1,25-Dihydroxyvitamin D(3) Inhibits Podocyte uPAR Expression and Reduces Proteinuria

Background Accumulating studies have demonstrated that 1,25-Dihydroxyvitamin D(3) (1,25(OH)2D3) reduces proteinuria and protects podocytes from injury. Recently, urokinase receptor (uPAR) and its soluble form have been shown to cause podocyte injury and focal segmental glomerulosclerosis (FSGS). Here, our findings showed that 1,25(OH)2D3 did inhibit podocyte uPAR expression and attenuate proteinuria and podocyte injury. Methodology/Principal Findings In this study, the antiproteinuric effect of 1,25(OH)2D3 was examined in the lipopolysaccharide mice model of transient proteinuria (LPS mice) and in the 5/6 nephrectomy rat FSGS model(NTX rats). uPAR protein expression were tested by flow cytometry, immune cytochemistry and western blot analysis, and uPAR mRNA expression by real-time quantitative PCR in cultured podocytes and kidney glomeruli isolated from mice and rats. Podocyte motility was observed by transwell migration assay and wound healing assay. Podocyte foot processes effacement was identified by transmission electron microscopy. We found that 1,25(OH)2D3 inhibited podocyte uPAR mRNA and protein synthesis in LPS-treated podocytes, LPS mice and NTX rats, along with 1,25(OH)2D3 reducing proteinuria in NTX rats and LPS mice.1,25(OH)2D3 reduced glomerulosclerosis in NTX rats and alleviated podocyte foot processes effacement in LPS mice. Transwell migration assay and wound healing assay showed that LPS-induced podocyte motility, irrespective of random or directed motility, were substantially reduced by 1,25(OH)2D3. Conclusions/Significance Our results demonstrated that 1,25(OH)2D3 inhibited podocyte uPAR expression in vitro and in vivo, which may be an unanticipated off target effect of 1,25(OH)2D3 and explain its antiproteinuric effect in the 5/6 nephrectomy rat FSGS model and the LPS mouse model of transient proteinuria.


Introduction
Proteinuria is a key feature of kidney glomerular dysfunction, and it is a risk factor for both renal and extrarenal diseases [1]. Emerging clinical and animal studies have demonstrated that vitamin D and its analog reduce proteinuria in patients with IgA nephropathy [2], non-dialysed chronic kidney disease stage 4-5 [3,4], diabetic nephropathy [5], and animal models such as subtotally nephrectomized model [6], diabetic nephropathy model [7], adriamycin-induced nephropathy [8], puromycin-induced nephropathy [9,10]. However, the mechanisms underlying the antiproteinuric effect of vitamin D remains to be fully elucidated.
Several studies showed that 1,25(OH)2D3,an active form of vitamin D [29], could down-regulate breast tumor cells invasion via inhibiting uPAR expression [30][31]. However, in this study, we showed that 1,25(OH) 2 D 3 inhibited the expression of podocyte uPAR, a recently confirmed pathogenic factor causing podocyte injury and proteinuria [16]. These findings that 1,25(OH) 2 D 3 inhibited podocyte uPAR may provide a new insight into the mechanisms underlying its well-known antiproteinuric effect.

