ( 5, 10 ), fawn hooded rats ( 20 ), or SNX rats ( 1 ). This consideration and the fact that podocytes possess receptors for 1,25-dihydroxyvitamin D 3 ( 49 ) prompted the present study to assess the action of 1,25(OH) 2 D 3 on podocytes in the SNX rat. As signs of podocyte injury, we assessed podocyte number, podocyte volume, and podocyte ultrastructure as well as changes in the pattern of structural molecules and de novo expression of desmin. De novo expression of desmin has recently been recognized as a sensitive early marker of podocyte injury ( 8, 16 ). Expression of proliferating cell nuclear antigen (PCNA) in podocytes as postmitotic cells does not necessarily indicate cell proliferation but rather activation of the cyclin cascade ( 15 ). We also studied expression of the cyclin-dependent kinase (CDK) inhibitory protein p27 because active vitamin D has been shown to upregulate expression of p27 in numerous cell lines, e.g., retinoic acid-resistant acute promyelotic leukemia cell lines (UF-1) ( 28 ), squamous carcinoma cell lines of the head and neck ( 12 ), and in human breast cancer MCF-7 cells ( 50 ). Furthermore, it has recently been shown that subtle injury to postmitotic podocytes leads not only to an abortive upreguation of the cyclin cascade but also to strong upregulation of p21 and p27 ( 27, 36 ). The importance of these proteins in preventing podocyte injury is illustrated by the observation in standard models of renal injury of more rapid progression in p21 and p27 knockout mice ( 18, 26 ).
MATERIALS AND METHODS
Animals
Twenty-nine male Sprague-Dawley rats (SD; 200 g; Ivanovas, Kisslegg, Germnay) were housed in single cages at a constant room temperature (20°C) and humidity (25%). The animals received a high-protein (25%) diet with 0.5% NaCl (Altromin, Lage, Germany). After 3 days of adaptation, the animals were randomly allocated to a surgical two-step SNX of moderate intensity or to a sham operation (sham). In the first operation, the right kidney was removed, and 7 days later cortical tissue of the hypertrophied left kidney was resected (see below). Treatment with either solvent (ethanol) or 1,25(OH) 2 D 3 (3 ng·100 g body wt -1 ·day -1 ) was started on the day of the second operation by subcutaneous implantation of osmotic minipumps. After 4 wk the minipumps had to be exchanged under general anesthesia (see below). Blood pressure was measured at the beginning of the experiment and subsequently every second week by tail plethysmography in conscious animals. After 2, 4, and 8 wk, urinary albumin excretion was measured in solvent- and 1,25(OH) 2 D 3 -treated sham and SNX animals using a sandwich ELISA system ( 45 ). The experiment was terminated using perfusion fixation (see below).
Partial Renal Ablation by Surgical SNX
The right kidney was removed and weighed under ketamine/diazepam anesthesia (100 mg/kg or 2.5 µg/kg, respectively). After 7 days, we partially resected the cortex of the left kidney, corresponding to two-thirds of the weight of the right kidney. Control animals were sham resected by removal of the kidney capsule. Special care was taken to avoid injury to the adrenals (for details, see Ref. 45 ).
Experimental Protocol
The animals were randomly allocated to the following experimental groups:
Sham-operated+solvent (sham+solvent; n = 8)
Sham-operated+1,25(OH) 2 D 3 [sham+1,25(OH) 2 D 3; n = 7]
SNX+solvent (SNX+solvent; n = 6)
SNX+1,25(OH) 2 D 3 [SNX+1,25(OH) 2 D 3; n = 8]
For immunohistochemical investigations (see below), five additional SNX+solvent and five SNX+1,25(OH) 2 D 3 animals were examined.
Perfusion fixation and tissue sampling. Sixteen weeks after the second operation, the experiment was terminated by retrograde perfusion fixation. The abdominal aorta was catheterized under ketamine/diazepam anesthesia (doses as above), and blood samples were taken for determination of serum parameters. The viscera were rinsed with 10% dextran solution containing 0.5 g/l procain hydrochloride for 2 min. Ten seconds after aortic perfusion was started, the vena cava was incised to drain the blood. After dextran infusion, the vascular system was perfused with 0.2 M phosphate buffer containing 3% glutaraldehyde (12 min) for morphometric and stereological measurements. For immunohistochemistry, five additional SNX+solvent and SNX+1,25(OH) 2 D 3 animals were perfused using ice-cold NaCl as the perfusion medium. The kidneys were taken out, weighed, and dissected in a plane perpendicular to the interpolar axis, yielding 1-mm-thick slices. Ten small pieces of each kidney (2 x 2 x 1 mm) were selected by area weight, sampling for embedding in Epon-Araldite. Five of the resin blocks were randomly chosen, from which semithin sections (0.8 µm) were prepared and stained with methylene blue and basic fuchsin. For qualitative electron microscopic investigations, several ultrathin sections (0.08 µm) per animal were prepared and stained with uranyl acetate and lead citrate. Sections were then qualitatively investigated using a Zeiss EM 10 (Zeiss, Oberkochen, Germany) at various magnifications.
