Fibulin7 aggravates calcium oxalate-induced acute kidney injury by binding to calcium oxalate crystals.

Fibulin7 (Fbln7) is a matricellular protein that is structurally similar to short fibulins but does not possess elastogenic abilities. Fbln7 is localized on the cell surface of the renal tubular epithelium in the adult kidney. We previously reported that Fbln7 binds artificial calcium phosphate particles in vitro, and that heparin counteracts this binding by releasing Fbln7 from the cell surface. Fbln7 gene (Fbln7) deletion in vivo decreased interstitial fibrosis and improved renal function in a high phosphate diet-induced chronic kidney disease mouse model. However, the contribution of Fbln7 during acute injury response remains largely unknown. We hypothesized that Fbln7 serves as an exacerbating factor in acute kidney injury (AKI). We employed three AKI models in vivo and in vitro, including unilateral ureteral obstruction (UUO), cisplatin-induced AKI, and calcium oxalate (CaOx)-induced AKI. Here, we report that Fbln7KO mice were protected from kidney damage in a CaOx-induced AKI model. Using HEK293T cells, we found that Fbln7 overexpression enhanced the CaOx-induced upregulation of EGR1 and LAMB3, and that heparin treatment canceled this effect. Interestingly, the protective function observed in Fbln7KO kidneys was limited to the CaOx-induced AKI model, while Fbln7KO mice were not protected against UUO-induced renal fibrosis or cisplatin-induced renal tubular damage. Taken together, our study indicates that Fbln7 mediates the local deposition of CaOx and damages the renal tubular epithelium. Releasing Fbln7 from the cell surface via heparin/heparin derivatives or Fbln7 inhibitory antibodies may provide a general strategy to mitigate calcium crystal-induced kidney injuries.

Risk factors for cytomegalovirus reactivation in patients with kidney disease under immunosuppressive therapy.

BACKGROUND: Cytomegalovirus (CMV) is a herpes virus that causes latent infections, and its reactivation due to immunosuppression can cause fatal complications. CMV reactivation is a complication frequently occurring in patients with kidney disease who require immunosuppressive therapy, and, therefore, this study retrospectively examined its risk factors. METHODS: Patients who received immunosuppressive therapy and underwent the CMV antigenemia test (CMV antigenemia: C7-HRP) for the treatment of primary nephritis (minimal change disease, membranous nephropathy, membranoproliferative glomerulonephritis, focal glomerulosclerosis, and IgA nephropathy) and anti-neutrophil cytoplasmic autoantibody (ANCA)-associated nephritis diagnosed at Saiseikai Kurihashi Hospital from January 2014 to December 2019 were recruited as study participants. Risk factors of CMV reactivation were examined using univariable and multivariable analyses. RESULTS: Among the 64 patients (36 men and 28 women; median age, 72 years) included, 34 had primary nephritis (20 minimal disease changes, 10 membranous nephropathy, 1 membranoproliferative glomerulonephritis, 1 focal glomerulosclerosis, and 2 IgA nephropathy) and 30 had ANCA-associated nephritis. Regarding glucocorticoid (GC), 43 patients received oral GC therapy, whereas 21 received GC pulse therapy. CMV reactivation participants showed significant differences in age, ANCA-associated nephritis, hemoglobin level, lymphocyte count, maximum GC dosage, and hemodialysis in univariable analysis. Multivariate analysis showed significantly lower lymphocyte counts in CMV-reactivated patients, but no significant difference in other factors. CONCLUSION: In patients with kidney disease, who require immunosuppressive therapy, CMV reactivation risk is high in patients with low lymphocyte count, and monitoring CMV during the treatment course could lead to early diagnosis and treatment of CMV disease.

A case of medullary cystic kidney disease with rapid progressive renal dysfunction and kidney enlargement.

