Apolipoprotein Erelated glomerular disorders , Takao Saito, Akira Matsunaga, Megumu Fukunaga , Kiyotaka Nagahama , , Shigeo Hara and Eri Muso OPEN Of the glomerular disorders that occur due to apolipoprotein E (apoE) mutations, apoE2 homozygote glomerulopathy and lipoprotein glomerulopathy (LPG) have been characterized. ApoE2 homozygote glomerulopathy has been found in individuals expressing homozygous apoE2/2. This was characterized histologically by glomerulosclerosis with marked infiltration of foam cells derived from macrophages, and occasionally with non-lamellated lipoprotein thrombi. Recently, several cases of apoE Toyonaka (Ser197Cys) combined with homozygous apoE2/2 have been reported, in which non-immune membranous nephropathy-like features were observed in glomeruli. Interestingly, in these cases, apoE accumulation was identified by tandem mass spectrometry. Therefore, it is speculated that these findings may arise from apoE molecules without lipids, which result from hinge damage by apoE Toyonaka and may cross the glomerular basement membrane as small molecules. LPG is primarily associated with heterozygous apoE mutations surrounding the low-density lipoproteinreceptor binding site, and it is histologically characterized by lamellated lipoprotein thrombi that lack foam cells. Recent studies have suggested that LPG can be induced by thermodynamic destabilization, hydrophobic surface exposure, and the aggregation of apoE resulting from the incompatibility of apoE mutated residues within helical regions. Additionally, apoE5 may play a supporting role in the development of LPG and in lipid-induced kidney diseases via hyperlipoproteinemia. Thus, it is interesting that many apoE mutations contribute to characteristic glomerular disorders through various mechanisms. In particular, macrophages may uptake lipoproteins into the cytoplasm and contribute to the development of apoE2 homozygote glomerulopathy as foam cells, and their dysfunction may contribute to the accumulation of lipoproteins in the glomerulus, causing lipoprotein thrombi in LPG. Kidney International 97, 279288; https://doi. org/10. 1016/j. kint. 2019. 10. 031https://doi. org/10. 1016/ https://doi. org/10. 1016/j. kint. 2019. 10. 031j. kint. 2019. 10. 031 Correspondence: Takao Saito, Sanko Clinic, 4-9-3 Ropponmatsu Chuoku, Fukuoka 810-0044, Japan. E-mail: mailto:tsaito@fukuoka-u. ac. jptsaito@fukuoka-u. ac. jp Received 7 August 2019; revised 25 October 2019; accepted 28 October 2019; published online 22 November 2019 KEYWORDS: apolipoprotein E2 homozygote glomerulopathy; apolipopro-tein E5; apolipoprotein E Toyonaka; apolipoprotein mutations; lipoprotein glomerulopathy; macrophage Copyright ª 2019, International Society of Nephrology. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license ( To explain the relationship between lipid abnormalities and chronic kidney disease, Moorhead et al. 1 proposed the hypothesis of lipid nephrotoxicity in 1982. Additionally, Faraggiana and Churg 2 conducted a pathology analysis of renal lipidoses and found that abnormal lipid storage in a number of diseases occurred either due to an inborn problem with metabolism or as a consequence of complex metabolic alterations. Few studies, however, have provided clear evidence regarding the causal relationship between abnormal lipid metabolism and renal disorders. Meanwhile, various glomerular disorders caused by abnormalities in apolipoprotein E (apoE) composinglipoproteins have recently been identified. Therefore, these disorders should be investigated to elucidate the mechanisms of pathological conditions related to abnormal lipid metabolism in the kidney. In this review, we describe the apoE-related glomerular disorders that have been reported so far and outline the role of various apoE abnormalities in these disorders. Apolipoprotein E (ApoE) Apolipoprotein molecules bind to lipids, form lipoproteins, and are involved in lipoprotein metabolism as lipids transport proteins, ligands possessing chylomicron remnants, and low-density lipoprotein (LDL) receptors and coenzymes that activate lipid-related enzymes. 3 In particular, apoE composes very-low-density lipoproteins (VLDLs), intermediate-density lipoproteins (IDLs), and high-density lipoproteins (HDLs) together with triglyceride and cholesterol, and this protein plays a central role in the metabolism of lipoproteins in the blood. 4 It is known that apoE-related lipoprotein abnormalities occur in not only cardiovascular diseases but also various neurologic and infectious diseases. 