Rapid detection of lactic acid bacteria by lateral flow immunochromatographic assay.

Lactic acid bacteria (LAB) cause quality deterioration including acidification and production of off-flavor components in food. To rapidly detect LAB, a lateral flow immunochromatographic assay (LFIA) was developed. An antibody (Ab) was obtained using Lactiplantibacillus plantarum as an immunogen, and the LFIA was performed based on this Ab. This showed positive reactions with LAB and a wide range of Gram-positive bacteria. An acid producing ability test using a semiconductor pH sensor was performed in combination with the LFIA. The bacteria testing positive in both the pH test (pH-positive) and the LFIA (LFIA-positive) were LAB. Twenty-three species of LAB in 13 genera could be detected specifically. Fresh foods and their equivalent spoiled foods were cultured for 0, 12, and 24 h, and the time required for the cultures to show LFIA-positive and pH-positive results was investigated. Of the 51 foods, 42 showed shorter culture times for the spoiled food than for the fresh food (spoilage detection rate: 82%). Amplicon analysis detected LAB including Leuconostoc citreum and Leuconostoc gelidum in spoiled foods. As this novel method can detect LAB in approximately 1 h, its use should make it possible to judge the freshness of food before shipping or sale.

TGF-ß1 is involved in senescence-related pathways in glomerular endothelial cells via p16 translocation and p21 induction.

p16 inhibits cyclin-dependent kinases and regulates senescence-mediated arrest as well as p21. Nuclear p16 promotes G1 cell cycle arrest and cellular senescence. In various glomerular diseases, nuclear p16 expression is associated with disease progression. Therefore, the location of p16 is important. However, the mechanism of p16 trafficking between the nucleus and cytoplasm is yet to be fully investigated. TGF-ß1, a major cytokine involved in the development of kidney diseases, can upregulate p21 expression. However, the relationship between TGF-ß1 and p16 is poorly understood. Here, we report the role of podocyte TGF-ß1 in regulating the p16 behavior in glomerular endothelial cells. We analyzed podocyte-specific TGF-ß1 overexpression mice. Although p16 was found in the nuclei of glomerular endothelial cells and led to endothelial cellular senescence, the expression of p16 did not increase in glomeruli. In cultured endothelial cells, TGF-ß1 induced nuclear translocation of p16 without increasing its expression. Among human glomerular diseases, p16 was detected in the nuclei of glomerular endothelial cells. In summary, we demonstrated the novel role of podocyte TGF-ß1 in managing p16 behavior and cellular senescence in glomeruli, which has clinical relevance for the progression of human glomerular diseases.

Rapid detection of total bacteria in foods using a poly-l-lysine-based lateral-flow assay.

Food safety and freshness are evaluated according to microbiological load. To analyze this load rapidly, a poly-l-lysine-based lateral-flow assay (PLFA) was developed. A total of 90 strains of bacteria that are often detected in spoiled foods, including Enterobacteriaceae, lactic acid bacteria, Pseudomonas, and Bacillus were detected using the PLFA. A positive signal was obtained when the bacterial concentration was ≥6 log10 (cfu/test). A total of 36 fresh foods (meats, pastries, lettuces, cabbages, radishes, and sprouts) and corresponding spoiled foods were cultured for 0, 3, 6, and 9 h to investigate how many hours were required for microbial detection using PLFA. The higher the number of bacteria in a food, the shorter was the culture time required for PLFA-positive results to be obtained, so the distinction between fresh and spoiled food could be made based on the time taken for the culture to become PLFA-positive. The coefficient of determination of the least squares regression between the time to become PLFA-positive and the initial log10 (cfu/g) bacterial count for the food was 0.9888. The test time for the PLFA, including pretreatment, was approximately 15-30 min. This novel method will enable the detection of total bacteria on the food processing site.