Medullary cystic kidney disease (MCKD) is usually associated with slowly progressive kidney injury. However, we encountered a case of MCKD with rapidly progressive kidney injury and irreversible renal dysfunction. A 63-year-old woman presented with a 4-month history of hypertension and rapidly progressive renal dysfunction. On admission, her blood pressure was slightly elevated (158/85 mmHg). The scrum creatinine (11.57 mg/dL) was markedly elevated. Urinalysis showed occult hematuria and proteinuria(1.06 g/gCr). /ß2- microglobulin 45,000 µg/ L, N-acetyl-/ß-D-glucosaminidase 5.6 U/L. Neither ultrasonography nor computed tomography revealed any evidence of renal medullary cysts. Both kidneys showed an irregular surface and enlargement. Microscopic evaluation of the renal biopsy revealed extensive tubular dilatation and atrophy with interstitial fibrosis. Often glomeruli, one had global sclerosis and the others were normal. The tubular dilatation was more marked in the distal than in the proximal tubules, according to the immunohistochemical findings of positivity for epithelial membrane antigen (EMA), a marker of distal tubules, and negativity for CD 10, a marker of proximal tubules. No immunoglobulin or complement deposition was detected in either the glomeruli or the tubules. Electron microscopy revealed disintegration of the tubular basement membrane with fragile thinning and lamination of the membrane. These pathological findings were compatible with MCKD. This was a case of MCKD diagnosed incidentally in an elderly patient who presented with rapidly progressive kidney injury accompanied by hypertension. Renal biopsy was necessary for the diagnosis.

Citric acid-based bicarbonate dialysate attenuates aortic arch calcification in maintenance hemodialysis patients: a retrospective observational study.

BACKGROUND: Progression of aortic calcification is associated with all-cause and cardiovascular mortality in hemodialysis patients. Blood calciprotein particle (CPP) levels are associated with coronary artery calcification and were reported to be inhibited when using citric acid-based bicarbonate dialysate (CD). Therefore, this study aimed to examine the effect of CD on the progression of the aortic arch calcification score (AoACS) and blood CPP levels in hemodialysis patients. METHODS: A 12-month retrospective observational study of 262 hemodialysis patients was conducted. AoACS was evaluated by calculating the number of calcifications in 16 segments of the aortic arch on chest X-ray (minimum score is 0; maximum score is 16 points). The patients were divided into the following groups according to their baseline AoACS: grade 0, AoACS = 0 points; grade 1, AoACS 1-4 points; grade 2, AoACS 5-8 points; grade 3, AoACS 9 points or higher. Patients on bisphosphonates or warfarin or with AoACS grade 3 were excluded. Progression, defined as ΔAoACS (12-month score - baseline score) > 0 points, was compared between the CD and acetic acid-based bicarbonate dialysate (AD) groups before and after adjusting the background using propensity score matching. RESULTS: The AoACS progression rate was significantly lower in the CD group than in the AD group (before matching: P = 0.020, after matching: P = 0.002). Multivariate logistic regression analysis showed that CD was significantly associated with AoACS progression (odds ratio 0.52, 95% confidence interval 0.29‒0.92, P = 0.025). CONCLUSION: CD may slow the progression of vascular calcification in hemodialysis patients.

Reduced Klotho expression level in kidney aggravates renal interstitial fibrosis.

Renal expression of the klotho gene is markedly suppressed in chronic kidney disease (CKD). Since renal fibrosis is the final common pathology of CKD, we tested whether decreased Klotho expression is a cause and/or a result of renal fibrosis in mice and cultured renal cell lines. We induced renal fibrosis by unilateral ureteral obstruction (UUO) in mice with reduced Klotho expression (kl/+ mice) and compared them with wild-type mice. The UUO kidneys from kl/+ mice expressed significantly higher levels of fibrosis markers such as α-smooth muscle actin (α-SMA), fibronectin, and transforming growth factor-ß(1) (TGF-ß(1)) than those from wild-type mice. In addition, in cultured renal fibroblast cells (NRK49F), the levels of α-SMA and PAI1 expression were significantly suppressed by addition of recombinant Klotho protein to the medium. The similar effects were observed by a TGF-ß(1) receptor inhibitor (ALK5 inhibitor). These observations suggest that low renal Klotho expression enhances TGF-ß(1) activity and is a cause of renal fibrosis. On the other hand, TGF-ß(1) reduced Klotho expression in renal cultured epithelial cells (inner medullary collecting duct and human renal proximal tubular epithelium), suggesting that low renal Klotho expression is a result of renal fibrosis. Taken together, renal fibrosis can trigger a deterioration spiral of Klotho expression, which may be involved in the pathophysiology of CKD progression.

