An adjustment in BMP4 function represents a treatment for diabetic nephropathy and podocyte injury

Podocyte injury has been proposed to play an important role in diabetic nephropathy; however, its pathological mechanism remains unclear. We have shown that bone morphogenetic protein 4 (BMP4) signaling leads to the glomerular changes characteristic of this disorder. To analyze the molecular mechanism of podocyte injury, the effect of BMP4 was investigated using streptozotocin (STZ)-induced, Bmp4 heterozygous knockout (Bmp4+/−) and podocyte-specific Bmp4 knockout mice. Mice with STZ-induced diabetes exhibited glomerular matrix hyperplasia and decreased numbers of podocyte nucleus-specific WT1-positive cells. The number of podocytes and proteinuria were improved in both diabetic Bmp4 knockout mouse models compared to the effects observed in the control mice. The effect of BMP4 overexpression on Bmp4-induced or podocyte-specific transgenic mice was examined. Tamoxifen-induced Bmp4-overexpressing mice exhibited mesangial matrix expansion and decreased numbers of WT1-positive cells. Podocyte-specific Bmp4-overexpressing mice displayed increased kidney BMP4 expression and mesangial matrix expansion but decreased nephrin expression and numbers of WT1-positive cells. Both lines of Bmp4-overexpressing mice exhibited increased albuminuria. In cultured podocytes, BMP4 increased phospho-p38 levels. BMP4 decreased nephrin expression but increased cleaved caspase-3 levels. p38 suppression inhibited caspase-3 activation. Apoptosis was confirmed in STZ-diabetic glomeruli and Bmp4-overexpressing mice. Bmp4 +/− mice with diabetes displayed reduced apoptosis. Based on these data, the BMP4 signaling pathway plays important roles in the development of both podocyte injury and mesangial matrix expansion in diabetic nephropathy.

the main pathways contributing to podocyte loss, which leads to glomerulosclerosis 11 . Podocytes are subjected to various stresses, including oxidative stress, endoplasmic reticulum stress, metabolic stress, inflammation, toxins, and physical-mechanical stimuli 12,13 . These factors may be involved in the complex mechanism underlying the development of kidney damage. For example, high glucose and advanced glycated proteins induce podocyte apoptosis, a process mediated by transforming growth factor beta (TGFβ), in conjunction with p38 14 . Specifically, the activation of the p38 MAP kinase and caspase-3 in cells exposed to stress or cytokines triggers the apoptotic response in podocytes.
BMPs are expressed during kidney development and affect tissue morphogenesis. BMP4 is an important molecule that induces the stem cell differentiation required for the development of various organs, including the kidney [15][16][17] . BMP4 also induces Smad1/5/8 phosphorylation and p38 activation during embryonic tissue formation, triggering apoptosis 18 . Additionally, the BMP4-Smad1 pathway mediates senescence in injured cells 19 . Consistent with podocyte injury in nephrotic syndrome, which is caused by apoptosis and cell cycle dysfunction, we have reported that BMP4 constitutes a critical molecule that induces glomerulosclerosis in subjects with DN. In addition, the expression of p16 and p21 in response to BMP4 has been shown to be mediated by the Smad1/5/8 signaling pathway 20 . Smad1 upregulates the transcription of type IV collagen, the primary protein component of the extracellular matrix in mesangial cells 6 . Notably, mesangial matrix hyperplasia in DN mice is suppressed by a BMP4 neutralizing antibody 21 . Thus, BMP4/Smad1 signaling plays a central role in the development of mesangial matrix hyperplasia in subjects with DN; however, the precise molecular mechanisms by which BMP4 induces podocyte injury remain unclear. In this study, we used Bmp4 heterozygous knockout mice (Bmp4+/−), podocyte-specific Bmp4 knockout mice, induced Bmp4 tgm, and podocyte-specific Bmp4 tgm to examine the molecular mechanism by which the BMP4/Smad1/p38 signaling pathway mediates glomerular injury in a mouse model of DN.

Results
Diabetic Bmp4 heterozygous knockout mice exhibit reduced podocyte injury. We first examined the development of DN in STZ-induced diabetes models using Bmp4 +/− mice. High blood glucose levels were observed at 24 weeks after the induction of diabetes in either wild-type or Bmp4 +/− mice, as we have previously reported 7 . Glycoalbumin levels were also significantly increased in the diabetic group (Supplemental Table 1A). A significant difference in blood glucose levels was not observed between the wild-type and Bmp4 +/− mice. The diabetic mice exhibited significantly increased mesangial matrix expansion and BMP4 levels compared to those in the control mice ( Fig. 1C-a and c). The area of BMP4 expression partially overlapped podocytes, but BMP4 expression was also observed elsewhere, including primarily mesangial areas ( Fig. 1A-b).