For immunohistochemical investigations, the kidney was fixed with buffered formalin and embedded in paraffin, and 3-µm-thick sections were cut.
Morphometric and Stereological Measurements
Mean glomerular tuft area. Area ( A A ) and volume density (V V ) of the renal cortex and medulla as well as the number of glomeruli per area ( N A ) were measured using a Zeiss eyepiece (Integrationsplatte II; Zeiss) and the point-counting method (P P = A A = V V ) at a magnification of x 400 ( 1, 54, 55 ). The total area of the glomerular tuft ( A T ) was determined as A T = A AT x A cortex ( 1 ). The number of glomeruli per volume ( N V ) and the volume density (V V ) of glomeruli was determined using the formula N V = k/ x N A 1.5 /V V 0.5 with k = 1.1 and = 1.382 ( 1 ). The total number of glomeruli was derived from the total volume of the renal cortex and the number of glomeruli per cortex volume: N glom = N V x V cortex. The mean glomerular tuft volume (v glom ) was determined according to v glom = / k x A T 1.5, with = 1.382 and k = 1.1 ( 1, 45 ).
Glomerular capillaries and cells. All investigations of semithin sections were performed in a blinded manner using a magnification of x 1,000 and oil immersion as described ( 1 ). In brief, the length density ( L V ) of glomerular capillaries was determined according to the standard stereological formula L V = 2 Q A (with Q A being the number of capillary transsects per area of the glomerular tuft). The total area of glomerular capillaries was derived from A A x A T. The filtration area (FF) was calculated according to the total area of glomerular capillaries times total the number of glomeruli per kidney. The total number of glomeruli per kidney was determined in paraffin sections as described above.
Glomerular cellularity (podocytes, mesangial, and endothelial cells) was assessed using a 100-point Zeiss eyepiece (Integrationsplatte II) and the point-counting method as described by Weibel ( 54 ) in at least 20 glomeruli/animal at a magnification of x 1,000. Thus V V and N A were determined and the number of cells per volume ( N V ): N V = k / x N A 1.5 /V V 0.5 with = 1.5 for podocytes and =1.4 for mesangial and endothelial cells and k =1 ( 1, 9, 11, 40, 54, 55 ).
The number of cells per glomerulus ( N glom ) was then derived from N glom = N V x v glom. The mean cell volume (v C ) was determined from volume density, mean glomerular volume, and mean number of cells per glomerulus: v C = V V x v glom / N glom.
Immunohistological Investigations
Immunohistochemistry was performed in solvent- and in 1,25(OH) 2 D 3 -treated SNX animals with the following antibodies: PCNA (dilution 1:500; DAKO, Hamburg, Germany), p27 (rabbit polyclonal antibody; dilution 1:500; Santa Cruz Biotechnology, Heidelberg, Germany), and WT1 (rabbit polyclonal; dilution 1:300; Santa Cruz Biotechnology). The streptavidin-biotin method with fast red or diaminobenzidin (DAB) as chromogen was used as described in detail ( 10, 45 ). Negative controls were performed by omitting the primary antibody. To reduce staining variability, all sections are stained in one procedure. In 50 glomeruli/kidney, the number of PCNA+, p27+, and WT1+ cells of the entire glomerulus as well as the number of PCNA+ cells per glomerular and per tubulointerstitial area were counted (magnification x 400). In addition, immunohistochemical staining for desmin was performed (mouse monoclonal antibody, dilution 1:400; DAKO) and investigated as a marker of podocyte degeneration or activation, respectively. For analysis of desmin immunohistochemistry, the capillary tuft was divided into 4 quarters and the following scoring system was used: 0, no expression; 1, desmin-positive cells in 1 quarter; 2, desmin-positive cells in 2 quarters; 3, desmin-positive cells in 3 quarters; and 4, desmin-positive cells all over the capillary tuft. The damage score was then calculated as {[(0 - 0 score) + score 1 + score 2 + score 3 + score 4]:5} +15 ( 10 ).