Effect of single-dose rituximab on primary glomerular diseases.

BACKGROUND: A paradigm shift from such toxic 'nonspecific' therapies to selective immunomodulating regimens is necessary for glomerular diseases. Rituximab, which acts by inhibiting CD20-mediated B cell proliferation and differentiation, could be effective in the treatment of nephrotic syndrome as shown in recent reports. DESIGN: To assess the effects of rituximab in patients with primary glomerular diseases, including minimal-change disease, immunoglobulin A (IgA) nephropathy, focal segmental glomerulonephritis, membranous nephropathy and membranoproliferative glomerulonephritis, we performed a prospective trial of the effects of single-dose rituximab therapy in 24 patients. We prospectively evaluated the serum and urinary biochemical parameters before and after 6 months of therapy. RESULTS: In all of the patients studied, depletion of CD19 and CD20 cells was noted, with significant reduction in the degree of proteinuria from 3.7 ± 3.4 g/day at baseline to 1.3 ± 2.0 g/day at 6 months after the drug administration (p = 0.002). However, no significant changes of the serum creatinine, urinary RBC sediment, serum CD4/8 or serum IL-4 levels were observed at 6 months after the drug administration. In subjects with IgA nephropathy, while depletion of CD19 and CD20 cells was noted, no significant change in the severity of proteinuria was observed at 6 months after the drug administration as compared with the level at the baseline. CONCLUSION: For the treatment of primary glomerular diseases, the use of a single dose of rituximab is demonstrated with no serious adverse events. Further study of the mechanism of action of rituximab in successfully treated patients could encourage new perspectives in the treatment of primary glomerular diseases.

Effect of the antiplatelet agent cilostazol on endovascular inflammatory biochemical parameters and the clinical symptoms of peripheral artery disease and restless legs syndrome in hemodialysis patients.

BACKGROUND: Peripheral artery disease (PAD) is a common complication in patients receiving hemodialysis (HD). Cilostazol is used for the treatment of ischemic symptoms in patients with PAD, based on its antiplatelet and vasodilating effects. In addition to these beneficial effects on clinical symptoms in PAD patients, cilostazol has been proposed to have additional effects on clinical symptoms in patients with restless legs syndrome (RLS) via the upregulation of dopamine. We performed an observational, prospective study to evaluate the effect of cilostazol on several clinical and biochemical parameters in HD patients with PAD and RLS. METHODS: All the study patients received cilostazol treatment for 12 months. During the study period, several biochemical parameters, such as high-sensitivity CRP, von Willebrand antigen (VW-Ag), triglyceride (TG), high-density lipoprotein (HDL) and malondialdehyde-modified low-density lipoprotein, were monitored. A questionnaire on the physical status of PAD and RLS was also completed. 45 HD patients who received cilostazol were compared with a control group of 22 patients. RESULTS: The patients who continued cilostazol treatment exhibited a improvement in their serum inflammatory and biochemical parameters (VW-Ag, TG, HDL). Although PAD and RLS scores were not improved by multivariate analysis, several patients showed improvement of signs and symptoms which were included in the PAD or RLS scores. CONCLUSION: The treatment of HD patients with cilostazol improved some of the lipid-related and endovascular inflammatory biochemical parameters associated with PAD, and relieved the clinical symptoms and physical status of PAD in some cases.

Recombinant human erythropoietin mitigates reductions in renal klotho expression.