Next, we investigated whether a decrease in BMP4 expression attenuated any renal changes in diabetic mice. Mesangial matrix expansion, BMP4 expression, and albuminuria were attenuated in the diabetic Bmp4 +/− mice compared with the diabetic wild-type mice (Fig. 1B,C-g-h and D). The results of periodic acid methenamine silver (PAM)-stained areas in the wild-type mice were compatible with findings from our previous report 7 . A statistically significant difference in the albuminuria levels was not observed between the diabetic and non-diabetic Bmp4 +/− mice. The podocyte number, as determined by the number of WT1-positive cells, was maintained in the diabetic Bmp4 +/− mice compared to the diabetic wild-type mice ( Fig. 1C-k,l and E). Furthermore, the diabetic Bmp4 +/− mice expressed higher levels of nephrin than the diabetic wild-type mice ( Fig. 1C-o,p and F). BMP4-positive staining was only observed in the wild-type mice, and the area was increased in the diabetic animals (Fig. 1G). Western blot analyses revealed decreased expression of podocin in the diabetic mice. The diabetic Bmp4 +/− mice exhibited similar levels of podocin as the nondiabetic mice (Fig. 1H). In addition, the creatinine clearance rate (Ccr) was slightly reduced in the diabetic Bmp4 +/− mice (Supplemental Table 1A).
Podocyte-specific Bmp4 knockout mice with diabetes exhibit reduced podocyte injury. We investigated whether the podocyte-specific reduction in levels of secreted BMP4 attenuated any renal changes in diabetic mice. Bmp4 loxP × Podocin-Cre mice exhibited decreased Bmp4 levels in the kidneys, as detected by quantitative PCR. Bmp4 loxP induction (−) in control mice has not been crossbred with Podocin-Cre mice. Mesangial matrix expansion was attenuated in the diabetic Bmp4 loxP × Pod-Cre mice compared with the diabetic Bmp4 loxP induced (−) mice ( Fig. 2A-c-d and B). The reduction in WT1-and nephrin-positive areas was alleviated in these mice compared to the diabetic non-induced mice ( Fig. 2 A-k-l,o-p,C,and D). Electronic microscopy analyses of diabetic Bmp4 loxP × Pod-Cre mice showed normal podocyte foot processes and glomerular basement membranes (GBMs) ( Fig. 2A-t). Diabetic Bmp4 loxP induced (−) mice displayed marked alterations such as GBM thickening and podocyte foot process effacement ( Fig. 2A-s). In addition, Ccr and albuminuria were not different between diabetic Bmp4 loxP × Pod-Cre mice and non-diabetic mice (Supplemental Table 1B).

Bmp4 overexpression induces podocyte injury and mesangial matrix expansion in vivo.