5 Additionally, several renal diseases associated with apoE mutations have been described, and the function and role of apoE within the kidney has been studied. ApoE is a glycoprotein composed of 317 amino acids, and it is coded by a gene on chromosome 19. A signal peptide of 18 amino acids at the N-terminus is diverged, and a mature protein of 299 amino acids (molecular weight: approximately 34, 000) is secreted extracellularly,(Figure 1). ApoE can be separated into 2 primary domains, each possessing a different structure and function., The N-terminal domain (amino acids 1 to 191) consists of 4 a-helices including an LDL-receptor binding region (amino acids 136 to 150) and a heparan sulfate proteoglycans (HSPG) binding region (amino acids 142 to 147). The C-terminal domain, composing one-third of the apoE molecule (amino acids 216 to 299), is critical for lipid binding. In particular, residues within the regions of amino acids 244 to 272 form amphipathic a-helices that mediate the binding of apoE to lipoprotein. Additionally a protease-sensitive loop within the hinge region (amino acids 192 to 215) may play an indispensable role in the binding of both domains(Figure 1). The human APOE gene has 3 alleles (2, 3, and 4), and these are expressed as the apoE isoforms E2, E3, and E4, respectively., ApoE3 is the most common isoform, present in more than 70% of the general population. Compared to the amino acid residues of apoE3, a cysteine is substituted for arginine at position 158 in apoE2, and an arginine for cystein at position 112 in apoE4. ApoE1, apoE5, and apoE7are known as minor isoforms and possess several variants with different amino acid substitutions. In particular, apoE5 is known to be involved in kidney damage and is discussed later (see ApoE5 involvement section). ApoE3 carriers are typically normolipi-demic, although apoE4 is a risk factor for Alzheimer disease, and apoE2 homozygosity occasionally induces type III hyperlipoproteinemia (HLP) as a result of its inability to bind to the LDL receptor. ApoE is a multifunctional protein that is synthesized primarily within the liver. However, it is also synthesized in several other tissues and cell types including the brain, kidney, adipocytes, and macrophages. This protein serves as a ligand for the receptor-mediated uptake of lipoproteins and plays a key regulatory role in the clearance of lipoproteins from the plasma. In the brain, apoE is generated in astrocytes and glia cells and is assembled in the lipoproteins of cere-brospinal fluid. Various functions of the central nervous system are related to apoE, and Alzheimer disease associated with apoE4 may occur due to the failure of one or more of these functions. The amount of apoE derived from macrophages is small but sufficient to reduce atherosclerosis without lowering hypercholesterolemia., It is important to elucidate these functions when considering the role of apoE in the renal lesions associated with macrophages. ApoE-related glomerular diseases ApoE2 homozygote glomerulopathy. In 1974, Amatruda et al. detailed glomerular lesions with foam cells in a case of type III HLP. Thereafter, the apoE2 homozygocity was determined to be responsible for type III HLP, and 10 cases exhibiting glomerular lesions due to this apoE isoform were reported worldwide. Kawanishi and colleaguessummarized these cases as apoE2 homozygote glomerulopathy in a review article. The histologic features of these cases are glomerulosclerosis with marked foamy macrophage infiltra-tion (Figure 2a), and it is often difficult to differentiate from diabetic nephropathy when accompanied by diabetes mellitus. As some patients do not necessarily suffer from diabetes mellitus, however, it is conceivable that abnormal lipidosis with type III HLP causes glomerular lesions characterized by foamy macrophages. This pathology can be explained through the concept of glomerulosclerosis, as suggested by Diamond and Karnovsky, who applied the mechanism of atherosclerosis to kidney disease in the 1980s. As reported by Sakatsume et al., however, some cases of apoE2 homozygote glomerulopathy displayed lipoprotein thrombi or non-immune subendothelial and subepithelial electron-dense deposits (EDDs), although lamella formation was not detected in lipoprotein thrombi as it was in those in lipoprotein glo-merulopathy (LPG). Considering that apoE2 homozygote glomerulopathy is based on type III HLP with increased serum triglycerides, VLDL, and IDL, apoE2 is speculated to be specifically involved in glomerular lesions possessing lipo-protein thrombi without lamella influenced by type III HLP, and this occurs in addition to the mechanism of atherosclerosis by LDL cholesterol. Indeed, 2 papers reported the relationship between heterozygous apoE2 and characteristic findings, as follows. One casewith apoE2/3 was associated with marked non-immune subendothelial EDDs. The other casehad the