Xiao-Liang Zhang,* Yin-Feng Guo,* Zhi-Xia Song, and Min Zhou

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1 RENAL-CARDIAC-VASCULAR Vitamin D Prevents Podocyte Injury via Regulation of Macrophage M1/M2 Phenotype in Diabetic Nephropathy Rats Xiao-Liang Zhang,* Yin-Feng Guo,* Zhi-Xia Song, and Min Zhou Institute of Nephrology, Zhong Da Hospital, Southeast University School of Medicine, Nanjing, Jiangsu , China Increasing evidence suggests the heterogeneity of macrophage phenotype and function ultimately determines the outcome of diabetic nephropathy (DN). This study aimed to investigate the effects of vitamin D on macrophage M1/M2 phenotype and its role in preventing podocyte impairment in streptozotocin-induced DN rats. Calcitriol, a bioactive 1,25-dihydroxyvitamin D 3, ameliorated proteinuria and renal damage as well as reversed the decline of both nephrin and podocin, crucial structural proteins in podocytes. DN rats showed increased infiltrating macrophages with M1 phenotype characterized by elevated expression of inducible nitric oxide synthase and TNF- in glomeruli and interstitium, which were inhibited after calcitriol treatment. Interestingly, calcitriol promoted M2 macrophage activation with enhanced expression of CD163, arginase-1, and mannose receptor at week 18 but not at week 8 or 14. The ratio of CD163 to CD68, considered as the proportion of M2 macrophages, was about 2.9-fold higher at week 18 after calcitriol treatment. Furthermore, the protein expression of inducible nitric oxide synthase, a crucial marker of M1 macrophages, was negatively correlated with the expression of either nephrin or podocin, whereas CD163, indicating M2 macrophages, was positively correlated. In vitro, 1,25-dihydroxyvitamin D 3 switched high-glucose induced M1 macrophages toward an M2 phenotype in either U937-derived macrophages or RAW264.7 cells. Our results suggest that vitamin D not only reduces macrophage infiltration and inhibits M1 macrophage activation but also enhances M2 macrophage phenotype to protect against podocyte injury. (Endocrinology 155: , 2014) Diabetic nephropathy (DN) is a common complication of diabetes mellitus, and it remains a leading cause of end-stage renal disease. Podocyte injury is a key event in the progression of DN, which results in proteinuria and often leads to progressive diabetic kidney disease (1). Because podocytes have limited ability to regenerate, the extent of podocyte injury is an important prognostic determinant in DN (2). Therefore, therapies aimed at preventing or limiting podocyte injury have major potential clinical and economic benefits. Mounting evidence has suggested that inflammation plays a crucial role in the pathogenesis of DN (3). Macrophages are key inflammatory cells mediating kidney ISSN Print ISSN Online Printed in U.S.A. Copyright 2014 by the Endocrine Society Received January 8, Accepted August 1, First Published Online September 4, 2014 damage in human and experimental diabetes. In human DN, macrophage accumulation within glomeruli and interstitium is correlated strongly with serum creatinine, proteinuria, and interstitial fibrosis and inversely with renal function (4). Likewise, macrophage infiltration is also associated with prolonged hyperglycemia, glomerular immune complex deposition, increased chemokine production, and progressive fibrosis in experimental diabetic animals (5). Macrophage-derived products including TNF-, IL-1, IL-6, and reactive oxygen species markedly suppress activity of the nephrin gene promoter in podocytes, leading to the development of proteinuria (6). In addition, we have previously demonstrated that mono- * X.-L.Z. and Y.-F.G. contributed equally to this work. Abbreviations: Arg-1, arginase-1; BG, blood glucose; BUN, blood urea nitrogen; CKD, chronic kidney disease; 1,25(OH) 2 D 3, 1,25-dihydroxyvitamin D 3 ; DN, diabetic nephropathy; FGF-23, fibroblast growth factor-23; IGN-, interferon- ; MR, mannose receptor; inos, inducible nitric oxide synthase; PAS, periodic acid Schiff; Scr, creatinine; STZ, streptozotocin. doi: /en Endocrinology, December 2014, 155(12): endo.endojournals.org 4939