Author Correction: Circulating Apolipoprotein L1 is associated with insulin resistance-induced abnormal lipid metabolism.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Circulating Apolipoprotein L1 is associated with insulin resistance-induced abnormal lipid metabolism.

Circulating ApolipoproteinL1 (ApoL1) is a component of pre-ß-high-density lipoprotein (HDL), however little is known about the relationship of ApoL1 with cardiometabolic factors. Considering previous studies reporting the correlation of ApoL1 to triglyceride, we have hypothesized that ApoL1 associates with insulin-related metabolism. The current study examined their associations in 126 non-diabetic subjects and 36 patients with type 2 diabetes (T2DM). Non-diabetic subjects demonstrated triglyceride (standardized coefficients [s.c.] = 0.204, p < 0.05), body mass index (s.c. =0.232, p < 0.05) and HDL cholesterol (s.c. = -0.203, p < 0.05) as independent determinant of ApoL1 levels, and the significant elevation of ApoL1 in metabolic syndrome. Lipoprotein fractionation analysis revealed the predominant distribution of ApoL1 in large HDL fraction, and the significant increase of ApoL1 in large LDL fraction in high ApoL1 samples with insulin resistance. In T2DM, ApoL1 was higher in T2DM with metabolic syndrome, however ApoL1 was lower with ß cell dysfunction. Insulin significantly promotes ApoL1 synthesis and secretion in HepG2 cells. In conclusion, circulating ApoL1 may be associated with abnormal HDL metabolism in insulin resistant status. This may suggest a regulation of insulin signal on the ApoL1 level, leading to offer a novel insight to the ApoL1 biology.

Involvement of Elf3 on Smad3 activation-dependent injuries in podocytes and excretion of urinary exosome in diabetic nephropathy.

Diabetic nephropathy (DN) is among the most serious complications of diabetes mellitus, and often leads to end-stage renal disease ultimately requiring dialysis or renal transplantation. The loss of podocytes has been reported to have a role in the onset and progression of DN. Here, we addressed the activation mechanism of Smad3 signaling in podocytes. Expression of RII and activation of Smad3 were induced by AGE exposure (P<0.05). Reduction of the activation of RII-Smad3 signaling ameliorated podocyte injuries in Smad3-knockout diabetic mice. The bone morphogenetic protein 4 (BMP4) significantly regulated activation of RII-Smad3 signalings (P<0.05). Moreover, the epithelium-specific transcription factor, Elf3was induced by AGE stimulation and, subsequently, upregulated RII expression in cultured podocytes. Induction of Elf3 and activation of RII-Smad3 signaling, leading to a decrease in WT1 expression, were observed in podocytes in diabetic human kidneys. Moreover, AGE treatment induced the secretion of Elf3-containing exosomes from cultured podocytes, which was dependent on the activation of the TGF-ß-Smad3 signaling pathway. In addition, exosomal Elf3 protein in urine could be measured only in urinary exosomes from patients with DN. The appearance of urinary exosomal Elf3 protein in patients with DN suggested the existence of irreversible injuries in podocytes. The rate of decline in the estimated Glomerular Filtration Rate (eGFR) after measurement of urinary exosomal Elf3 protein levels in patients with DN (R2 = 0.7259) might be useful as an early non-invasive marker for podocyte injuries in DN.

Rapid detection of coliform bacteria using a lateral flow test strip assay.