Results
1,25(OH)2D3 Treatment Inhibited Proteinuria, Glomerulosclerosis and Podocyte uPAR Induction in 5/6 Nephrectomy (NTX Rats) Rats We tested the anti-proteinuric effect of 1,25(OH)2D3 in 5/6 nephrectomy rats (NTX rats). This model resembled FSGS, a podocyte-related proteinuric kidney disease in human being and is featured nephron loss leading to proteinuria, podocyte dysfunction, glomerulosclerosis, and progressive renal dysfunction [6,32]. We fed male Sprague-Dawley 5/6 nephrectomy rats once daily with vehicle or 1,25(OH)2D3.and left sham-operated rats with vehicle. Vehicle-treated NTX rats developed heavy proteinuria compared with vehicle-treated sham-operated rats ( Figure 1A). Meanwhile, 1,25(OH)2D3 attenuated proteinuria at time points of 8 weeks and 12 weeks ( Figure 1A Figure1A). We then wondered whether uPAR expression was elevated in the NTX rats. Morphologically, there was low expression of uPAR in glomeruli from the sham rats ( Figure 2A). uPAR was partially localized in podocytes, as indicated by colabeling with synaptopodin. In contrast, expression of uPAR protein in the NTX rats ( Figure 2A) was substantially increased in podocytes. After 1,25(OH)2D3 treatment, we found a substantial reduction of uPAR protein expression in NTX rats (Figure 2A,B and C). We then performed real-time quantitative PCR with kidney cortex isolated from these rats. We analyzed PLAUR expression in RNA samples from NTX rats. We found low level PLAUR mRNA expression in sham rats. In contrast, NTX rats had a significant increase in PLAUR mRNA expression ( Figure 2D). Notably, we found that, 1,25(OH)2D3 treatment inhibited podocyte uPAR induction in NTX rats.
As NTX rats in the advanced-stage show marked glomerulosclerosis [32], we then sought to examine the effect of 1,25(OH)2D3 on glomerulosclerosis at the time point of 12 weeks. As expected, TNX rats showed significant glomerulosclerosis ( Figure 1B). Treatment of TNX rats with 1,25(OH)2D3, reduced glomerulosclerosis ( Figure 1B).
It is well known that LPS-induced proteinuric mice are featured with changing in altered podocyte foot process dynamics result in foot process effacement and proteinuria [33][34]. To explore whether 1,25(OH)2D3 has a role in regulating podocyte foot process structure and function, we observed podocyte foot process structure by transmission electron microscopy. Compared with untreated LPS mice, LPS treated mice shows significant foot process effacement ( Figure 3B). Treatment of LPS treated mice with 1,25(OH)2D3, reduced foot process effacement ( Figure 3B), indicating the anti-proteinuric effect of 1,25(OH)2D3 may be associated with its altering podocyte foot process dynamics and structure action.
We then asked whether uPAR expression was elevated in the LPS mice. Morphologically, there was low expression of uPAR in glomeruli from the control mice ( Figure 4A). uPAR was partially localized in podocytes, as indicated by colabeling with the podocyte marker synaptopodin [31]. In contrast, expression of uPAR protein in the LPS mice ( Figure 4A) was substantially increased in podocytes. Interestingly, after 1,25(OH)2D3 treatment, we found a substantial reduction of uPAR protein expression in the LPS mice (Figure 4 A,B,C). We then performed real-time quantitative PCR with kidney cortex isolated from these mice. We analyzed PLAUR (encoding uPAR) expression in RNA samples from LPS mice. We found low level PLAUR mRNA expression in control mice. In contrast, the LPS mice had a significant increase in PLAUR mRNA expression ( Figure 4D). Of note, we found that 1,25(OH)2D3 inhibited podocyte uPAR induction in LPS-induced proteinuric mice.
As uPAR is a motility-associated molecule [21,22] and podocyte motility is regarded as a surrogate indicator for proteinuria and effacement of podocyte foot processes in vivo [16][17][18][19][20], we next explore whether 1,25(OH)2D3 has a role in inhibiting cell motility of podocytes in vitro. We first studied podocyte motility before and after 1,25(OH)2D3 treatment using transwell migration assay to assess the random migration of podocytes on vitronectin, a known binding partner of uPAR. LPS treatment for 24 h significantly promoted the migration of podocytes (Figure 6 A,B). In contrast, after plus treatment with 1,25(OH)2D3, the number of migrating podocytes decreased ( Figure 6A, B).
We also analyzed the effect of 1,25(OH)2D3 on the spatial motility of podocytes with a scrape-wound assay. As compared with the control, LPS treatment significantly promoted podocyte wound closure (Figure 6 C,D). In contrast, treatment with 1,25(OH)2D3 reduced podocyte-directed motility ( Figure 6C,D). Together, these data show that1,25(OH)2D3 inhibits podocyte motility as well as podocyte uPAR expression.