BACKGROUND: Erythropoietin (EPO) and Klotho expression have both been detected in the kidney. Since a recent study suggested that both EPO and Klotho mitigate kidney damage, we explored the relation between EPO and Klotho in a doxorubicin hydrochloride (DXR)-induced rat nephropathy model treated with recombinant human erythropoietin (rhEPO). METHODS: Male Sprague-Dawley rats were subjected to DXR-induced nephropathy. The rhEPO group was intracutaneously injected with rhEPO twice weekly at 4-16 weeks after the DXR injection. The rats were sacrificed at 16 weeks after the DXR administration. Expression of renal Klotho, HSP70, alpha-smooth-muscle actin and E-cadherin were assessed using real-time PCR or western blotting. The hematocrit, plasma creatinine and phosphate levels were also determined. Immunohistochemical studies and Masson-trichrome staining were performed. RESULTS: The renal Klotho mRNA and Klotho protein expressions were significantly reduced in the DXR nephropathy group. Treatment with rhEPO improved the serum creatinine, phosphate level and histological changes observed in the DXR nephropathy group. The reduction in Klotho expression induced by DXR nephropathy was mitigated by rhEPO administration. CONCLUSION: rhEPO is involved in the pathophysiology of DXR nephropathy. rhEPO mitigated elevated plasma phosphate concentrations in an experimental model of chronic kidney disease via the expression of Klotho.

Klotho reduces apoptosis in experimental ischaemic acute kidney injury via HSP-70.

BACKGROUND: High Klotho expression has been detected in the kidney, and since the results of a recent study suggested that Klotho induction mitigates ischaemic damage in the kidney, in the present study we explored the mechanism by which Klotho expression reduces renal ischaemia-reperfusion injury (IRI). METHODS: Male mice were subjected to bilateral renal ischaemia for 30 min and reperfusion for 24 h, or to a sham operation. Both the IRI group and the sham group were intravenously injected with an adenovirus harbouring the mouse Klotho gene (ad-kl) before renal IRI. In addition, mIMCD3 cells induced to overexpress Klotho by transferring the Klotho gene with ad-kl were analysed by DNA microarray and real-time PCR. Renal expression of Klotho and several genes selected by DNA microarray were assessed by real-time PCR or Western blotting, and TUNEL staining was performed to assess apoptosis. RESULTS: Prior administration of ad-kl to the mice resulted in robust induction of Klotho mRNA in the kidney and liver. Ad-kl transfer improved the plasma creatinine values and mitigated the histological damage and apoptosis induced by IRI. Expression of several genes was altered in mIMCD3 cells as a result of the change in Klotho expression, and expression of heat shock protein 70 (HSP70), in particular, was up-regulated in ad-kl mouse kidneys and HK2 cells. CONCLUSION: The results suggest that Klotho is involved in the pathophysiology of IRI. Klotho mitigates apoptosis in experimental ischaemic acute kidney injury via expression of HSP70.

Frequency of renal disease in Japan: an analysis of 2,404 renal biopsies at a single center.

BACKGROUND: Evaluating the frequency of renal disease according to sex, age, time period and ethnicity is of considerable importance. METHODS: Disease frequency was evaluated in a total of 2,404 cases that had undergone a renal biopsy at Tokyo Women's Medical University between 1979 and 2008. RESULTS: The overall frequencies of primary glomerulonephritis (GN) and secondary GN were 77.8 and 14.4%, respectively. Primary GN and nephrosclerosis occurred more frequently among men, while secondary GN was more frequent among women. Primary GN decreased and secondary GN increased with advancing age. Immunoglobulin A nephropathy was the most common form of primary GN during each time period and also gradually increased over time (44.4-57.4%). The most common form of secondary GN was lupus nephritis (59.0%); this disease was commonly observed in women (79.3%) but not as frequently among men (27.9%). Our data regarding the frequencies of each form of primary GN were almost the same as data from other regions of Japan and East Asia but were quite different from data originating in West Asia and South America. CONCLUSIONS: Our epidemiological results may be useful for analyzing the morbidity of renal disease.

Association between clinical characteristics and AL amyloid deposition in the kidney.

Renal involvement is very common in AL amyloidosis. Our aim was to investigate associations between the clinical characteristics and renal pathology of patients with AL amyloidosis. The data of 11 adult Japanese patients with AL amyloidosis and renal involvement were analyzed retrospectively. To assess the difference based on the pattern of distribution of amyloid deposition, we divided the patients into a group with the capillary form and a group with the small vessel form, and compared the clinical characteristics of the two groups. Concerning AL amyloidosis, the small vessel form group was associated with cardiac involvement and left ventricular thickening compared with the capillary form group (p = 0.03). There were no significant differences between the groups in the rates of patient survival and renal survival. There was a negative correlation between grade of amyloid deposition and renal survival duration (p = 0.04, r² = 0.66). The degree of amyloid deposition was not correlated with the extent of proteinuria or renal function. These findings suggest that the vascular deposition pattern of amyloid in the kidney is important for determining patient survival and renal outcome.