Coliform bacteria in foods are enumerated at food processing plants and are used as sanitary and quality indicators. To detect coliform bacteria rapidly, seven Lateral Flow Test Strips (LFTSs) that can detect the genera Aeromonas, Citrobacter, Enterobacter, Hafnia, Klebsiella/Raoultella, Pantoea and Serratia were developed. For 55 tested food isolates, the detection rate of each individual LFTS assay was only 38% to 76%, but the detection rate of the 7 combined assays was 100%. For 38 culture collection strains, including clinical isolates, each individual LFTS assay had a detection rate of only 18% to 76%, but the 7 assays in combination had a detection rate of 89%. A feasibility study conducted on 20 types of meat (beef, chicken and pork) indicated that the LFTS assays detected coliform bacteria from 3 types of meat without incubation and from all other meats after 8 h of incubation. LFTS assays showed a positive signal when the meat was spoiled by more than 4.9 log10 (cfu/g) coliform bacteria. A longer incubation time led to increased bacterial counts, more positive LFTSs (1.8 at 8 h and 4.6 at 24 h) and a greater maximal signal intensity (1, 366 at 8 h and 2, 678 at 24 h). Thus, LFTSs of coliform bacteria have great potential for the rapid determination of food freshness as well as food sanitation status.

All-trans retinoic acid suppresses bone morphogenetic protein 4 in mouse diabetic nephropathy through a unique retinoic acid response element.

Diabetic nephropathy (DN) causes mesangial matrix expansion, which results in glomerulosclerosis and renal failure. Collagen IV (COL4) is a major component of the mesangial matrix that is positively regulated by bone morphogenetic protein 4 (BMP4)/suppressor of mothers against decapentaplegic (Smad1) signaling. Because previous studies showed that retinoids treatment had a beneficial effect on kidney disease, we investigated the therapeutic potential of retinoids in DN, focusing especially on the regulatory mechanism of BMP4. Diabetes was induced with streptozotocin in 12-wk-old male Crl:CD1(ICR) mice, and, 1 mo later, we initiated intraperitoneal injection of all-trans retinoic acid (ATRA) three times weekly. Glomerular matrix expansion, which was associated with increased BMP4, phosphorylated Smad1, and COL4 expression, worsened in diabetic mice at 24 wk of age. ATRA administration alleviated DN and downregulated BMP4, phosopho-Smad1, and COL4. In cultured mouse mesangial cells, treatment with ATRA or a retinoic acid receptor-α (RARα) agonist significantly decreased BMP4 and COL4 expression. Genomic analysis suggested two putative retinoic acid response elements (RAREs) for the mouse Bmp4 gene. Chromatin immunoprecipitation analysis and reporter assays indicated a putative RARE of the Bmp4 gene, located 11,488-11,501 bp upstream of exon 1A and bound to RARα and retinoid X receptor (RXR), which suppressed BMP4 expression after ATRA addition. ATRA suppressed BMP4 via binding of a RARα/RXR heterodimer to a unique RARE, alleviating glomerular matrix expansion in diabetic mice. These findings provide a novel regulatory mechanism for treatment of DN.

Myo-inositol oxygenase accentuates renal tubular injury initiated by endoplasmic reticulum stress.

Besides oxidant stress, endoplasmic reticulum (ER) stress has been implicated in the pathogenesis of various metabolic disorders affecting the kidney. These two forms of stresses are not mutually exclusive to each other and may operate by a feedback loop in worsening the cellular injury. To attest to this contention, studies were performed to assess whether in such a setting, there is worsening of tubulointerstitial injury. We employed tunicamycin as a model of ER stress and used tubular cells and mice overexpressing myo-inositol oxygenase (MIOX), an enzyme involved in glycolytic events with excessive generation of ROS. Concomitant treatment of tunicamycin and transfection of cells with MIOX-pcDNA led to a marked generation of ROS, which was reduced by MIOX-siRNA. Likewise, an accentuated expression of ER stress sensors, GRP78, XBP1, and CHOP, was observed, which was reduced with MIOX-siRNA. These sensors were markedly elevated in MIOX-TG mice compared with WT treated with tunicamycin. This was accompanied with marked deterioration of tubular morphology, along with impairment of renal functions. Interestingly, minimal damage and elevation of ER stressors was observed in MIOX-KO mice. Downstream events that were more adversely affected in MIOX-TG mice included accentuated expression of proapoptogenic proteins, proinflammatory cytokines, and extracellular matrix constituents, although expression of these molecules was unaffected in MIOX-KO mice. Also, their tunicamycin-induced accentuated expression in tubular cells was notably reduced with MIOX-siRNA. These studies suggest that the biology of MIOX-induced oxidant stress and tunicamycin-induced ER stress are interlinked, and both of the events may feed into each other to amplify the tubulointerstitial injury.