Discussion
Our results demonstrated that 1,25(OH)2D3 inhibited podocyte uPAR mRNA and protein synthesis in vitro and in vivo, which may be an unanticipated effect of 1,25(OH)2D3 and explain its antiproteinuric effect in the 5/6 nephrectomy rat FSGS model and the LPS mouse model of transient proteinuria.
Our findings provided a new insight into the mechanisms for 1,25(OH)2D3's well known anti-proteinuric effect, which is independent of its regulation of calcium, phosphate metabolism. In this study, 1,25(OH)2D3 ameliorated proteinuria and inhibited podocyte uPAR induction, a pathogenic pathway activated in podocytes during the development of podocyte damage and proteinuria [16]. uPAR is a proteinase receptor and is also involved in nonproteolytic pathways, mainly through interactions with other plasma membrane proteins such as integrins, caveolin and G-protein-coupled receptors [21,22]. uPAR, together with b3 integrin and vitronectin, mediates podocytes dysfunction and development of proteinuria in mice [16,17]. It has been reported that uPAR-deficient mice(Plaur2/2 mice) were protected from proteinuria in response to LPS and most notably, when uPAR was reconstituted, Plaur2/2 mice developed heavy proteinuria after LPS injection [16]. Recently, the soluble form of uPAR has also been identified as the circulating FSGS factor leading to proteinuria [17]. Previous studies have shown that active vitamin D inhibits uPAR expression in breast tumor cells [31]. Here, we provide a new evidence in support of 1,25(OH)2D3's inhibitory action on uPAR expression not only in LPS-treated podocytes but also in two animal models of proteinuria, suggesting that the antiproteinuric effect of 1,25(OH)2D3 may be related to a podocyte uPAR-related mechanism. In the NTX rats, a model resembling FSGS in human beings [6,32], our results showed that 1,25(OH)2D3 reduced proteinuria and glomerulosclerosis as well as inhibited podocyte uPAR expression. The expression of podocyte uPAR protein in the NTX rats was substantially increased, when treated with 1,25(OH)2D3, uPAR expression was substantially reduced. We also found that 1,25(OH)2D3 inhibited podocyte uPAR expression and induced remission of proteinuria in LPS-induced proteinuric mice, indicating that the antiproteinuric action of 1,25(OH)2D3 may be partially related to its inhibition of uPAR. However, the two animal models, used in this study, induce proteinuria via different mechanisms; one is due to inflammation and the other one by hemodynamic changes. The results of the current study did not rule out the possibility that the anti-proteinuric effect of 1,25(OH)2D3 is non-specific.
We also noted that LPS-induced proteinuric mice(LPS mice) is a tool to investigate podocyte-specific proteinuria in other studies [33][34]. A new concept is that LPS causes proteinuria by targeting podocytes and not other cell types in the kidney, which is in keeping with the observation that podocyte-specific expression of cathepsin L-resistant dynamin [14,36] or synaptopodin [33] is sufficient to safeguard against LPS-induced proteinuria. Our evidences demonstrated 1,25(OH)2D3 inhibited uPAR induction in podocytes in LPS-induced proteinuric mice, indicating that the antiproteinuric effect of 1,25(OH)2D3 may be directly attributable to its action on podocytes. However, due to the broad effects of LPS on inflammation induction and LPS can cause a variety of immune-and cellular disorders [37], we could not rule out the possibility that the effect of 1,25(OH)2D3 on LPS-induced proteinuria may target to other cells.
uPAR, a molecule associated with cell motility, is highly expressed on cell surface of diseased podocytes [21,22]. Most cases of proteinuria are associated with effacement of podocyte foot processes, which represents podocyte dynamics in vivo or motility of podocytes [15,16]. There, podocytes stay attached to the GBM, but altered motility of podocytes results in podocyte foot process effacement and proteinuria. Previous studies have shown that active vitamin D inhibits tumor cells metastases [30][31]. To wonder whether 1,25(OH)2D3 inhibits cell motility of podocytes in vitro, we examined motility of podocytes when treated with 1,25(OH)2D3. Our data showed that 1,25(OH)2D3 reduced podocyte-directed motility and random migration. To observe the effects of 1,25(OH)2D3 on podocyte motility in vivo or podocyte foot processes effacement in LPS mice, ultrastructural analysis were performed by transmission electron microscopy. Our results showed that 1,25(OH)2D3 ameliorated foot process effacement in LPS mice, which may be interpreted that in vivo podocyte motility was inhibited by 1,25(OH)2D3.
The mechanisms underlying vitamin D's inhibition proteinuria remain to be fully elucidated. Recently, experimental data suggested that vitamin D may protect podocytes by targeting multiple pathways, including the renin-angiotensin system, Wnt/ b-catenin pathway and pro-apoptotic pathway [29]. And vitamin D has also been shown to reduce proliferating cell nuclear antigen, cyclin-dependent kinase inhibitor p27, desmin (a marker of early podocyte damage), local renin-angiotensin system [6,7,29,38,39]. The active Vitamin D also prevents podocyte apoptosis by inhibiting p38 MAPK (mitogen-activated protein kinase) and activating the PI3K (phosphatidylinositol 3-kinase)/AKT signaling pathway [9]. Although diabetic vitamin D receptor knockout mice developed more severe proteinuria and glomerulosclerosis due to increased glomerular basement membrane thickening and podocyte effacement [7],the present study could not show that the effect of 1,25(OH)2D3 on uPAR is through the vitamin D receptors and this renoprotective effect might be an off-target effect. Together, our findings provided a new insight into the renoprotective effect of 1,25(OH)2D3, and might offer a potential target in preventing the progression of kidney diseases.