Clinical effects of calcium channel blockers and renin-angiotensin-aldosterone system inhibitors on changes in the estimated glomerular filtration rate in patients with polycystic kidney disease.

BACKGROUND: In the tubular cells of patients with polycystic kidney disease (PKD), a reduced intracellular Ca(2+) level accelerates cell proliferation, resulting in cyst formation. Thus, whether calcium channel blockers (CCB) are useful for the treatment of hypertension in patients with PKD is questionable. METHODS: Thirty-two outpatients with autosomal dominant PKD (ADPKD) were treated at Tokyo Women's Medical University between 2003 and 2008; these patients were studied retrospectively. Periods during which the antihypertensive drug prescriptions for CCB and/or renin-angiotensin-aldosterone system inhibitors (RAAS-I; including angiotensin converting enzyme inhibitor and angiotensin II receptor blocker) had not been changed for at least 1 year and during which time a diuretic agent had not been prescribed were selected from among the clinical histories of the 32 outpatients. Consequently, 31 periods of 31 patients were analyzed, and mean treatment duration was 2.4 years in this study. The estimated glomerular filtration rate (eGFR) was used to evaluate renal function. To evaluate the influence of CCB and RAAS-I with respect to the decrease of the eGFR, analysis of covariance (ANCOVA), including confounding factors [baseline eGFR, mean systolic blood pressure (SBP), mean diastolic blood pressure (DBP)], was used. Only CCB significantly contributed to a reduction in ∆eGFR in both a univariable ANCOVA and a multivariable ANCOVA. None of the confounding factors, RAAS-I, the baseline eGFR, or blood pressure, contributed to reductions in ∆eGFR. CONCLUSION: These results suggest that from a renoprotective perspective, CCB should possibly be avoided in patients with PKD unless treatment for resistant hypertension is necessary.

Pharmacokinetics of orally administered tacrolimus in lupus nephritis patients.

The pharmacokinetics of orally administered tacrolimus were examined in six female lupus nephritis patients (mean age 43 years, range 24-55 years). Tacrolimus (3 mg) was administered after supper, and blood tacrolimus concentrations were measured just prior to dosing and 1, 2, 4, 6, 8, 12 and 24 h after administration. The maximum blood concentration (C(max)) was observed 4-8 h (mean: 6.7 h) after administration. The mean C(max) and area under the tacrolimus concentrationti-me curve (AUC(0-24 h)) were 12.7 ng/ml and 163.1 ng x h/ml, respectively. Although there was a weak correlation between AUC(0-24 h) values and tacrolimus concentrations 2, 4, and 6 h after administration, concentrations at 12 h and 24 h were highly correlated with AUC(0-24 h) values, suggesting that the trough concentration (C(24 h)) and C(12 h) are valid markers for therapeutic tacrolimus monitoring. Enzyme-linked immunoabsorbent assay (ELISA) and microparticle enzyme immunoassay (MEIA) measurements of blood tacrolimus concentrations were similar. We recommend that monitoring should be carried out by C(12 h) in lupus nephritis outpatients.

[Case of MMF monotherapy for membranous nephropathy].

We report the case of a 58-year-old male patient who visited our hospital for the management of edema and proteinuria. He was diagnosed as having nephrotic syndrome, with serum total protein and albumin levels of 4.6 g/dL and 2.1 g/dL, respectively, and a urinary protein excretion level of 6.0 g/day. A percutaneous renal biopsy showed features of membranous glomerulonephritis, with capillary-wall granular deposits of IgG and C3 on immunofluorescence and subepithelial immune complex deposits on electron microscopy. No other secondary cause of membranous glomerulopathy was found even after extensive investigations. The patient was started on mycophenolate mofetil (MMF) monotherapy (1,500 mg/day), and 18 months after the start of this therapy, the proteinuria decreased to 0.5 g/day, with return to a normal serum albumin level. No digestive symptoms, kidney function worsening or increase in blood pressure were noted during treatment. These findings suggest that MMF monotherapy is effective and safe for the treatment of membranous nephropathy.