combination of heterozygous apoE2 and apoE Tokyo/Maebashi, isoforms and showed infiltration of foamy macrophages in addition to lipoprotein thrombi. As apoE Tokyo/Maebashi,, an in-frame deletion of Leu141 to Lys143, is one of the representative mutations for LPG, atypical histopathologic changes may be induced by a compound heterozygote. Several studies indicated that the apoE2 isoform was a significant risk factor for the development of diabetic ne-phropathy in both type 1and type 2diabetes, although some papers have presented evidence against such a relationship. Therefore, it is likely that diabetic ne-phropathy is influenced by abnormal lipid metabolism via apoE2. Additionally, Yorioka et al. reported that serum apoE2 levels were correlated with the severity of IgA ne-phropathy, and some studies, have suggested that the apoE2 allele may be involved in the progression to end-stage renal disease. In contrast, the proportion of apoE2 homozygote carriers is 0. 5% to 1% within the entire population, and in these carriers, type III HLP is found in less than 10% of individuals. Additionally, as reports of apoE2 homozygote glomerulopathy are limited to 10 cases in the world, specific etiologic factors other than the apoE2 homozygote may conribute to the development of this disease. Membranous nephropathy-like apoE deposition disease (MN-like apoE disease). Recently, a new glomerular disease was identified in non-consanguineous carriers possessing the combination of apoE Toyonaka (Ser197Cys), a novel mutation, and apoE2 homozygote(Figure 1). The first case reported by Fukunaga et al. was a 20-year-old female Japanese patient who underwent renal biopsy because she was found to have chance proteinuria and hematuria. In silver-methenamine-stained sections of a biopsy specimen, spike formations as seen in MN were found in the majority of glomeruli without mesangial proliferation or matrix expansion (Figure 2b). Electron microscopy revealed massive EDDs located primarily in the subepithelial area of the glomerular basement membrane and in the subendothelial and mesangial areas (Figure 3a). These findings were similar to those observed in secondary MN associated with autoimmune disease, such as lupus nephritis type V, rather than idiopathic MN. The appearance of microbubbles or microcysts on higher-magnification electron microscopy images (Figure 3b) and non-specific IgG and C3 depositions in the immuno-fluorescence study, however, refuted the immunogenicity of this disease. Meanwhile, glomerular apoE accumulation was identified by immunohistochemical studies (Figure 3c) and tandem mass spectrometry, and the apoE levels were determined to be extremely high in the serum, although hyperlipidemia was not identified. These findings suggest the involvement of apoE mutation in this disease, and analysis of the apoE gene detected apoE Toyonaka (Ser197Cys) in the hinge region of one allele in combination with apo E2 homozygosity (Figure 3d). Other than several noninvasive single-nucleotide polymorphisms, mutations in the hinge region have not yet been reported. Therefore, apoE Toyonaka may be the first mutation within the hinge region that plays a pathologic role in apoE-related diseases. A deformed hinge region may markedly affect the 3-dimensional structure of apoE and cause disconnections not only between the N-terminal domain and LDL receptors but also between the C-terminal domain and lipids. This is likely due to the fact that the structural and functional stability of apoE is mediated by the hinge region connecting both terminals. When apoE Toyonaka is involved in hinge damage, type III HLP induced by defective binding to the LDL receptor in the N-terminal domain of the apoE2 homozygote may be negated due to the dysfunction of the C-terminal domain that connects with lipids. In contrast, altered apoE without lipids may accumulate within the glomerulus and induce EDD in the sub-epithelial region, as the apoE molecule is relatively small (34 kDa) and rich in arginine, a positively-charged amino acid that may cross the glomerular basement membrane. Thus, apoE Toyonaka may play a pivotal role in non-immune MN-like lesions with spike formation. The other 2 cases exhibiting apoE Toyonaka and apo E2 homozygosity, were of type III HLP with marked hyper-triglyceridemia, which was different from the case presented by Fukunaga et al. The serum apoE levels, however, were high in all 3 cases. Spike formations were recognized by light microscopy, and subepithelial and subendothelial EDDs including microbubbles were both observed by electron microscopy. Immunoglobulin and complement depositions were not specifically obsereved in immunofluorescence studies. Unlike the case reported by