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.

Novel Interplay Between Smad1 and Smad3 Phosphorylation via AGE Regulates the Progression of Diabetic Nephropathy.

Diabetic nephropathy (DN) is the major cause of end-stage renal failure and is associated with increased morbidity and mortality compared with other causes of renal diseases. We previously found that Smad1 plays a critical role in the development of DN both in vitro and in vivo. However, functional interaction between Smad1 and Smad3 signaling in DN is unclear. Here, we addressed the molecular interplay between Smad1 and Smad3 signaling under a diabetic condition by using Smad3-knockout diabetic mice. Extracellular matrix (ECM) protein overexpression and Smad1 activation were observed in the glomeruli of db/db mice but were suppressed in the glomeruli of Smad3+/-; db/db mice. Smad3 activation enhanced the phosphorylation of Smad1 C-terminal domain but decreased the phosphorylation of linker domain, thus regulating Smad1 activation in advanced glycation end product-treated mesangial cells (MCs). However, forced phosphorylation of the Smad1 linker domain did not affect Smad3 activation in MCs. Phosphorylation of the Smad1 linker domain increased in Smad3+/-; db/db mice and probucol-treated db/db mice, which was consistent with the attenuation of ECM overproduction. These results indicate that Smad3 expression and activation or probucol treatment alters Smad1 phosphorylation, thus suggesting new molecular mechanisms underlying DN development and progression.

Urinary type IV collagen excretion is involved in the decline in estimated glomerular filtration rate in the Japanese general population without diabetes: A 5-year observational study.

Urinary type IV collagen (U-Col4) and albumin excretion is evaluated to monitor the development of diabetic kidney disease. However, U-Col4 excretion in the general population without diabetes has not yet been fully elucidated. In this study, 1067 participants without diabetes and with urinary albumin-creatinine ratio <300 mg/gCr (normo- or microalbuminuria) who underwent an annual health examination in 2004 were enrolled and observed for 5 years. They were divided according to the amount of U-Col4 or urinary albumin excreted. The decline in estimated glomerular filtration rate (eGFR) was calculated. In participants with eGFR ≥80 mL/min, abnormal U-Col4 excretion was indicated as a significant independent risk factor for 10% eGFR change per year, which is one of the prognostic factors for the development of end-stage kidney disease. Moreover, in contrast to urinary albumin excretion, U-Col4 excretion was not related to age or kidney function, suggesting that some individuals with abnormal U-Col4 excretion can have an independent hidden risk for the development of kidney dysfunction. In conclusion, it is important to measure U-Col4 excretion in the general population without diabetes to determine changes in renal features in every individual and help detect future complications such as diabetic kidney disease. If U-Col4 excretion is abnormal, kidney manifestation should be carefully followed up, even if the kidney function and urinalysis findings are normal.

Urinary IgG4 and Smad1 Are Specific Biomarkers for Renal Structural and Functional Changes in Early Stages of Diabetic Nephropathy.

Diabetic nephropathy (DN) is the major cause of end-stage kidney disease, but early biomarkers of DN risk are limited. Herein we examine urinary IgG4 and Smad1 as additional early DN biomarkers. We recruited 815 patients with type 2 diabetes; 554 patients fulfilled the criteria of an estimated glomerular filtration rate (eGFR) >60 mL/min and no macroalbuminuria at baseline, with follow-up for 5 years. Patients without macroalbuminuria were also recruited for renal biopsies. Urinary IgG4 and Smad1 were determined by enzyme-linked immunoassays using specific antibodies. The specificity, sensitivity, and reproducibility were confirmed for each assay. Increased urinary IgG4 was significantly associated with lower eGFR. The level of urinary IgG4 also significantly correlated with surface density of peripheral glomerular basement membrane (Sv PGBM/Glom), whereas Smad1 was associated with the degree of mesangial expansion-both classic pathological findings in DN. Baseline eGFR did not differ between any groups; however, increases in both urinary IgG4 and Smad1 levels at baseline significantly predicted later development of eGFR decline in patients without macroalbuminuria. These data suggest that urinary IgG4 and Smad1 at relatively early stages of DN reflect underlying DN lesions and are relevant to later clinical outcomes.