2 4940 Zhang et al Vitamin D Regulates Macrophage to Protect Podocyte Endocrinology, December 2014, 155(12): cytes could also stimulate renal resident cells to synthesize TGF- 1 via direct cell contact (7), which is a key marker of progressive renal fibrosis (8). Present investigations raise the possibility that it is the effector phenotype of the recruited macrophages rather than their presence alone that determines the extent of renal parenchymal injury (9). In response to renal microenvironments, macrophages give rise to different polarization states with distinct functions that are categorized as classically activated macrophages, or M1 macrophages, and alternatively activated macrophages, or M2 macrophages. The M1 macrophages are mainly involved in the expansion of inflammation and tissue damage, whereas the M2 macrophages have anti-inflammatory and immunoregulatory functions (10). Therefore, understanding the role of macrophages at different time points in renal inflammation and development of strategies for modulating macrophage phenotype and function are critical to early prevention of podocyte injury in DN. Vitamin D is characterized as a regulator of homeostasis of bone and mineral metabolism. In addition to its classical actions on mineral homeostasis, the putative benefits of vitamin D also have been implicated in various diseases, including muscle disorders (11), inflammatory bowel disease (12), cardiovascular disease (13), and DN (14). During the past years, the potent renoprotective action of vitamin D has been confirmed in a large number of randomized clinical trials (15). Emerging experimental data suggested that podocytes were major target of the renoprotective efficacy of vitamin D in DN (16). Although previous study has indicated the protective effect vitamin D on podocytes by inhibiting macrophages infiltration (17), the influence of vitamin D on macrophages M1/M2 polarization in DN has not been reported. Calcitriol, a selective vitamin D receptor (VDR) activator, not only is responsible for downregulating interferon- (IFN- ), IL- 1, IL-12, and TNF-, which are key products of M1 macrophages, but also upregulating IL-10 and IL-4, important inducers for M2 macrophage polarization (18). In addition, the peroxisome proliferator-activated receptor- gene, a specific factor for M2 macrophage maturation (19), is a primary target of vitamin D (20). Therefore, we hypothesized whether vitamin D protected against podocyte impairment via regulation of macrophage M1/M2 phenotype in streptozotocin (STZ)-induced DN rats. Materials and Methods Animal experiments The experimental protocol was approved by the Ethics Committee of Southeast University. Healthy male Sprague-Dawley rats weighing 200 to 220 g were purchased from Shanghai Slac Laboratory Animal Co. Ltd. All rats were housed in an air-conditioned room at 23 C 1 C with alternating 12-hour cycles of light and dark and were allowed free access to food or water. After 1 week of acclimatization, the rats were randomly divided into 4 groups: 1) control (n 8); 2) control calcitriol (control rats treated with calcitriol, n 8); 3) DN (n 24, divided into 3 time points; n 8 for each time point); and 4) DN calcitriol (DN rats treated with calcitriol, n 24, divided into 3 time points; n 8 for each time point). DN was induced with a single ip injection of STZ (Sigma) dissolved in 0.1M citrate buffer (ph 4.5) at 58 mg/kg, whereas the control rats received only the 0.1M citrate buffer solution. Three days later, the diabetic state was confirmed by measuring tail blood glucose (BG) level. Rats with a BG level over 16.7 mmol/l were considered as diabetic rats. Calcitriol (1,25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ] soft capsules) was purchased from the Shanghai Roche Company. Calcitriol was administered orally at a daily dose of 0.1 g/kg after the induction of DN. During the treatment period, body weight was measured weekly. BG was monitored with the BG monitoring system (Bayer) using 1 drop of tail blood, and 24-hour urine samples were collected in metabolic cages at various time points (weeks 4, 6, 8, 12, 14, and 18). Rats were killed at 3 time points (week 8, 14, and 18) after treatment. Blood samples were taken for measuring biochemical parameters, and kidneys were collected for histological examination and molecular assays. Cellular experiments The human monocytic cell line U937 was graciously provided by Professor Williams (University of Cardiff, Cardiff, United Kingdom), and the RAW264.7 cells were purchased from Shanghai Institutes for Biological Sciences. The U937 cells and RAW264.7 cells were maintained in RPMI 1640 media (containing 11.1mM glucose) supplemented with 10% fetal bovine serum, 100 U/mL penicillin (HyClone), and 100 g/ml streptomycin (HyClone) and incubated at 37 C in 5% CO 2. First, U937 cells were differentiated into macrophages by culturing them in the presence of phorbol-12-myristate-13-acetate (100 ng/ml; Sigma) for 48 hours. Second, to mimic macrophage phenotype in the state of DN in vivo, U937-derived macrophages were stimulated with various glucose doses (11.1mM, 20mM, 30mM, and 35mM) and times (0, 6, 12, 24, and 48 hours). Regular concentration