Statistics
All data are expressed as means ± SD. All variables were tested for normal distribution by the Kolmogorov-Smirnov test. One-way ANOVA was performed, followed by the least significant difference test to assess differences between groups. All statistical tests were two tailed, and a significance level of = 0.05 or less was used.
RESULTS
Description of the Animal Model
Body weight was significantly higher in sham+1,25(OH) 2 D 3 than in sham+solvent and SNX+1,25(OH) 2 D 3 rats ( Table 1 ). The weight of the left kidney (or remnant kidney) was significantly higher in SNX+solvent than in sham+solvent rats. Systolic blood pressure was comparable in all animals before the operation (115-120 mmHg). At the end of the study, it was significantly higher in SNX than in sham rats but did not differ in SNX+solvent and SNX+1,25(OH) 2 D 3 rats, indicating that 1,25(OH) 2 D 3 had no effect on blood pressure. The relative heart weight (heart weight-to-body weight ratio) was also significantly higher in SNX+solvent (3.02 ± 0.54 mg/g) compared with both sham-operated groups [sham+solvent: 2.60 ± 0.3 mg/g and sham+1,25(OH) 2 D 3 : 2.56 ± 0.33 mg/g, respectively]; it was not significantly different in SNX+1,25(OH) 2 D 3 compared with SNX+solvent rats. The urinary albumin excretion rate rose progressively over time in solvent- and 1,25(OH) 2 D 3 -treated SNX compared with stable values in sham-operated rats (values <0.1 mg/24 h); after 8 wk, it was significantly higher ( P < 0.05) in SNX+solvent (5.4 ± 3.89 mg/24 h) than in SNX+1,25(OH) 2 D 3 rats (0.95 ± 0.45 mg/24 h). Serum creatinine and urea concentrations were significantly higher in both SNX groups than in sham rats; there was no difference between solvent- and 1,25(OH) 2 D 3 -treated SNX rats. Serum Ca was significantly lower in SNX+solvent than in the three other groups. The highest serum Ca value in one animal of the SNX+1,25(OH) 2 D 3 group was still in the normal range, i.e., 2.56 mmol/l. Serum 1,25(OH) 2 D 3 concentration was three times higher ( P < 0.01) in SNX+ 1,25(OH) 2 D 3 than in SNX+solvent rats. Hemoglobin and hematocrit were not significantly different between the experimental groups (data not shown).
Table 1. Animal data
Effect of 1,25(OH) 2 D 3 Treatment on Glomerular Capillary Density
The length density of glomerular capillaries, i.e., the length of all capillaries per glomerular tuft volume, was significantly lower in SNX+solvent compared with both sham groups. It was higher and no longer different from sham when SNX rats were treated with 1,25(OH) 2 D 3 ( Fig. 1 ). This finding is in agreement with the previous finding ( 45 ) of less glomerulosclerosis after treatment with 1,25(OH) 2 D 3 ( Fig. 2 ), because less glomerulosclerosis implies more open capillaries per area of glomerular tuft. Higher glomerular capillary length density was seen in SNX+1,25(OH) 2 D 3 compared with SNX+solvent rats ( Fig. 1 ). The total length of glomerular capillaries was significantly less in SNX+1,25(OH) 2 D 3 than in sham+solvent and SNX+solvent rats ( Table 2 ). This was due to the fact that glomerular enlargement in SNX+solvent rats was partly prevented by 1,25(OH) 2 D 3 treatment ( Fig. 2 ).
Fig. 1. Effect of 1,25(OH) 2 D 3 treatment on length density ( L V ) of glomerular capillaries. L V was significantly lower in subtotally nephrectomized (SNX)+ solvent-compared with sham-operated (sham) and SNX+1,25(OH) 2 D 3 -treated Sprague-Dawley rats. * P < 0.05 vs. sham groups. + P < 0.05 vs. SNX+solvent rats.
Fig. 2. Effect of 1,25(OH) 2 D 3 treatment on glomerulosclerosis. Note that mesangial matrix expansion ( * ), enlargement of podocytes with protein deposition (arrowhead), and adhesion to Bowman's capsule (arrow) in solvent-treated SNX rats ( A ) were prevented when SNX rats were treated with 1,25(OH) 2 D 3 ( B ). Magnification: x 1,000.