To investigate the role of BMP4 in adult mice, we generated Bmp4 tgm using the tamoxifen-regulated Cre-loxP system. As previously reported (ref. 7 ). The Bmp4 tgm (+) is a tamoxifen-induced BMP4-expressing adult mouse strain. Bmp4 tgm (−) control mice were not induced with tamoxifen. Bmp4 tgm (+) displayed increased BMP4 expression in whole glomeruli, as shown in red ( Fig. 3A-d) and glomerulosclerosis ( Fig. 3A-b). These mice exhibited a significant decrease in the number of WT1-positive cells and nephrin-positive area compared to Bmp4 tgm (−) control mice (Fig. 3A-f and h). Bmp4 tgm (+) exhibited a decreased number of WT1-positive cells compared with Bmp4 tgm (−) (Fig. 3B). We confirmed the podocin levels using western blotting in vivo. Bmp4 tgm (+) displayed decreased podocin levels compared with those in Bmp4 tgm (−) (Fig. 3C).  control mice were not crossbred with Podocin-Cre mice. CAG-B4 × Pod-Cre mice showed mesangial matrix hyperplasia at 30 weeks, and progressive mesangial matrix hyperplasia at 40 weeks of age ( Fig. 4G-g and h). The 60-week-old mice developed severe mesangial sclerosis accompanied by interstitial fibrosis. In particular, the CAG-B4 × Pod-Cre mice showed increased BMP4 expression in the glomerulus. These BMP4-expressing areas colocalized with podocytes, although staining was also observed in the area expressing collagen IV ( Fig. 4E-b). These mice displayed fewer podocytes at 40 weeks of age than the reference mice (Fig. 4C), and they also exhibited significantly reduced nephrin expression (Fig. 4D). Furthermore, an increased number of pSmad1-positive cells was observed not only in partially WT1-positive cell areas but also among areas of predominantly mesangial cells ( Fig. 4A-l). The electron microscopy analyses of these mice revealed GBM thickening, podocyte foot process effacement, and mesangial expansion, similar to the findings of DN mice ( Fig. 4A-n). The CAG-B4 × Pod-Cre mice exhibited decreased podocin levels compared with those in the CAG-B4 control mice (Fig. 4F). In addition, the Ccr was decreased in the CAG-B4 × Pod-Cre mice compared with the level in the CAG-B4 cont mice. Albuminuria was increased in the CAG-B4 × Pod-Cre mice compared with the level in the CAG-B4 control mice. Systolic blood pressure levels were not different between the CAG-B4 × Pod-Cre mice and the CAG-B4 control mice (Supplemental Table 1D).

Changes in the levels of cleaved caspase-3 and apoptosis in vivo.
As these findings suggest that BMP4/p38/caspase-3 signaling may play a key role in establishing podocyte injury, we investigated the involvement of these molecules in vivo. Cleaved caspase-3 levels were increased in the glomeruli of STZ-induced DN mice (Fig. 6A-c). The area expressing cleaved caspase-3 overlapped with both mesangial cells and podocytes. STZ-induced Bmp4 knockout mice exhibited reduced levels of cleaved caspase-3 (Fig. 6A-c and d), whereas increased levels of cleaved caspase-3 were detected in the tamoxifen-induced Bmp4 and CAG-B4 × Pod-Cre mice (Fig. 6A-f and h). Diabetic-induced nuclear damage in wild-type mice was reflected by increased apoptosis, as highlighted by TUNEL staining. The diabetic Bmp4 +/− mice exhibited notably reduced apoptotic changes, as assessed by TUNEL staining (Fig. 6B-c and d). Bmp4 tgm (+) and CAG-B4 × Pod-Cre mice displayed nuclear damage due to apoptosis (Fig. 6B-j red color and l green color). The green color in images of Bmp4 tgm is GFP, which is derived from the recombinant gene.

Discussion
Podocyte injury and loss have been indicated as the crucial markers of the pathogenesis of glomerular injury; however, the detailed molecular mechanisms and cell-to-cell responses remain to be elucidated, because multiple factors may cause podocyte injury. In the glomerulus, three types of cells (i.e., endothelial, mesangial, and parietal epithelial) respond to podocyte injury. Endothelial and mesangial cells are connected to podocyte cells across the glomerular basement membrane. However, the detailed mechanisms underlying the relation between the mesangium and podocyte injury are unclear. DN is characterized by mesangial matrix expansion, which is caused by the excess deposition of extracellular matrix proteins in the mesangial area, as detected by increased expression of type IV collagen. The initial finding of the present study was that BMP4 secreted by glomerular cells, including mesangial cells and podocytes, directly caused podocyte injury and correlated with urinary albumin excretion. BMP4 secreted from cells adheres to collagen IV 22 . We postulate that BMP4 secreted from podocytes accumulates in glomerular mesangial cells, in which collagen IV is a primary component. Concomitantly, BMP4 accumulates in mesangial cells, activates Smad1, and increases the production of extracellular matrix proteins in mesangial cells. BMP4 expression was primarily observed in mesangial cells, as well as in podocytes. BMP4 is a secreted protein that may act on various cells in a paracrine manner. Therefore, BMP4 signaling pathways play central roles in the development of podocyte injury and mesangial matrix expansion in subjects with DN. P38 MAPK signaling mediates BMP4-induced apoptosis and senescence in cancer cells 23 , whereas BMP4-induced Smad1 signaling activates apoptotic cell death in embryonic stem cells 24 . Although the p38 MAPK and Smad1 pathways have been reported to synergistically induce apoptosis and autophagy in tumor cells 25 , other studies have reported that the p38 MAPK pathway is separate from Smad1 signaling because it mediates NRAGE-p38 signaling 26 . In particular, the mechanism underlying podocyte damage depends on BMP4/BMP receptor signaling in the early stage of diabetes. BMP4 induced podocyte apoptosis mediated by p38 activation and cleaved caspase-3 (Fig. 5B). This pathway induced podocyte loss, as detected by the number of WT1-positive cells, and subsequent foot process injury, as evidenced by nephrin staining (Fig. 4A-h,j and n).