[Clinico-pathological features and outcome in adult patients with Henoch-Schönlein purpura nephritis].

We examined the data of 24 patients with Henoch-Schönlein purpura nephritis (HSPN) over a 5-year follow-up period. Proteinuria, sediment RBC and CRP significantly decreased between the time of diagnosis and the end of the 5-year period. In the steroid usage group (n = 16), proteinuria was significantly higher, and crescent formation was significant higher at the time of diagnosis than in the non-steroid usage group (n = 8). However, there was no significant difference in the decrease in eGFR from the baseline at the end of the 5-year period between the two groups. Furthermore, to clarify the factors influencing the risk of renal function deterioration, we divided the patients into two groups, the (delta eGFR/pre eGFR) <0.25 group (n = 13) and (delta eGFR/pre eGFR) >0.25 group (n = 11), and compared the clinico-pathophysiological characteristics between the two groups. In the (delta eGFR/pre eGFR) >0.25 group, the ratio of glomerular obsolescence at the time of diagnosis was significantly higher than in the (delta eGFR/pre eGFR) <0.25 group. Glomerular obsolescence was identified as an independent risk factor for renal function deterioration. In this study, the prognosis of HSPN was related to glomerular obsolescence rather than to the disease activity. It may be necessary to consider the decrease in nephrons, in accordance with non-immunological glomerular obsolescence, in addition to immunological treatment to clarify the prognosis.

ATF3 protects against renal ischemia-reperfusion injury.

Oxidative stress-induced cell death plays a major role in the progression of ischemic acute renal failure. Using microarrays, we sought to identify a stress-induced gene that may be a therapeutic candidate. Human proximal tubule (HK2) cells were treated with hydrogen peroxide (H2O2) and RNA was applied to an Affymetrix gene chip. Five genes were markedly induced in a parallel time-dependent manner by cluster analysis, including activating transcription factor 3 (ATF3), p21(WAF1/CiP1) (p21), CHOP/GADD153, dual-specificity protein phosphatase, and heme oxygenase-1. H2O2 rapidly induced ATF3 approximately 12-fold in HK2 cells and approximately 6.5-fold in a mouse model of renal ischemia-reperfusion injury. Adenovirus-mediated expression of ATF3 protected HK2 cells against H2O2-induced cell death, and this was associated with a decrease of p53 mRNA and an increase of p21 mRNA. Moreover, when ATF3 was overexpressed in mice via adenovirus-mediated gene transfer, ischemia-reperfusion injury was reduced. In conclusion, ATF3 plays a protective role in renal ischemia-reperfusion injury and the mechanism of the protection may involve suppression of p53 and induction of p21.

Late-Onset Neutropenia after Rituximab Treatment for Adult-Onset Nephrotic Syndrome.

A 41-year-old woman developed nephrotic syndrome at the age of 32 and was diagnosed with minimal change nephrotic syndrome based on a renal biopsy. Although remission was achieved with administration of prednisolone (PSL) and cyclosporine, the nephrotic syndrome recurred. She was also started on rituximab (RTX). She developed late-onset neutropenia after RTX treatment (R-LON) and improved 17 days later. Although the majority of R-LON cases undergo spontaneous remission, cases of death have been reported. This report is intended to warn about R-LON, since the use of RTX for adult-onset nephrotic syndrome is expected to increase in the future.

[Effects of steroid therapy on membranous nephropathy].

BACKGROUND: Although membranous nephropathy is a common cause of nephrotic syndrome in adults, its treatment remains under debate. METHODS: To clarify the effects of steroid therapy, the data of 51 Japanese adult patients with idiopathic membranous nephropathy who received treatment at our department were analyzed retrospectively. We divided the patients with nephrotic syndrome and a serum creatinine level <1.7 mg/dL, into two groups: the steroid therapy group (n=20) and the non-steroid therapy group (n=7), and compared the clinical characteristics between the two groups. RESULTS: Significantly decreased proteinuria levels (p<0.05) after 2 and 5 years were observed in the steroid therapy group as compared to the non-steroid therapy group. There was no significant difference in the serum creatinine levels after 2 and 5 years between the steroid therapy group and the non-steroid therapy group. CONCLUSION: Steroid therapy in idiopathic membranous nephropathy showed good efficacy in patients with nephrotic syndrome.