Table 2. Effect of treatment with 1,25(OH) 2 D 3 on glomerular capillaries
Effect of 1,25(OH) 2 D 3 Treatment on Podocytes
Mean podocyte number per glomerulus was significantly lower in SNX+solvent than in both sham groups indicating loss of podocytes ( Table 3 ). This was not seen when SNX rats were treated with 1,25(OH) 2 D 3. The decrease in podocyte number per glomerulus was paralleled by a significantly ( P < 0.001) lower number of WT1-positive cells per glomerulus in SNX+solvent (4.92 ± 0.11) compared with SNX+1,25(OH) 2 D 3 rats (7.0 ± 0.13). In parallel, mean podocyte volume was significantly higher in SNX+solvent than in both sham groups and in SNX+1,25(OH) 2 D 3 rats. This finding indicates that hypertrophy of podocytes occurs after SNX. This was prevented by treatment with 1,25(OH) 2 D 3.
Table 3. Effect of 1,25(OH) 2 D 3 on glomerular cells
The number of PCNA-positive glomerular cells was significantly ( P < 0.05) higher in solvent-treated SNX (2.10 ± 1.64) compared with SNX+1,25(OH) 2 D 3 rats (0.47 ± 0.29). In contrast, the number of cells, mainly podocytes, staining positive for the cell cycle-inhibitory protein p27 was significantly ( P < 0.01) higher in SNX+1,25(OH) 2 D 3 (4.44 ± 1.24) than in SNX+solvent rats (2.6 ± 1.08). The immunohistological staining score of podocytes for desmin, a marker of podocyte damage, was significantly ( P < 0.05) lower in SNX+ 1,25(OH) 2 D 3 (13.4 ± 4.94) than in SNX+solvent rats (18.3 ± 4.22). These findings are illustrated in Fig. 3.
Fig. 3. Glomerular desmin staining as a marker of podocyte damage. Desmin staining is much more intensive in solvent-treated SNX ( A ) compared with 1,25(OH) 2 D 3 -treated SNX rats ( B ), indicating preservation of podocyte morphology after 1,25(OH) 2 D 3 treatment. Magnification: x 1,000.
Light microscopy and in particular semithin sections ( Fig. 4 ) documented hypertrophy and degeneration of podocytes with more frequent synechia, i.e., adhesion of the capillary tuft to Bowman's capsule. Electron microscopic investigations using ultrathin sections ( Fig. 5 ) confirmed podocyte hypertrophy and revealed signs of podocyte degeneration (i.e., cytoplasmic vacuolization, pseudocyst formation, loss or flattening of foot processes, bridging of podocyte cytoplasm) in solvent-treated SNX ( Fig. 5, B and E ) compared with sham-operated animals ( Fig. 5, A and D ). As a consequence, denudation of glomerular basement membrane and tuft adhesion to Bowman's capsule were noted ( Fig. 5 E ). These alterations were not seen in the treated SNX animals ( Fig. 5, C and F ). In addition, mesangial cell hyperplasia with mesangial matrix deposition was present in solvent-treated SNX ( Fig. 5 B ) compared with sham rats ( Fig. 5 A ), which were prevented by 1,25(OH) 2 D 3 ( Fig. 5 C ). Thus, in summary, glomerular ultrastructure was markedly preserved when SNX rats were treated with 1,25(OH) 2 D 3 ( Fig. 5 D ).
Fig. 4. Effect of 1,25(OH) 2 D 3 treatment on glomerular cell morphology and capillary length density in sham+solvent ( A ), sham+ 1,25(OH) 2 D 3 ( B ), SNX+solvent ( C ), and SNX+1,25(OH) 2 D 3 rats ( D ). Note glomerular enlargement with mesangial cell hyperplasia, mesangial matrix expansion ( * ), as well as hypertrophy and degenerative changes in podocytes (arrow) in SNX+solvent ( C ) compared with sham+solvent ( A ), sham+ 1,25(OH) 2 D 3 ( B ), and SNX+1,25(OH) 2 D 3 rats ( D ). Semithin sections are shown. Magnification: x 750.
Fig. 5. Representative electronmicrographs of sham+solvent ( A and D ), SNX+solvent ( B and E ), and SNX+1,25(OH) 2 D 3 ( C and F ) animals. A - C : overview of glomerular ultrastructure. Mesangial cell hyperplasia, mesangial matrix deposition ( * ), and podocyte changes are present in SNX+solvent ( B ) rats compared with the normal morphology in sham-operated animals ( A ) and were almost completely prevented by treatment of SNX with 1,25(OH) 2 D 3 ( C ). D and E : details of podocyte changes (original magnification: x 3,000). Enlargement and degenerative changes in podocytes (foot process fusion, pseudocyst formation, cytoplasmic inclusions, etc.) are shown, leading to retraction of podocytes and denudation of the glomerular with subsequent adhesion to Bowman's capsule ( E ) compared with the normal morphology of podocytes in sham-operated animals ( D ). Podocyte morphology is largely prevented in 1,25(OH) 2 D 3 -treated SNX animals ( F ).