of glucose (11.1mM) in RPMI 1640 media was used as control. Third, to examine the effect of vitamin D on macrophage polarization, U937-derived macrophages were preincubated with 30mM high glucose for 12 hours in the presence or absence of 1,25(OH) 2 D 3 (Sigma) or IL-4 (Perotech) for another 48 hours. In addition, 2 classical activation models of M1 and M2 macrophages in vitro were established by treating cells with 100 U/mL IFN- (Perotech) (M1 differentiation) or 20 ng/ml IL-4 (M2 differentiation). Fourth, RAW264.7 cells were also preincubated with 30mM high glucose for 12 hours in the presence or in the absence of 1,25(OH) 2 D 3 for another 48 hours. The supernatants were collected and cells were washed twice with PBS followed by RNA harvest for quantitative RT-PCR. Serum and urine chemistry analyses Blood urea nitrogen (BUN), serum creatinine (Scr), calcium, and phosphorus were analyzed by an automatic biochemistry analyzer (Hitachi, Japan). Serum 1,25(OH) 2 D 3 and fibroblast

3 doi: /en endo.endojournals.org 4941 growth factor-23 (FGF-23) were detected using ELISA kits (Uscn Life Science Inc). Urinary proteinuria was measured using an ELISA Kit (Jiancheng) according to the manufacturer s instructions (21). Renal histology analyses Kidneys were fixed in 10% formalin, processed, and paraffinembedded for slicing into 2- m sections. Sections were stained with periodic acid Schiff (PAS) reagent. Digital images of glomeruli areas were obtained from light microscopy (magnification, 400). For glomerular damage analysis, we evaluated mesangial matrix expansion of glomeruli. We analyzed 20 randomly selected PAS-stained glomeruli from each rat. An area computation method was used to quantify mesangial matrix deposition. The Image-Pro Plus analysis software was used to calculate the area of pink or red mesangial matrix deposition within each glomeruli and the entire glomeruli area. The ratio of pink or red area to the entire glomeruli area is the percentage of mesangial matrix deposition in a given glomeruli (22). Immunohistochemistry Immunohistochemistry was performed on paraffin sections using a microwave-based antigen retrieval technique. Sections were incubated with primary mouse anti-cd68 (Santa Cruz Biotechnology) followed by appropriate secondary antibody incubation. The immunostaining was visualized using diaminobenzidine tetrahydrochloride, and the slides were counterstained with hematoxylin. The number of CD68-positive macrophages was evaluated by counting in at least 20 randomly selected glomerular and tubulointerstitial sections. Western blot Tissue protein was separated by SDS-PAGE and transferred to a nitrocellulose membrane. The membranes were then incubated overnight at 4 C with the primary antibodies against nephrin (Abcam; ab136894), inducible nitric oxide synthase (inos) (Abcam; ab15323), mannose receptor (MR) (Abcam; ab64693), CD68 (Santa Cruz; SC-59103), podocin (Bioss; bs-6597r), TNF- (Santa, SC-1350), CD163 (Santa Cruz; SC-58965), arginase-1 (Arg-1) (Santa Cruz; SC-20150), and -actin (Immunoway Biotechnology) followed by incubation with horseradish peroxidase-conjugated secondary antibodies for 1 hour. Finally, the membranes were visualized with an enhanced chemiluminescence advanced system (GE Healthcare) and captured on x- ray film. Immunoreactive bands were quantified with densitometry using Image J software (National Institutes of Health). ELISA analyses The inos activity was assayed using an inos assay kit (Jiancheng). The IL-6, TNF-, and IL-12 levels in culture supernatants were assayed using an ELISA kit (R&D Systems). Quantitative RT-PCR Total RNA was extracted from U937-derived macrophages and RAW264.7 cells separately by RNAiso plus reagent (Takara). RNA (1 g) was reverse transcribed using a reverse transcription system kit (Takara). All the PCR primers were synthesized by TaKaRa Ltd. The following primer sequences were used: for human inos, forward primer (fp) 5 -AGGAGATGCT- GAACTACG-3 and reverse primer (rp) 5 -GGATGGT- GACTCTGACTC-3 ; human MR, fp5 -GGACGGATGGAC- GAGGAG-3 and rp 5 -CTGGAGGATTAGTCAAGGAAGG- 3 ; human GAPDH,fp5 -GAGTCCACTGGCGTCTTC-3 and rp 5 -GATGATCTTGAGGCTGTTGTC-3 ; mouse inos, fp 5 -ACAGCCTCAGAGTCCTTC-3 and rp 5 -GGTAGTAGTA GAATGGAGATAGG-3 ; mouse Arg-1, fp5 -GCTCACACT- GACATCAACACTC-3 and rp 5 -CTCTTCCATCACCTTG CCAATC-3 ; mouse MR, fp 5 -TTCGGGATTGTGGAGCA GATG-3 and rp 5 -TTGTCGTAGTCAGTGGTGGTTC-3 ; mouse IL-10,fp5 -CAACATACTGCTAACCGACTCC-3 and rp 5 -ACTCTTCACCTGCTCCACTG-3 ; mouse GAPDH, fp 5 -GCCTTCCGTGTTCCTACCC-3 and rp 5 -TGAAGTCG- CAGGAGACAACC-3. Real-time PCR was performed on an ABI PRISM 7300 real-time PCR system (Applied Biosystems). The protocol included