Animals
The animal study has been approved by Research Ethics Committee, Guangdong General Hospital, Guangdong Academy of Medical Sciences (No.GDREC 2010071A). We purchased Sprague-Dawley rats and C57BL/6 mice from Laboratory Animal Center, Sun Yat-sen University, China.
The extent of glomerulosclerosis was determined in 3 mm kidney sections and a glomerulosclerotic index was then calculated, as described previously [17]. In brief, 60 glomeruli from each rat were examined in a masked protocol. The degree of sclerosis in each glomerulus was graded on a scale of 0-4 as described previously with Grade 0, normal; Grade 1, sclerotic area up to 25% (minimal); Grade 2, sclerotic area 25-50% (moderate); Grade 3, sclerotic area 50-75% (moderate to severe) and Grade 4, sclerotic area 75-100% (severe).

Transmission Electron Microscopy (TEM)
For transmission electron microscopy, ultrathin sections (60 to 100 nm) were cut from cortical kidney tissue samples embedded in epon resin, using an ultramicrotome (Leica), collected on copper grids, and stained with uranyl acetate and lead citrate. Ultrathin sections were stained with uranyl acetate for 10 min and subsequently in Reynolds lead citrate for 2 min. Ultrastructural analysis was performed by transmission electron microscopy.

Cell Culture
Conditionally immortalized mouse podocytes were kindly provided by Dr. FR Danesh. (Baylor Medical Hospital, Houston, USA ) and cultured as reported previously [35,40].

Flow Cytometry
Flow cytometry assay is used to assess uPAR on cell surface of podocytes after 24 h of these treatment, approximately 10 6 / ml cells were trypsinized, washed with PBS(Ca 2+ free), and incubated with Phycoerythrin (PE)-conjugated rat monoclonal anti-mouse uPAR (FAB531P,R&D Systems, USA ) for 20 minutes at 4uC. Cells were then washed in PBS (Ca 2+ free) twice. As a control for this analysis, cells in a separate tube were treated with PE-labeled rat IgG 2A antibody. Data were collected by Cell Lab Quanta TM SC Flow Cytometry System and analysed using Cell Lab Quanta TM SC analyse (Beckman Coulter, Inc, USA).