R-Smad has been reported to affect the cell cycle; accordingly, Smad1 may impact senescence in podocytes 27 . Alternatively, we considered that Smad1 upregulated the extracellular matrix expansion in mesangial cells, based on our observation that phosphorylated Smad1 directly induces collagen IV expression in mesangial cells 6 . The Smad1 inhibitor did not affect the levels of cleaved caspase-3 in cultured podocytes. However, the induction of apoptosis was suppressed by p38 inhibitors (Fig. 5C). BMP4 exerts two direct effects on glomeruli injury, including the effect of BMP4/Smad1 signaling on mesangial matrix expansion and the effect of BMP4/p38 signaling on podocyte injury. As shown in our previous study, a BMP4 neutralizing antibody blocked Smad1-mediated mesangial matrix expansion 21 . Based on these findings, BMP4 signaling represents a promising target for developing a therapeutic approach for DN.
Podocyte-specific Bmp4 tgm showed typical mesangial sclerosis accompanied by podocyte loss and increased phosphorylated Smad1 levels in mesangial cells. Thus, podocyte loss itself is responsible for mesangial sclerosis, and the increased BMP4 expression observed in podocytes induces Smad1 activation in mesangial cells in a paracrine fashion. Based on these findings, BMP4/p38 signaling in podocytes and BMP4/Smad1 signaling in mesangial cells are potential therapeutic targets for DN. Because BMP4 regulates both podocyte loss and mesangial Smad1 activation in DN, BMP4 represents an essential molecule involved in the development of the typical pathological features of DN.
Both podocyte loss and mesangial expansion were concomitantly observed in most pathological cases of DN; however, microalbuminuria did not completely reflect the characteristic renal pathological changes, including mesangial matrix expansion and glomerular basement membrane thickening, in the early stages of human DN. Urinary albumin excretion is affected by the charge and size of the barrier of the glomerular basement membrane 28,29 . The mechanism of renal disease progression requires a subsequent in-depth analysis of the relationship between podocyte dysfunction and GBM disruption. Bmp4 knockout mice exhibited reduced podocyte injury and albuminuria levels. These initial observations provide sufficient evidence that BMP4 is a therapeutic target. Based on the findings of the present study, we speculate that BMP4 induces podocyte injury in an early stage of DN in which microalbuminuria is observed. Additional studies are necessary to confirm these observations.

Experimental Procedures
Experimental animals. All animal experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee of Chugai Pharmaceutical Co., Ltd. and the Tokushima University animal facility, and the Ethical Review Board of Tokushima University granted permission for the procedures performed in this study.
Bmp4-knockout mice (C57BL/6 Bmp4+/−) and conditional knockout mice (C57BL/6 Bmp4 loxP ) were gifts from B. Hogan and M. Saitou. The generation and characterization of these mutant mice have been described elsewhere 16,30 . The Bmp4 +/− mice were maintained on a C57BL/6 background. The Podocin-Cre (Pod-Cre) mice were obtained from The Jackson Laboratory (Bar Harbor, ME, USA). We crossed Pod-Cre mice with Bmp4 loxP mice to generate the podocyte-specific Bmp4 knockout mice. Diabetes was induced in 8-week-old male C57BL/6 mice weighing 22-24 g by the intraperitoneal injection of 50 mg per kg body weight streptozotocin (STZ) in The BMP4-expressing areas are shown in green, and the nephrin area is shown in red. Yellow indicates the area of BMP4 and nephrin overlap (arrowhead). (F) Podocin levels were determined by western blotting. Podocin levels were decreased in CAG-B4xPod-Cre mice compared with those in CAG-Bmp4 cont mice. G) Glomerular PAS staining in each mouse from 10 weeks to 40 weeks of age. PAS staining was increased in CAG-B4xPod-Cre mice aged more than 30 weeks (g and h). * P < 0.01, (t-test).