melting for 15 minutes at 37 C, 5 seconds at 95 C, and 40 cycles of 2-step PCR including melting for 5 seconds at 95 C and annealing for 31 seconds at 60 C. The 2 cycle threshold method was used to determine the relative amounts of product using GAPDH as endogenous control. Statistical analysis All data are expressed as mean SD. Statistical differences among experimental groups were determined by one-way ANOVA followed by Tukey s post hoc test using SPSS version 16.0 statistical software. Data from all groups were collected then subjected to Pearson correlation analysis to clarify the correlation of macrophage phenotype and podocytes in different conditions. P.05 was considered to be statistically significant. Results Effects of calcitriol on BG, body weight, renal function, and proteinuria in STZ-induced DN rats After the injection of STZ, DN rats developed overt diabetes with higher BG concentration and lower body weight. However, both BG and body weight were not improved after calcitriol treatment (Table 1 and Figure 1). Scr and BUN significantly increased by around 50% to 60% and 160% to 180%, respectively, in DN rats, which was inhibited by calcitriol by about 20% to 40% at either week 14 or 18, although the difference was close at week 8. Similarly, DN rats exhibited increased proteinuria from week 4 to 18, which was attenuated after 6 weeks of calcitriol treatment (Figure 1). We also measured the plasma levels of 1,25(OH) 2 D 3, phosphorus, calcium, and FGF- 23. As shown in Table 1, oral gavage of calcitriol increased the level of 1,25(OH) 2 D 3 in the circulation without affecting serum calcium and phosphorus in normal rats. The level of 1,25(OH) 2 D 3 in serum in DN rats was decreased from week 8 to 18, and calcitriol treatment significantly inhibited the reduction. DN rats showed reduced serum calcium but elevated serum phosphorus at week 18, both of which were mildly improved after calcitriol treatment. Calcitriol treatment also increased serum FGF-23 concentration in normal rats. The serum FGF-23 concentration

4 4942 Zhang et al Vitamin D Regulates Macrophage to Protect Podocyte Endocrinology, December 2014, 155(12): Table 1. Effects of Calcitriol on the General Parameters in Experimental Animals a BG, mmol/l Serum Ca, mmol/l Serum P, mmol/l 1,25(OH) 2 D 3, pg/ml FGF-23, pg/ml Control Control calcitriol b b DN (8 wk) b b b DN calcitriol (8 wk) b b,c b DN (14 wk) b b b DN calcitriol (14 wk) b c b DN (18 wk) b b b b b DN calcitriol (18 wk) b c c c b a Data are presented as mean SD (n 8). b P.05 vs control group. c P.05 vs DN group at the same time point. was significantly increased in DN rats at all time points, especially at week 18, whereas calcitriol slightly but not significantly increased the level of serum FGF23 (Table 1). Calcitriol ameliorates renal histopathologic changes in STZ-induced DN rats As shown in Figure 1, DN rats displayed severe morphological kidney lesions. The glomerular mesangial matrix was increased in DN rats at week 8 and further exacerbated until week 18 compared with that in non-dn rats (control rats and control rats treated with calcitriol). The above renal histopathologic lesions were significantly attenuated by calcitriol treatment. Calcitriol alleviates podocytes injury and upregulates nephrin and podocin expression in STZ-induced DN rats Podocytes are a key structure of the glomerular filtration barrier. We quantified expression of critical proteins in podocytes, including nephrin and podocin, by Western blot. Consistent with these above functional and histological changes, the protein expression of both nephrin and podocin were significantly reduced in DN rats compared with that in non-dn rats, especially at week 18, and was significantly increased by calcitriol treatment to different extents at all time points (Figure 2). Calcitriol decreases macrophage infiltration within glomeruli and interstitium in STZ-induced DN rats To assess the potential effect of calcitriol on renal inflammation, we examined renal infiltration of the CD68- positive macrophages in STZ-induced DN rats. As shown in Figure 3, compared with non-dn rats, DN rats exhibited significant macrophages infiltration in the glomeruli and interstitium at week 8 after STZ induction, as illustrated by immunohistochemical staining for CD68 antigen. The increase in the number of infiltrating macrophages was even more remarkable at week 18 (Figure 3). Consistent with this immunohistochemical staining, protein expression of CD68 was also increased in DN rats compared with non-dn rats (Figure 4). Treatment with calcitriol substantially reduced CD68-positive macrophage infiltration as well as CD68 protein