Immunocytochemistry
Murine kidneys and cultured podocytes were harvested and snap-frozen according to standard protocols and fixed after sectioning in ice-cold acetone for 10 minutes. For immunofluorescent labeling, sections were washed once with PBS, permeabilized with 0.5% Triton X-100 in PBS and incubated with blocking solution (5% BSA) for 20 minutes at room temperature before further incubation with one of the primary antibodies (synaptopodin (N-14), sc-21536; uPAR(FL-290), sc-10815) for 2 hours at room temperature. For double labeling, sections were washed three times with PBS for 5 minutes, and one of the secondary antibodies (Alexa FluorH 488 monkey anti-goat IgG (H+L), A11055; Alexa FluorH 635 goat anti-mouse IgG (H+L), A31575; Alexa FluorH 546 goat anti-rabbit IgG (H+L), A11010, Invitrogen, USA; rabbit anti-goat IgG-FITC, sc-2777, Santa Cruz Biotech, USA; NorthernLights TM anti-mouse IgG-NL637,NL008, R&D Systems, USA) was applied for 2 hours. Pictures were captured with confocal microscopy (Leica Microsystems). All images were analyzed by two investigators blinded to the identity of the samples.

Transwell Migration Assay
Transwell cell culture inserts (pore size 5 mm; Costar Corporation, USA) were coated with vitronectin, rinsed once with DPBS and placed in DMEM medium (10%FBS) in the  21 cultured differentiated podocytes were seeded in the inserts and allowed to migrate for 24 h while being incubated at 37uC. Nonmigratory cells were removed from the upper surface of the membrane, and migrated cells were fixed with 4% paraformal-dehyde and stained with Crystal Violet Solution (Sigma-Aldrich, USA). The number of migrated cells was counted using phasecontrast microscopy with a 620 objective on an microscope (Olympus) in the centre of a membrane (one field). The

Wound Healing Assay
Cultured differentiated podocytes (each 1610 5 ml 21 ) were seeded overnight on vitronectin-coated coverslips in six-well plates. Each coverslip was then scratched with a sterile 200 ml pipette tip, washed with PBS and placed into fresh medium. After 24 h, cells were fixed with cold methanol, permeabilized with 0.5% Triton X-100 in PBS and cell nuclei were stained with DAPI (Roche Diagnostics). Pictures were captured by phase-contrast microscopy under a 610 objective on an microscope (Leica Microsystems) at 0 and 24 h after scratching, and the number of cells that had migrated into the same-sized square fields were counted. The data presented represent the mean6sd of five independent experiments.

Western Blot Analysis
Protein expression of uPAR were determined by Western blot analysis. Briefly, kidney cortex isolated from rodent was homogenized in 1 ml of tissue lysis buffer. Samples were centrifuged at 3,000 g for 15 min, and the supernatants were assayed. After being mixed with SDS-PAGE (NuPAGE) sample buffer and boiled for 5 min, samples were electrophoresed on 10% SDS polyacrylamide gels and transferred to PVDF membranes (Millipore) for 2 h at 40 V. Membranes were blocked for 30 min with Trisbuffered saline that contained 5% BSA (5% BSA/TBS) and incubated with diluted primary antibody including anti-uPAR(FL-290), sc-10815 (1:1000, Santa Cruz Biotechnology), anti-GAPDH (1:1000, Santa Cruz Biotechnology) overnight at room temperature in 5% BSA/TBS that contained 0.05% Tween 20. The membranes were washed and developed using the enhanced chemiluminescence system (Applygen Technology).

Statistical Analysis
We assessed statistical significance by one-way ANOVA analysis of variance followed by LSD test for comparison between two groups. P,0.05 was considered significant. All values are expressed as mean6s.d.