Rapid detection of Klebsiella pneumoniae, Klebsiella oxytoca, Raoultella ornithinolytica and other related bacteria in food by lateral-flow test strip immunoassays.

Bacteria of the genera Klebsiella and Raoultella, which are present in foods and the natural environment, are associated with health hazards in humans. In the present study, two types of strips-based methods were developed to detect these bacteria simply and quickly. One method used lateral-flow test strips (LFTS) in combination with anti-Klebsiella antibodies labeled with palladium nanoparticles that bind to target bacteria, allowing their visualization. In the other, the antibodies were immobilized on nitrocellulose membranes, and urease activity was measured using an ion sensitive field effect transistor pH sensor. Testing of a combination of these two methods on 72 cultured strains successfully identified all 25 strains of Klebsiella pneumoniae, Klebsiella oxytoca and Raoultella ornithinolytica. This approach also accurately identified 76 of 77 (99%) strains isolated from meats and pastries. When combined with preculture, this method was accurate in identifying 19 of 26 (73%) target bacteria in food. These results suggest that the novel combination of strip-based assays may be effective for the on-site monitoring of food production plants, and thereby enhance food safety.

A Novel Method of DAPI Staining for Differential Diagnosis of Renal Amyloidosis.

Amyloidosis is often overlooked because its clinical manifestations can mimic those of more-common diseases. It is important to get a precise diagnosis as early as possible for the prevention of further organ damages. Amyloidosis is a disorder caused by deposition of insoluble abnormal amyloid. The kidney is a frequent site of amyloid deposition. The amyloid fibrils have a characteristic appearance and generate birefringence under polarized light when stained with the Congo red dye. Classification of amyloidosis is based on the precursor protein that forms the amyloid fibrils and the distribution of amyloid deposits as either systemic or localized. Involvement of amyloid fibrils in kidneys mainly occurs as amyloid light-chain (AL) or amyloid A (AA) amyloidosis. The potassium permanganate method with Congo red staining was once used widely to discriminate AL and AA amyloidoses, but this method has a problem of false positive results. We found that extracellular and cytoplasmic glomerular 4', 6-diamidino-2-phenylindole (DAPI)-positive areas were clearly consistent with amyloid deposition in AL amyloidosis. In contrast, the overlapping staining was not seen in AA amyloidosis. Therefore, we propose that DAPI staining readily distinguishes AL renal amyloidosis from AA renal amyloidosis as a simple and reproducible histochemical method. J. Med. Invest. 64: 217-221, August, 2017.

Mesangial Cell Mammalian Target of Rapamycin Complex 1 Activation Results in Mesangial Expansion.