expression at all time points (Figures 3 and 4). Calcitriol inhibits proinflammatory M1 macrophages and enhances an M2 macrophage phenotype in STZ-induced DN rats To clarify whether calcitriol-mediated attenuation of DN results from a phenotypic regulation of renal M1/M2 macrophages, we analyzed several specific markers of M1 and M2 macrophage phenotype by Western blot using protein extracted from kidney tissue. As shown in Figure 4, renal concentrations of inos and TNF-, representative proinflammatory cytokines of M1 macrophages, were significantly increased in DN rats at week 8 after STZ injection and continued to increase until week 18. Calcitriol treatment significantly suppressed the expression of inos and TNF- at weeks 8 to 18. On the other hand, although no differences in the expression of CD163, Arg-1, and MR, specific markers for M2 macrophages, were observed at weeks 8 to 14 between DN rats and calcitriol-treated DN rats, the expressions of CD163, Arg-1, and MR were significantly elevated at week 18 after calcitriol treatment (Figure 4). Furthermore, the protein expression ratio of CD163 to CD68, considered as the proportion of M2 macrophages, was also calculated. As shown in Figure 4, the ratio of CD163 to CD68 was about 2.9-fold higher at week 18 after calcitriol treatment. Together, these results suggested that macrophages infiltrated within glomeruli and interstitium were predominantly proinflammatory M1 macrophage phenotype in the state of DN. Calcitriol initially reduces macrophage infiltration, inhibits M1 macrophage activation, and then plays an anti-inflammatory role in subsequent phases by

5 doi: /en endo.endojournals.org 4943 Figure 1. Effect of calcitriol on body weight, Scr, BUN, proteinuria. and renal histopathological change in DN rats. A, Body weight. B, Scr. C, BUN. D, Proteinuria. E, Mesangial matrix index of all groups. F, PAS stains of tissue as indicated ( 400). Data are presented as mean SD (n 8). *, P.05 vs control group; #, P.05 vs DN group at the same time point. promoting M2 macrophage phenotype in inflamed kidneys in STZ-induced DN rats. 1,25(OH)2D3 switches high-glucose induced M1 macrophages toward an M2 phenotype in vitro As shown in Figure 5, after stimulation with glucose, the activity of inos in U937-derived macrophages was increased in a glucose dose- and time-dependent manner and reached a maximum when given 30mM high glucose and cultured for 12 hours, equivalent to that in the IFN- stimulation group. Hence, we stimulated U937-derived macrophages with high glucose at a concentration of 30mM for 12 hours in the subsequent experiments. U937derived macrophages stimulated with 30mM high glucose exhibited increased mrna expression of inos and decreased MR compared with that in cells stimulated with regular glucose (11.1mM). Furthermore, the inflammatory cytokines produced by U937-derived macrophages incubated with 30mM high glucose, such as IL-6, TNF-, and IL-12 were also significantly increased (Figure 5).

6 4944 Zhang et al Vitamin D Regulates Macrophage to Protect Podocyte Endocrinology, December 2014, 155(12): Figure 2. Effect of calcitriol on nephrin and podocin protein expression in DN rats at different time points. A and B, Representative Western blotting analysis of nephrin (A) and podocin (B). -Actin was used as an internal control. C and D, Quantification of nephrin (C) and podocin (D) protein expression. Data are presented as mean SD (n 8). *, P.05 vs control group; #, P.05 vs DN group at the same time point. Therefore, these results indicated that high glucose induced proinflammatory M1 macrophage activation. On the other hand, 1,25(OH) 2 D 3 robustly enhanced mrna expression of MR and lowered inos by 1.8- and 8-fold, respectively, in U937-derived macrophages preincubated with 30mM high glucose, which was parallel to that in IL-4 stimulation and suggested an M2 macrophage activation. Accordingly, 1,25(OH) 2 D 3 also dramatically reduced IL-6, TNF-, and IL-12 production in U937-derived macrophages preincubated with 30mM high glucose (Figure 5). Taken together, the above data demonstrated that 1,25(OH) 2 D 3 switched high-glucose induced inflammatory M1 macrophages toward an M2 phenotype, which was further confirmed in RAW264.7 cells with elevated mrna expression of MR, Arg-1, IL-10, and reduced inos after 1,25(OH) 2 D 3 stimulation (Figure 5). Correlations between M1 marker (inos), M2 marker (CD163), and either nephrin or podocin expression As shown in Figure 6, there was a strong negative correlation between protein expression of inos and either nephrin or podocin (r 0.707, P.01; and r 0.712, P.01, respectively). In contrast, protein expression of CD163 