Fibroblast growth factor 23 is upregulated in the kidney in a chronic kidney disease rat model.

The hormone fibroblast growth factor 23 (FGF23) is secreted from bone and is involved in phosphorus (P) metabolism. FGF23 mainly binds the FGF receptor, which interacts with αKlotho in the kidney or parathyroid and regulates Na-dependent phosphate co-transporter type IIa (NaPi-IIa) and type IIc (NaPi-IIc) expression, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) activity, and parathyroid hormone (PTH) secretion. In this study, we utilized hemi-nephrectomized rats fed a high-P diet (HP Nx), rats subjected to a partial nephrectomy (PN) and rats with doxorubicin-induced renal failure (DXR) as chronic kidney disease (CKD) animal models and analyzed the P metabolism and FGF23 expression in the kidneys in each CKD model. We cultured HK2 cells with a high level of P, 1,25(OH)2D3 or transforming growth factor-ß1 (TGFß1) to investigate the FGF23 expression mechanism. In both the HP Nx and PN rats, the blood FGF23 and PTH levels were increased. However, the 1,25(OH)2D3 level was increased in the HP Nx rats and decreased in the PN rats. In all three animal models, the mRNA expression of αKlotho, NaPi-IIa and NaPi-IIc was decreased, and the mRNA expression of TGFß1, collagen1a1, osteopontin and FGF23 was elevated in the kidney. FGF23 protein and mRNA were expressed at high levels in the extended tubule epithelium, which was an osteopontin-positive region in the HP and PN rats. FGF23 and osteopontin mRNAs were expressed in HK2 cells incubated with TGFß1; however, these levels were not altered in HK2 cells incubated with 1,25(OH)2D3 and high P levels in vitro. Altogether, FGF23 is expressed in the kidneys in CKD model rats. Following stimulation with TGFß1, the injured renal tubular epithelial cells are strongly suspected to express both FGF23 and osteopontin. FGF23 produced in the kidney might contribute to P metabolism in subjects with CKD.

Fibulin-7, a heparin binding matricellular protein, promotes renal tubular calcification in mice.

Ectopic calcification occurs during development of chronic kidney disease and has a negative impact on long-term prognosis. The precise molecular mechanism and prevention strategies, however, are not established. Fibulin-7 (Fbln7) is a matricellular protein structurally similar to elastogenic short fibulins, shown to bind dental mesenchymal cells and heparin. Here, we report that Fbln7 is highly expressed in renal tubular epithelium in the adult kidney and mediates renal calcification in mice. In vitro analysis revealed that Fbln7 bound heparin at the N-terminal coiled-coil domain. In Fbln7-expressing CHO-K1 cells, exogenous heparin increased the release of Fbln7 into conditioned media in a dose-dependent manner. This heparin-induced Fbln7 release was abrogated in CHO-745 cells lacking heparan sulfate proteoglycan or in CHO-K1 cells expressing the Fbln7 mutant lacking the N-terminal coiled-coil domain, suggesting that Fbln7 was tethered to pericellular matrix via this domain. Interestingly, Fbln7 knockout (Fbln7-/-) mice were protected from renal tubular calcification induced by high phosphate diet. Mechanistically, Fbln7 bound artificial calcium phosphate particles (aCPP) implicated in calcification and renal inflammation. Binding was decreased significantly in Fbln7-/- primary kidney cells relative to wild-type cells. Further, overexpression of Fbln7 increased binding to aCPP. Addition of heparin reduced binding between aCPP and wild-type cells to levels of Fbln7-/- cells. Taken together, our study suggests that Fbln7 is a local mediator of calcium deposition and that releasing Fbln7 from the cell surface by heparin/heparin derivatives or Fbln7 inhibitory antibodies may provide a novel strategy to prevent ectopic calcification in vivo.