Effect of 1,25(OH) 2 D 3 on Mesangial and Endothelial Cells
The number of mesangial cells and endothelial cells per glomerulus was significantly higher in SNX+solvent compared with both sham groups, indicating mesangial and endothelial cell hyperplasia. Both mesangial and endothelial cell hyperplasia were prevented in SNX+1,25(OH) 2 D 3 rats. The mean mesangial and endothelial cell volumes were not significantly different among the four groups [mesangial cell volume: 2,407 ± 248 µm 3 in sham+solvent, 2,443 ± 474 µm 3 in sham+1,25(OH) 2 D 3, 2,204 ± 560 µm 3 in SNX+solvent, and 2,228 ± 209 µm 3 in SNX+1,25(OH) 2 D 3; endothelial cell volume: 1,250 ± 120 µm 3 in sham+solvent, 1,283 ± 499 µm 3 in sham+1,25(OH) 2 D 3, 1,034 ± 250 µm 3 in SNX+solvent, and 1,145 ± 266 µm 3 in SNX+1,25(OH) 2 D 3 ]. The changes in glomerular cellularity and capillarization are illustrated in Fig. 4.
DISCUSSION
The present study confirms previous findings of this ( 25, 45 ) and other laboratories ( 14, 24 ) of less glomerular damage in 1,25(OH) 2 D 3 -treated rats with renal injury.
These findings are extended by using more sophisticated stereological methodology, e.g., length density of glomerular capillaries as an index of open capillaries per volume of the glomerular tuft. The in vivo relevance was also documented by a significant effect of 1,25(OH) 2 D 3 on the urinary albumin excretion rate. Our results confirm previous observations that the mesangial cell number (as a result of glomerular cell proliferation) is less in SNX rats treated with active vitamin D compared with solvent-treated SNX rats.
The novel finding of this study is that the decrease in podocyte number, the increase in podocyte volume, as well as immunohistochemical and ultrastructural indexes of podocyte injury were markedly less pronounced in SNX rats treated with 1,25(OH) 2 D 3 compared with solvent-treated SNX rats. These findings identify the podocyte as a direct or indirect target for the renoprotective action of 1,25(OH) 2 D 3.
The podocyte has recently been recognized as an important mediator in the progression of glomerular damage ( 21, 27 ). This is illustrated by clinical observations of podocyte loss in patients with progressive renal diseases, e.g., type 2 ( 32 ) or type 1 diabetes ( 47 ), and patients with lupus nephritis and focal segmental glomerulosclerosis ( 51 ). There is also abundant experimental evidence that the progression of renal injury is associated with progressive podocyte loss ( 10, 21 ). It is remarkable that even in the present short-term study, a significant effect of vitamin D on podocyte number could be observed.
Podocytes are known to express vitamin D receptors ( 48, 49 ). This observation would be consistent with a direct effect of 1,25(OH) 2 D 3 on podocytes. However, we cannot exclude the following alternative explanation: less expansion of glomerular volume diminishes the domain that has to be covered by postmitotic podocytes, leading to denudation of the basement membrane, formation of glomerular adhesions, and thus to the induction of progressive segmental glomerulosclerosis ( 9, 21, 41 ).