Fukunaga et al., foam cells characteristic of apoE2 homozygote glomerulopathy and non-lamellated lipoprotein thrombi as reported by Sakatsume et al. were conspicuous in the cases reported by Hirashima et al. and Kato et al., respectively. Therefore, in carriers exhibiting both apoE2 homozygote and apoE Toyonaka, the balance of non-immune MN-like lesions and apoE2 homo-zygote glomerulopathy may be dependent upon the influence of apoE Toyonaka within the hinge region. Lipoprotein glomerulopathy. LPG was identifiedas a glomerular disease associated with type III HLP in 1989. The genetic and pathologic features, however, differ from those of apoE2 homozygote glomerulopathy. As we have previously described this disease in several reviews and commentaries,, in this section we only briefly provide an overview and introduce recent studies. In contrast to apoE2 homozygote glomerulopathy, this disease was characterized by dilated glomerular capillaries with lipoprotein thrombi showing lamella formation instead of foam cells(Figure 2c). Similar cases were thereafter reported from some areas in Japan, and they were categorized into a single disease entity. Through apoE gene analyses, various mutations were discovered in patients who suffered from LPG,,(Figure 1). Although most were heterozygous apoE missense variants close to the LDL receptor binding site, mutants far from the LDL receptor binding site,, or involving the deletion of several amino acids,,,, were also reported. Additionally, Ishigaki et al. revealed that the virus-mediated transduction of apoE Sendai, first reported in human LPG, reproduced LPG-like lesions in the kidney of experimental apoE-knockout mice. Although Wen et al. reported that aged apoE knockout mice spontaneously developed LPG and that apoE Sendai was not necessarily required for onset of LPG, Ishimura et al. clearly demonstrated the presence of lipoprotein thrombi in LPG induced by apoE Sendai, differentiated them from the lesions in aged mice, and clarified that apoE Sendai was responsible for the onset of LPG. Approximately 150 LPG cases have been reported worldwide, the majority in Japan and China; however, there are also cases from the United States, Italy, France, Brazil, and Russia. Recent in-depth studies suggest that the incidences of APOE Sendai and APOE Kyoto, the major LPG mutants, have increased through a founder mutation in Japanand China, respectively. It is interesting, however, that LPG cases with apoE Sendai are restricted to eastern Japan. In contrast, cases with apoE Kyoto are found worldwidein Asia, the United States, and Europe. At the time of its discovery, type III HLP was considered to be prominently involved in LPG, as well as in apoE2 homo-zygote glomerulopathy. Currently, however, the apoE structural deformities caused by apoE mutations are considered to be direct risk factors that contribute to the onset of LPG, as normolipidemic cases are occasionally reported., In particular, the majority of the apoE mutants associated with LPG, including apoE Sendai, have substitutions of proline for arginine.,,, as this mutation loosens the a-helix structure and transforms the LDL receptor binding site (residues 140150). In this regard, Hoffmann et al. demonstrated a reduction in receptor binding activity of apoE Sendai. Additionally, Georgiadou et al. in Greecesuggested in their detailed biochemical analysis that LPG could be induced by thermodynamic destabilization, hydrophobic surface exposure, and aggregation of apoE resulting from the incompatibility of proline within helical regions. Meanwhile, in apoE Kyoto, a point mutation is different from the LDL-receptor binding site and does not appear to be involved in the reduced binding to the LDL receptor. Mat-sunaga et al., however, demonstrated that apoE Kyoto exhibits only 10% of the normal receptor-binding activity in an in vitro competition assay using apoE lysosomes. Furthermore, a recent study by a Greek groupsuggested that the non-proline substituted apoE mutants associated with LPG, including apoE Kyoto, may also contribute to protein aggregation within glomerular capillaries and that a common underlying mechanism may be responsible for the pathogenesis of LPG. ApoE5 involvement. ApoE5, one of the minor isoforms of apoE, is detected at a higher positive charge, compared to that of apoE4, in response to isoelectric focusing. Genetically, several variants have been reported worldwide, with apoE5 (Glu3Lys) alone observed at a relative frequency of approximately 0. 