Human glomerular diseases can be caused by several different diseases, many of which include mesangial expansion and/or proliferation followed by glomerulosclerosis. However, molecular mechanisms underlying the pathologic mesangial changes remain poorly understood. Here, we investigated the role of the mammalian target of rapamycin complex 1 (mTORC1)-S6 kinase pathway in mesangial expansion and/or proliferation by ablating an upstream negative regulator, tuberous sclerosis complex 1 (TSC1), using tamoxifen-induced Foxd1-Cre mice [Foxd1ER(+) TSC1 mice]. Foxd1ER(+) TSC1 mice showed mesangial expansion with increased production of collagen IV, collagen I, and α-smooth muscle actin in glomeruli, but did not exhibit significant mesangial proliferation or albuminuria. Furthermore, rapamycin treatment of Foxd1ER(+) TSC1 mice suppressed mesangial expansion. Among biopsy specimens from patients with glomerular diseases, analysis of phosphorylated ribosomal protein S6 revealed mesangial cell mTORC1 activation in IgA nephropathy and in lupus mesangial proliferative nephritis but not in the early phase of diabetic nephropathy. In summary, mesangial cell mTORC1 activation can cause mesangial expansion and has clinical relevance for human glomerular diseases. This report also confirms that the tamoxifen-induced mesangium-specific Cre-loxP system is useful for studies designed to clarify the role of the mesangium in glomerular diseases in adults.

Transcriptional and Translational Modulation of myo-Inositol Oxygenase (Miox) by Fatty Acids: IMPLICATIONS IN RENAL TUBULAR INJURY INDUCED IN OBESITY AND DIABETES.

The kidney is one of the target organs for various metabolic diseases, including diabetes, metabolic syndrome, and obesity. Most of the metabolic studies underscore glomerular pathobiology, although the tubulo-interstitial compartment has been underemphasized. This study highlights mechanisms concerning the pathobiology of tubular injury in the context of myo-inositol oxygenase (Miox), a tubular enzyme. The kidneys of mice fed a high fat diet (HFD) had increased Miox expression and activity, and the latter was related to phosphorylation of serine/threonine residues. Also, expression of sterol regulatory element-binding protein1 (Srebp1) and markers of cellular/nuclear damage was increased along with accentuated apoptosis and loss of tubular brush border. Similar results were observed in cells treated with palmitate/BSA. Multiple sterol-response elements and E-box motifs were found in the miox promoter, and its activity was modulated by palmitate/BSA. Electrophoretic mobility and ChIP assays confirmed binding of Srebp to consensus sequences of the miox promoter. Exposure of palmitate/BSA-treated cells to rapamycin normalized Miox expression and prevented Srebp1 nuclear translocation. In addition, rapamycin treatment reduced p53 expression and apoptosis. Like rapamycin, srebp siRNA reduced Miox expression. Increased expression of Miox was associated with the generation of reactive oxygen species (ROS) in kidney tubules of mice fed an HFD and cell exposed to palmitate/BSA. Both miox and srebp1 siRNAs reduced generation of ROS. Collectively, these findings suggest that HFD or fatty acids modulate transcriptional, translational, and post-translational regulation of Miox expression/activity and underscore Miox being a novel target of the transcription factor Srebp1. Conceivably, activation of the mTORC1/Srebp1/Miox pathway leads to the generation of ROS culminating into tubulo-interstitial injury in states of obesity.

A Novel Interaction between FLICE-Associated Huge Protein (FLASH) and E2A Regulates Cell Proliferation and Cellular Senescence via Tumor Necrosis Factor (TNF)-Alpha-p21WAF1/CIP1 Axis.

Dysregulation of the cell proliferation has been implicated in the pathophysiology of a number of diseases. Cellular senescence limits proliferation of cancer cells, preventing tumorigenesis and restricting tissue damage. However, the role of cellular senescence in proliferative nephritis has not been determined. The proliferative peak in experimental rat nephritis coincided with a peak in E2A expression in the glomeruli. Meanwhile, E12 (an E2A-encoded transcription factor) did not promote proliferation of Mesangial cells (MCs) by itself. We identified caspase-8-binding protein FLICE-associated huge protein (FLASH) as a novel E2A-binding partner by using a yeast two-hybrid screening. Knockdown of FLASH suppressed proliferation of MCs. This inhibitory effect was partially reversed by the knockdown of E2A. In addition, the knockdown of FLASH induced cyclin-dependent kinase inhibitor p21WAF1/CIP1 (p21) expression, but did not affect p53 expression. Furthermore, overexpression of E12 and E47 induced p21, but not p53 in MCs, in the absence of FLASH. We also demonstrated that E2A and p21 expression at the peak of proliferation was followed by significant induction of FLASH in mesangial areas in rat proliferative glomerulonephritis. Moreover, we revealed that FLASH negatively regulates cellular senescence via the interaction with E12. We also demonstrated that FLASH is involved in the TNF-α-induced p21 expressions. These results suggest that the functional interaction of E2A and FLASH play an important role in cell proliferation and cellular senescence via regulation of p21 expression in experimental glomerulonephritis.