was positively correlated with either nephrin or podocin (r 0.627, P.01; and r 0.613, P.01, respectively), strongly suggesting that calcitriol-mediated M1/M2 macrophage balance was associated with the amelioration of podocyte impairment. Discussion Podocyte injury plays a central role in the pathogenesis of progressive DN. Podocyte abnormalities include podocyte loss and disruption of slit diaphragm proteins such as nephrin, podocin, and CD2-associated protein, resulting in proteinuria and development of DN (1). Consistent with previous studies, our study demonstrated that expression of both nephrin and podocin were significantly reduced in DN rats and accompanied by increased proteinuria and deterioration of renal function. Vitamin D deficiency is associated with a higher incidence of albuminuria (23), progression to end-stage renal disease in the general population (24), and mortality in chronic kidney disease (CKD) cohorts (25). A high prevalence of vitamin D deficiency is quite common in patients with CKD (26), mainly as a result of renal dysfunction and abnormal vitamin D metabolism (27). Numerous randomized clinical trials have demonstrated the antiproteinuric efficacy of vitamin D analogs in diabetic patients with CKD (15, 28), which was also proved in a large number of

7 doi: /en endo.endojournals.org 4945 Figure 3. Effect of calcitriol on the number of CD68 macrophages in glomeruli and interstitium in DN rats at different time points. A and B, Immunohistochemical staining of CD68 macrophages in glomeruli (A) and interstitium (B) ( 400). Quantification of number of CD68 macrophages in glomeruli (C) and interstitium (D). Data are presented as mean SD (n 8). *, P.05 vs control group; #, P.05 vs DN group at the same time point. animal models of kidney disease. Treatment with vitamin D prevents podocyte dysfunction, albuminuria, and kidney damage in 5/6 nephrectomized rats (29) as well as adriamycin-induced nephropathy (17). A recently pub lished study also showed vitamin D ameliorated renal fibrosis in mice subjected to unilateral ureteral obstruction (30). In the present study, we indicated that vitamin D therapy reduced proteinuria and alleviated podocyte im-

8 4946 Zhang et al Vitamin D Regulates Macrophage to Protect Podocyte Endocrinology, December 2014, 155(12): Figure 4. Effect of calcitriol on M1/M2 macrophage-specific markers including inos, TNF-, CD163, Arg-1, MR, and the CD163 to CD68 ratio in DN rats at different time points. A H, Representative Western blotting analysis (A) and quantification of inos (B) and TNF- (C), Arg-1(D), MR (E), CD163 (F), CD68 (G), and CD163 to CD68 ratio (H). -Actin was used as an internal control. Data are presented as mean SD (n 8). *, P.05 vs control group; #, P.05 vs DN group at the same time point. pairment in STZ-induced DN rats. The renoprotective activity of vitamin D is mediated by vitamin D receptor and appears to act by regulating multiple classical pathways, including suppression of the renin-angiotensin system (31), inhibition of nuclear factor- B activation (32), blocking of the Wnt/ -catenin pathway (17), direct effects on expression of slit diaphragm proteins (33), and inhibition of TGF- -Smad signal transduction (30). Although the renoprotective role of vitamin D has been documented over the years, the underlying protective mechanism of vitamin D in DN remains elusive. Macrophage infiltration is a common feature in both human and experimental renal diseases (34). These infiltrating cells are strongly associated with the severity of kidney injury and progressive chronic renal failure (35). In experimental crescentic glomerulonephritis, inhibition of macrophage infiltration in glomeruli significantly alleviated crescent formation and proteinuria (36). Similarly, in our study, STZ-induced DN rats were clearly characterized by large macrophage infiltration in the glomeruli and interstitium at the early stage after disease induction, which was followed by the development of podocyte in-