1% within the total Japanese population(Figure 1). Interestingly, it has been postulated that carriers of apoE5 are at risk for hyperlipidemia and subsequent atherosclerosis, despite exhibiting 2-fold greater LDL receptorbinding activity than that of wild-type apoE3 carriers. To explain this paradox, it has beens suggested that hypercholesterolemia results from the downregulation of LDL receptor due to the high uptake of apoE5-containing lipoproteins. However, few studies have been conducted in the 2000s regarding apo E5, and the precise mechanism underlying its function remains unclear. The first renal disease associated with apoE5 was reported in a transplant case with LPG. LPG was diagnosed from renal biopsy of the original kidney, and this ultimately developed into end-stage renal disease. The patient underwent a cadaver renal transplant, and relapse of LPG was observed at the 1-year follow-up biopsy. Miyata et al. identified heterozygous apoE mutations with apoE2 and apoE5 (Glu3Lys) in this patient. In contrast, Kodera et al. recently reported another case of LPG that possessed apoE Chicago and apoE5 (Glu3Lys), and they clarified, by gene analysis of the family of the patient, that both mutations were expressed within the same allele. Additonally, apoE Chicago was also identified in a re-analysisof the case previously analyzed by Miyata et al.. Takasaki et al. also reported a case of LPG with a combination of apoE Sendai and apoE5 (Glu3Lys). Thus, the occurrence of LPG cases with heterozygous apo E5 appears to be increasing, and all of these cases exhibit hypertriglyceridemia of $400 mg/dl.,, It is unknown if apoE5 plays a pathogenetic role in LPG, as apoE5 in these cases has always been associated with LPG-specific mutants such as apoE Chicago or apoE Sendai. Hyper-triglyceridemia resulting from apoE5, however, is likely to be the trigger for LPG. In contrast, we have observed focal and segmental glomerulosclerosis (FSGS), with apoE5(Glu3Lys)/ E3(Figure 2d), that displays hypercholesterolemia and foamy macrophages. As the etiologic role of hyperlipidemia in FSGS has been studied experimentally, and clinically, it is possible that apoE5(Glu3Lys)-induced hyperlipidemia is involved in the development of FSGS, as shown in the next section. Collectively, these findings indicate that apoE5 may be one of the risk factors underlying lipid-induced kidney diseases. Role of macrophages Ever since the speculation by Brown and Goldsteinregarding the mechanisms underlying atherosclerosis, the role of macrophage infiltration associated with endothelial injury, hyperlipoproteinemia, and hypertension have been discussed in the context of FSGS and other glomerular diseases. Indeed, we have demonstrated that macrophages are increased in experimental and clinical FSGS, and in diabetic glomerulosclerosis. For apoE-related glomerular diseases, similar findings were observed in apoE2 homozygote glomerulopathy, and a case of FSGS that was associated with apoE5also illustrated the significance of macrophage infil-tration due to hyperlipoproteinemia. as mentioned earlier. In contrast, foamy macrophage infiltration is rarely observed in LPG, and presence of lipoprotein thrombi due to the accumulation of lipoprotein is one of the diagnostic criteria. Kanamaru et al. found that LPG-like glomerular lesions with lipooprotein thrombi were induced by graft-versus-host disease in Fcg receptorknockout mice possessing normal apoE, and they suggested that these findings resulted from the decrease of LDL uptake in macrophages. This speculation was later confirmed by an additional experiment using a similar model, which showeda drastic decline of scavenger receptor CD36. Additionally, Ito et al. generated LPG-like lesions in apoE and Fcg receptor double knockout mice through the injection of various vectors of apoE mutants, and they revealed that the impairment of macrophages plays a prominent role in the development of LPG associated with apoE abnormalities. In addition to these experiments, some studies have shown that macrophages produce a small amount of apoE, which is important for the suppression of hyperlipidemia and arteriosclerosis. 1113 Interestingly, one of these studies showed that the expression of macrophages producing apoE Sendai in mice that received a bone marrow transplant protected against atherosclerosis while inducing LPG. Collectively, these results indicate that macrophages play various roles in lipoprotein metabolism involving apoE, and that their hyperactivity or suppression can be an important factor in each different type of renal lipidosis. In particular, as carriers with apoE mutants of apoE2 homozygote glomerul-opathyand LPG, show low genetic penetrance, the functional variety of macrophages may be important as another etiologic factor of these diseases. Additionally, it is of interest that macrophage-derived apoE may be associated with mutations and may regulate the activity