SCIeNTIFIC REPORtS | (2018) 8:13011 | DOI:10.1038/s41598-018-31464-9 citrate buffer, pH 4.5, for 5 consecutive days. The diabetic state was confirmed 4 weeks after the final injection by measuring the blood glucose levels with an Accu-Chek monitor (Roche). All mice that were administered STZ had a blood glucose concentration exceeding 400 mg/dl and were considered diabetic. Reference mice were injected with citrate buffer. The mice were classified into four groups: (A) nondiabetic C57BL/6 (n = 7), (B) nondiabetic Bmp4 +/− (n = 7), (C) diabetic C57BL/6 (n = 9), and (D) diabetic Bmp4 +/− (n = 9) mice. These mice were euthanized 24 weeks after the final injection of STZ and citrate buffer. Glycoalbumin levels were measured using an enzyme assay (Lucica GA-L, Asahi Kasei Co., Ltd.). Microalbuminuria was measured using a DCA vantage analyzer (SIEMENS Healthineers). The creatinine concentration was measured with an enzyme assay (Serotec Co., Ltd.). The procedure for generating tamoxifen-inducible Bmp4 tgm is described in a previous report 7 . Eight-to 10-week-old transgenic mice were fed a diet (CE-2, CLEA Japan) containing 0.02% tamoxifen citrate (Sigma) to induce the expression of the Bmp4 gene. Control mice were fed a normal diet (CE-2). The CAG-CAT-Bmp4 transgenic mice (CAG-B4) were provided by RIKEN Bio Resource Center (Tsukuba, Japan) under an agreement with Dr. Y. Saga (National Institute of Genetics, Japan). We generated the double tgm by mating CAG-B4 mice with Pod-Cre mice. CAG-B4 x Pod-Cre mice overexpressed BMP4 in podocytes. Reference mice were not mated with Pod-Cre mice. These mice were monitored for 1 year. Blood pressure levels were measured with a non-invasive blood pressure device (BP-98A-L, Softron Co., Ltd.) attached to the animal's tail.
Morphology studies. Tissues used for light microscopy studies were fixed with 10% neutral buffered formalin. One-μm-thick sections were prepared from paraffin-embedded tissues and subjected to periodic acid-Schiff (PAS), periodic acid methenamine silver (PAM) and picrosirius red staining. Morphometric analyses of PAM-stained tissues were performed using ImageJ software. The glomerular surface area and the PAM-positive area/glomerular area (%) were measured for 30 glomeruli in each mouse.
The avidin-biotin complex (ABC) method was employed for immunohistochemistry to detect the expression of various proteins. Tissue sections were deparaffinized, rehydrated with PBS and treated with 3% H 2 O 2 . Subsequently, tissue sections were subjected to an antigen retrieval procedure in HistoVT one buffer according to the manufacturer's instructions (Nacalai Tesque, Inc., Japan). Sections were incubated with various primary antibodies. Briefly, 4-μm-thick cryostat sections were prepared, air-dried for an hour and fixed with 4% paraformaldehyde for 20 min at 4 °C. After equilibrating the sections with PBS, they were incubated with various primary antibodies overnight at 4 °C, washed with PBS and then incubated with FITC-conjugated secondary antibodies conjugated (Thermo Fisher Scientific) for 1 hr. Following a PBS wash, tissue sections were covered with a drop of buffered glycerol, mounted with a cover slip and examined with a fluorescence microscope (KEYENCE CORPORATION). Anti-BMP4 (Abcam), anti-WT1 (Santa Cruz Biotechnology), anti-nephrin (IBL), anti-cleaved caspase-3 (Cell Signaling Technology) antibodies were used for these procedures. Nephrin immunoreactivity was quantified by calculating the nephrin-positive areas in the glomeruli using ImageJ software. The WT1-positive cells were counted in the glomeruli.
Tissues used for electron microscopy were fixed with 2.5% glutaraldehyde. We entrusted electron microscopy analysis to a specialized company (BML, Inc.).
Cell culture experiment. Conditionally immortalized murine podocytes were obtained from a cell line service (Eppelheim, Germany). Podocytes were cultured in RPMI-1640 medium (Sigma) containing 10% fetal calf serum, 100 U/ml penicillin, 100 mg/ml streptomycin (Invitrogen) and 2 mM L-glutamine at 33 °C in a 5% CO 2 atmosphere. Cells were cultured on type I collagen-coated dishes at 37 °C for 1 week to induce differentiation.