9 doi: /en endo.endojournals.org 4947 Figure 5. Effect of 1,25(OH) 2 D 3 on M1/M2 macrophage phenotypes in either U937-derived macrophages or RAW264.7 cells preincubated with high glucose in vitro. U937-derived macrophages were stimulated with various glucose doses (11.1mM, 20mM, 30mM, and 35mM) and times (0, 6, 12, 24, and 48 hours). A and B, Activity of inos after U937-derived macrophages cultured with glucose at various doses (A) and times (B). U937-derived macrophages were stimulated with 11.1mM glucose, 30mM high glucose, or IFN- for 12 hours in the presence or in the absence of 1,25(OH) 2 D 3 or IL-4 for another 48 hours, followed by harvest of supernatants for ELISA and cell RNA for quantitative RT-PCR. C and D, mrna expression of inos and MR (C) of U937-derived macrophages and concentrations of TNF-, IL- 12, and IL-6 (D) in cell supernatants. RAW264.7 cells were cultured with 30mM high glucose for 12 hours in the presence or absence of 1,25(OH) 2 D 3 for another 48 hours. E, mrna expression of inos, MR, Arg-1, and IL-10 in RAW264.7 cells. Data are presented as mean SD (n 3 4 per group). *, P.05 vs 11.1mM glucose condition; #, P.05 vs 30mM glucose condition. jury and profound proteinuria, suggesting macrophages as a major constituent of the infiltrated inflammatory cells in DN. Although commonly recognized for the pathogenic role of macrophages in renal inflammation and fibrosis, macrophages also play a critical role in tissue remodeling and repair (9) as well as in immune regulation (37). Previous studies have demonstrated that macrophages comprised a heterogeneous population of cells with diverse functions and phenotypic plasticity (10). Classically activated M1 macrophages are induced by lipopolysaccharides and proinflammatory stimuli, such as IFN- or TNF- and IL-12. Therefore, M1 macrophages have antimicrobial and cytotoxic properties but also exacerbate inflammation and tissue injury. Studies have indicated that M1 macrophages are involved in various macrophage-dependent inflammation and several autoimmune diseases, such as rheumatoid arthritis and inflammatory bowel disease (38, 39). Recently, Ryu et al (40) found that M1 macrophages also accelerated podocyte loss and aggravated Alport nephropathy in type IV collagen-a3 (Col4a3)-deficient mice. In addition, M1 macrophage-derived TNF- directly downregulated podocyte nephrin and podocin expression in vitro (41). In the present study, we first showed that M1 macrophages are a dominant cell population with high expression of inos and TNF- in DN and contribute to podocyte impairment as illustrated by the negative correlation between inos and either nephrin or podocin, which was further confirmed by our in vitro studies, in which both U937-derived macrophages and RAW264.7 cells exhibited the M1 phenotype when incubated with high glucose. Calcitriol treatment notably reduced macrophage infiltration and meanwhile inhibited the proinflammatory cytokine production. Therefore, the protective effect of calcitriol on podocytes seems to be associated with inhibition of M1 macrophage activation. On the other hand, alternatively activated M2 macrophages play an important role in the resolution of inflammation and tissue repair. Three subsets of M2 macrophages have been proposed: those exposed to IL-4 and IL-13, called M2a; those exposed to immune complexes and IL-1 or lipopolysaccharide, called M2b; and those exposed to IL-10 and TGF- or glucocorticoids, called M2c. However, the activation and phenotype of macrophage diversity is obviously more complex in tissue environments of both animal and human and is likely to extend beyond the above M1/ M2a/M2b/M2c paradigm. In addition, the specific markers and functions of various subsets of M2 have not been clearly delineated. Overall, M2 macrophages all display similar biological characteristics in the resolution of in-

10 4948 Zhang et al Vitamin D Regulates Macrophage to Protect Podocyte Endocrinology, December 2014, 155(12): Figure 6. Correlations between M1/M2 macrophage phenotypes and nephrin or podocin expression. A D, Correlations between M1 macrophage marker inos and nephrin expression (r 0.707, P.01) (A), M1 macrophage marker inos and podocin expression (r 0.712, P.01) (B), M2 macrophage marker CD163 and nephrin expression (r 0.627, P.01) (C), and M2 macrophage marker CD163 and podocin expression (r 0.613, P.01) (D). flammation via a high endocytic clearance capacity and secretion of trophic factors and anti-inflammatory cytokines. Typical markers of M2 macrophage such as CD163, MR, and Arg-1 are detected during the repair phase (42). Nishida et al (43) first revealed the presence of 2 distinct macrophage phenotypes, M1 and M2, in kidney disease. They found that macrophage depletion during progressive inflammatory fibrosis in mice with unilateral ureteral obstruction ameliorated scarring and reduced the number of myofibroblasts, whereas macrophage depletion during the recovery phase led to a failure of matrix degradation and persistent scarring. Anti-inflammatory macrophages for renal healing responses also have been confirmed in an acute kidney injury study using an acute ischemia-reperfusion model. In the postischemic kidney, M1 macrophages subsequently underwent a phenotypic switch toward anti-inflammatory M2 macrophages, which stimulated epithelial proliferation