It is unclear why the number of podocytes decreases in the SNX animals, and by which mechanisms the decrease of podocyte number was abrogated by 1,25(OH) 2 D 3. In principle, podocyte loss may be caused both by apoptosis ( 44 ) and by detachment ( 27 ) through interruption of podocyte linkage to the basement membrane via 3 1 -integrins ( 4, 6 ) and dystroglycan ( 38, 39 ), respectively. Evidence for detachment is provided by the observation that vital podocytes can be cultured from the urine of animals ( 19 ) or patients ( 13 ) with progressive renal disease. With respect to apoptosis, a particular comment is appropriate: it is known that activation of the vitamin D receptor (VDR) by WT1 mediates apoptosis in mouse embryonic kidney cortex. Strong interaction between WT1 and VDR is possible because the VDR contains a WT1 consensus binding site ( 22, 52 ). The metanephric blastema and podocyte precursors in immature glomeruli as well as mature podocytes express WT1 ( 37 ). In view of the action of 1,25(OH) 2 D 3 in the embryonic kidney, where 1,25(OH) 2 D 3 promotes apoptosis, it is of particular note that in the adult rat kidneys investigated in this study, if anything, 1,25(OH) 2 D 3 prevented apoptosis (although we did not study apoptosis directly). The relevance of the 1,25(OH) 2 D 3 -reduced apoptosis in the embryonic kidney is particularily puzzling because in VDR knockout mice or vitamin D binding protein knockout mouse, no abnormality of renal structure was noted ( 17, 43 ). This discrepancy is unexplained, but there have been speculations on a role of nonreceptor-mediated mechanisms and potential alternative vitamin D metabolites.
Injury to podocytes in the solvent-treated animals is suggested by the findings of abnormal ultrastructure with signs of cell activation and degeneration (i.e., increased cytoplasmic vacuolization, pseudocyst formation, loss or flattening of foot processes, bridging of podocyte cytoplasm, etc.), increased podocyte volume, changed cytoskeleton expression pattern with appearance of desmin, and expression of PCNA. The latter observation may appear paradoxical in view of the fact that podocytes are postmitotic cells ( 30 ). However, it has recently been shown that PCNA expression may reflect cell activation rather than proliferation with full completion of the cell cyle ( 15 ). This interpretation is rendered more likely by the present observation of upregulation of p27 in glomerular cells. The increased glomerular expression of the cell cycle-inhibitory compound p27 after 1,25(OH) 2 D 3 treatment is in line with observations in lymphocytes ( 34 ). It further confirms several studies which showed that 1,25(OH) 2 D 3 inhibits cell proliferation in malignant ( 29 ) as well as nonmalignant cells ( 3 ). This effect of 1,25(OH) 2 D 3 ( 23 ) and 1,25(OH) 2 D 3 analogs ( 34, 46 ) is dependent on the presence of the VDR. It is mediated by an increased expression of the CDK-inhibitory proteins p21 and p27, respectively, inducing a G 1 block in the cell cycle secondary to reduction of CDK2 and CDK6 activity. This observation is in line with recent findings of Petermann and coworkers ( 36 ) that podocyte injury causes upregulation of cell cycle proteins, e.g., phosphorylated histone 3, cdc2, and cyclin B 1 and B 2, but at the same time also of CDK-inhibitory proteins particularly p21 and p27. As a result, podocytes do not complete the cell cycle and do not undergo cytokinesis. This has been attributed to the accumulation of DNA damage, inhibiting podocyte proliferation at G 1 /S and G 2 /M phases by increased p21.
Several points in the design and methodology of the present study deserve comment. The dose of 1,25(OH) 2 D 3 increased serum Ca in SNX rats to some extent, but not into the hypercalcemic range. We used a dose of 1,25(OH) 2 D 3 that restored plasma 1,25(OH) 2 D 3 levels to the physiological range. Consequently, the results reflect physiological actions rather than pharmacological consequences of 1,25(OH) 2 D 3. Intentionally, only a modest amount of cortical tissue was resected so that the number of nephrons was only reduced by 60% as documented for this resection protocol ( 1 ). This was done to avoid confounders such as severe hypertension, severe hyperparathyroidism, metabolic acidosis, anemia, etc. In particular, blood pressure as a potential confounder for glomerular injury was higher in SNX than in sham-operated controls but was not affected by treatment with 1,25(OH) 2 D 3.
In conclusion, the present study documents important direct or indirect effects of 1,25(OH) 2 D 3 on podocytes in a standard model of nonimmune progressive renal damage as illustrated by amelioration of morphological and immunohistochemical indicators of podocyte injury. These findings may explain blood pressure-independent attenuation of progression by active vitamin D.
GRANTS
This work was supported by the Deutsche Forschungsgemeinschaft (SFB 423, project B8).
ACKNOWLEDGMENTS
The skillful technical assistance of Z. Antoni, M. Klewer, P. Rieger, S. Söllner, M. Weckbach, and H. Ziebart is gratefully acknowledged.
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作者单位：Departments of 1 Pathology and 2 Internal Medicine, 5 Institute of Biomedicine, Universitiy of Erlangen-Nuernberg, D-91054 Erlangen; and Departments of 3 Pathology and 4 Internal Medicine, University of Heidelberg, 69115 Heidelberg, Germany