Bone Morphogenetic Protein 4 and Smad1 Mediate Extracellular Matrix Production in the Development of Diabetic Nephropathy.

Diabetic nephropathy is the leading cause of end-stage renal disease. It is pathologically characterized by the accumulation of extracellular matrix in the mesangium, of which the main component is α1/α2 type IV collagen (Col4a1/a2). Recently, we identified Smad1 as a direct regulator of Col4a1/a2 under diabetic conditions in vitro. Here, we demonstrate that Smad1 plays a key role in diabetic nephropathy through bone morphogenetic protein 4 (BMP4) in vivo. Smad1-overexpressing mice (Smad1-Tg) were established, and diabetes was induced by streptozotocin. Nondiabetic Smad1-Tg did not exhibit histological changes in the kidney; however, the induction of diabetes resulted in an ∼1.5-fold greater mesangial expansion, consistent with an increase in glomerular phosphorylated Smad1. To address regulatory factors of Smad1, we determined that BMP4 and its receptor are increased in diabetic glomeruli and that diabetic Smad1-Tg and wild-type mice treated with a BMP4-neutralizing antibody exhibit decreased Smad1 phosphorylation and ∼40% less mesangial expansion than those treated with control IgG. Furthermore, heterozygous Smad1 knockout mice exhibit attenuated mesangial expansion in the diabetic condition. The data indicate that BMP4/Smad1 signaling is a critical cascade for the progression of mesangial expansion and that blocking this signal could be a novel therapeutic strategy for diabetic nephropathy.

Dual involvement of growth arrest-specific gene 6 in the early phase of human IgA nephropathy.

BACKGROUND: Gas6 is a growth factor that causes proliferation of mesangial cells in the development of glomerulonephritis. Gas6 can bind to three kinds of receptors; Axl, Dtk, and Mer. However, their expression and functions are not entirely clear in the different glomerular cell types. Meanwhile, representative cell cycle regulatory protein p27 has been reported to be expressed in podocytes in normal glomeruli with decreased expression in proliferating glomeruli, which inversely correlated with mesangial proliferation in human IgA nephropathy (IgAN). METHODS: The aim of this study is to clarify Gas6 involvement in the progression of IgAN. Expression of Gas6/Axl/Dtk was examined in 31 biopsy proven IgAN cases. We compared the expression levels with histological severity or clinical data. Moreover, we investigated the expression of Gas6 and its receptors in cultured podocytes. RESULTS: In 28 of 31 cases, Gas6 was upregulated mainly in podocytes. In the other 3 cases, Gas6 expression was induced in endothelial and mesangial cells, which was similar to animal nephritis models. Among 28 podocyte type cases, the expression level of Gas6 correlated with the mesangial hypercellularity score of IgAN Oxford classification and urine protein excretion. It also inversely correlated with p27 expression in glomeruli. As for the receptors, Axl was mainly expressed in endothelial and mesangial cells, while Dtk was expressed in podocytes. In vitro, Dtk was expressed in cultured murine podocytes, and the expression of p27 was decreased by Gas6 stimulation. CONCLUSIONS: Gas6 was uniquely upregulated in either endothelial/mesangial cells or podocytes in IgAN. The expression pattern can be used as a marker to classify IgAN. Gas6 has a possibility to be involved in not only mesangial proliferation via Axl, but also podocyte injury via Dtk in IgAN.