and promoted tubular repair (44). An intriguing observation from our study is that calcitriol therapy selectively enhances antiinflammatory M2 macrophages at week 18 but not week 8 or 14 in STZ-induced DN rats. A positive correlation between M2 macrophages and nephrin or podocin protein in podocytes was also observed. In addition, in our in vitro study, we revealed that 1,25(OH) 2 D 3 shifted high-glucose induced inflammatory M1 macrophages toward an M2 phenotype in both U937-derived macrophages and RAW264.7 cells. To our knowledge, this is the first study to demonstrate that, in addition to the reduction of glomerular macrophage infiltration and inhibition of M1 macrophage activation, calcitriol also enhances the devel-

11 doi: /en endo.endojournals.org 4949 opment of M2 macrophages to attenuate podocyte impairment in DN. Strategies designed to block M1 generation and/or enhance M2 polarization may be available for treating renal diseases (45). Wang et al (9) provided direct evidence that adoptive transfer of macrophages primed ex vivo by exposure to IL-4 and IL-13 to induce M2 macrophages can reduce renal injury and facilitate repair in adriamycininduced nephropathy in mice. Therefore, we speculate that modulating the macrophage M1/M2 phenotype by vitamin D to produce protective macrophages is a straightforward and effective approach for early prevention of DN. However, it must be acknowledged that although an M2-skewed macrophage response is associated with inflammation resolution and tissue healing, these cells also promote fibrosis. The mechanisms that fine tune the M2- associated response to achieve repair without scarring and long-term consequences are unknown and need more exploration. Moreover, the molecular mechanism for vitamin D modulating macrophage M1/M2 phenotypes has not been discussed in this study and needs further investigation. In conclusion, we demonstrate in this study that vitamin D reduces proteinuria, improves renal function, and prevents podocyte injury in STZ-induced DN rats. Our findings indicate for the first time that vitamin D can not only reduce macrophage infiltration and inhibit M1 macrophage activation but also enhance the M2 macrophage phenotype, which is associated with the amelioration of podocyte impairment. Altogether, our findings provide new insight into the mechanisms underlying the renoprotective effect of vitamin D and supply a foundation for therapeutic strategies. Acknowledgments Address all correspondence and requests for reprints to: Xiao- Liang Zhang, Institute of Nephrology, Zhong Da Hospital, Southeast University School of Medicine, Nanjing, Jiangsu , China. tonyxlz@163.com. This study was supported by grants from the National Natural Science Foundation of China ( ), Medical Key Talents Programs of Jiangsu Province (RC ) and Fundamental Research Funds for the Central Universities of China (CXLX13 122) (to X.-L.Z.). Disclosure Summary: The authors declare that there are no conflicts of interest. References 1. Jefferson JA, Shankland SJ, Pichler RH. Proteinuria in diabetic kidney disease: a mechanistic viewpoint. Kidney Int. 2008;74(1): Mathieson PW. The podocyte as a target for therapies new and old. Nat Rev Nephrol. 2012;8(1): Nelson CL, Karschimkus CS, Dragicevic G, et al. Systemic and vascular inflammation is elevated in early IgA and type 1 diabetic nephropathies and relates to vascular disease risk factors and renal function. Nephrol Dial Transplant. 2005;20(11): Nguyen D, Ping F, Mu W, Hill P, Atkins RC, Chadban SJ. Macrophage accumulation in human progressive diabetic nephropathy. 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Curr Diabetes Rev. 2011; 7(1): He W, Kang YS, Dai C, Liu Y. Blockade of Wnt/ -catenin signaling by paricalcitol ameliorates proteinuria and kidney injury. JAmSoc Nephrol. 2011;22(1): Li X, Zheng W, Li YC. Altered gene expression profile in the kidney of vitamin D receptor knockout mice. J Cell Biochem. 2003;89(4): Odegaard JI, Ricardo-Gonzalez RR, Goforth MH, et al. Macrophage-specific PPARgamma controls alternative activation and improves insulin resistance. Nature. 2007;447(7148): Dunlop TW, Väisänen S, Frank C, Molnár F, Sinkkonen L, Carlberg C. The human peroxisome proliferator-activated receptor delta gene is a primary target of 1alpha,25-dihydroxyvitamin D3 and its nuclear receptor. J Mol Biol. 2005;349(2): Sun H, Yuan Y, Sun ZL. Cholesterol contributes to diabetic nephropathy through SCAP-SREBP-2 pathway. Int J Endocrinol. 2013;2013: Sugimoto H, LeBleu VS, Bosukonda D, et al. Activin-like kinase 3 is important for kidney regeneration and reversal of fibrosis. 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