of these diseases(Figure 4). Further studies, however, are needed to clarify these findings. Treatments and prognosis As described above, apoE-related glomerular diseases are caused by lipoprotein degeneration based on apoE gene mutations, even if pathologic types are different, and often they progress into end-stage renal disease. Accordingly, no suitable treatment has been recognized at present. LPG, however, is typically associated with type III HLP, of which triglyceride-rich lipoproteins such as VLDLs and IDLs, are the major components, and our studies have clarified that hypertriglyceridemia exacerbates LPG in human cases 54, 99 and in animal models., Given these findings, the prevention of hypertriglyceridemia may be important in LPG treatment, and the efficacy of lipid-lowering agents including fibrates, as shown clinically and by pathology studies, has been reported in several cases in Japan.,, Additionally, Hu et al. compared patient and renal survival rates over 3 years between fenofibrate-treated and control groups, and they confirmed the significant availability of fenofibrate for LPG treatment. In contrast, in apoE2 homozygote glomer-ulopathy, fibrates have been used in some cases, but the effect is unclear. In advanced chronic kidney disease, triglyceride-rich lipoproteins including VLDL and IDL are increased in a manner similar to that observed in LPG regardless of the origin of chronic kidney disease. This observation suggests that lipid-lowering treatment with fibrates in LPG may provide insights on the prevention of renal dysfunction in chronic kidney disease, as pemafibrate, a selective peroxisome proliferator-activated receptor-a modulator, exhibited an effective safety profile and efficacy in patients with chronic kidney disease. Although apheresis therapy has been attempted in some cases of LPG, its effect is unclear. Interesting case reports from Italyand Chinahave been useful when discussing the mechanism of LPG. In an Italian case, apheresis was performed using a heparin-induced extracorporeal lipoprotein precipitation system instead of the usual LDL-apheresis with dextran sulfate columns to avoid anaphylactoid reaction to angiotensin-converting enzyme inhibitors, and this treatment approach led to complete remission within a very short time. The authors suggested that heparin in the heparin-induced extracorporeal lipoprotein precipitation system activated li-poprotein lipase and hepatic triglyceride lipase and easily removed triglyceride-rich lipoproteins, such as VLDLs and IDLs, which are generally elevated in LPG. In China, 13 patients with LPG were treated with staphylococcal protein-A immunoadsorption. As a result, a rapid decline in urine protein and serum apoE levels, with the disappearance of LPT in a repeat biopsy, was observed. As protein-A possesses strong affinity to the Fc portion of IgG and acts as an Fcg receptor, this effect may compensate for Fcg receptor defi-ciency, as studied in rat LPG by Kanamaru et al., and may lead to the improvement of LPG. Renal transplantation has been performed in several cases of LPG, but recurrence is recognized in most of them, as indicated in previous reports, and reviews., The apoE abnormality in the recipients may induce lipoprotein thrombi in the transplanted kidney. Conclusions ApoE is an important apolipoprotein that plays a central role in lipoprotein metabolism. Abnormal lipoprotein metabolism caused by apoE mutations is involved in variety of disorders. Among these, in relation to kidney diseases, there are several notable pathologic conditions (Table 1,,,,), and these conditions can be categorized into apoE-related glomerular disorders. Meanwhile, recent studies have revealed that the hyperactivity and suppression of macrophages, in addition to apoE mutations, are involved in these disorders. Thus, it must be considered that various factors other than apoE mutations are responsible for their development. Many unresolved issues remain, however, and research in this field must be expanded. All the authors declared no competing interests. The authors thank Drs. Kenichi Kudo, Shinichi Oikawa, Hiroshi Sato, Hisako Hirashima, Toshiyuki Komiya, Tamayo Kato, Satoshi Takasaki, Hitoshi Kodera, Kenji Ito, Hitoshi Nakashima, and Maho Watanabe for their collaboration, and we thank members of the Japanese Society of Kidney and Lipids for their warm support. Some of our studies described in this article were supported in part by grants-in-aid (nos. 10670982, 14571043, 16590806, 18590917, and 21591049) from the Ministry of Education, Science, Technology, Sport and Culture of Japan, and a Progressive Renal Diseases Research Projects grant from the Ministry of Health, Labour and Welfare of Japan.