Takahito Ito

Fibrosis and adipogenesis originate from a common mesenchymal progenitor in skeletal muscle

Accumulation of adipocytes and collagen type-I-producing cells (fibrosis) is observed in muscular dystrophies. The origin of these cells had been largely unknown, but recently we identified mesenchymal progenitors positive for platelet-derived growth factor receptor alpha (PDGFRα) as the origin of adipocytes in skeletal muscle. However, the origin of muscle fibrosis remains largely unknown. In this study, clonal analyses show that PDGFRα+ cells also differentiate into collagen type-I-producing cells. In fact, PDGFRα+ cells accumulated in fibrotic areas of the diaphragm in the mdx mouse, a model of Duchenne muscular dystrophy. Furthermore, mRNA of fibrosis markers was expressed exclusively in the PDGFRα+ cell fraction in the mdx diaphragm. Importantly, TGF-β isoforms, known as potent profibrotic cytokines, induced expression of markers of fibrosis in PDGFRα+ cells but not in myogenic cells. Transplantation studies revealed that fibrogenic PDGFRα+ cells mainly derived from pre-existing PDGFRα+ cells and that the contribution of PDGFRα− cells and circulating cells was limited. These results indicate that mesenchymal progenitors are the main origin of not only fat accumulation but also fibrosis in skeletal muscle.

Production of adiponectin, an anti-inflammatory protein, in mesenteric adipose tissue in Crohn’s disease

Background and aims: A characteristic feature of Crohn’s disease (CD) is mesenteric adipose tissue hypertrophy. Mesenteric adipocytes or specific proteins secreted by them may play a role in the pathogenesis of CD. We recently identified adiponectin as an adipocyte specific protein with anti-inflammatory properties. Here we report on expression of adiponectin in mesenteric adipose tissue of CD patients. Methods and results: Mesenteric adipose tissue specimens were obtained from patients with CD (n = 22), ulcerative colitis (UC) (n = 8) and, for controls, colon carcinoma patients (n = 28) who underwent intestinal resection. Adiponectin concentrations were determined by enzyme linked immunosorbent assay, and adiponectin mRNA levels were determined by real time quantitative reverse transcription-polymerase chain reaction. Tissue concentrations and release of adiponectin were significantly increased in hypertrophied mesenteric adipose tissue of CD patients compared with normal mesenteric adipose tissue of CD patients (p = 0.002, p = 0.040, respectively), UC patients (p = 0.002, p = 0.003), and controls (p<0.0001, p<0.0001). Adiponectin mRNA levels were significantly higher in hypertrophied mesenteric adipose tissue of CD patients than in paired normal mesenteric adipose tissue from the same subjects (p = 0.024). Adiponectin concentrations in hypertrophied mesenteric adipose tissue of CD patients with an internal fistula were significantly lower than those of CD patients without an internal fistula (p = 0.003). Conclusions: Our results suggest that adipocytes in hypertrophied mesenteric adipose tissue produce and secrete significant amounts of adiponectin, which could be involved in the regulation of intestinal inflammation associated with CD.

Exacerbation of Albuminuria and Renal Fibrosis in Subtotal Renal Ablation Model of Adiponectin-Knockout Mice

Objective—Obesity is recognized increasingly as a major risk factor for kidney disease. We reported previously that plasma adiponectin levels were decreased in obesity, and that adiponectin had defensive properties against type 2 diabetes and hypertension. In this study, we investigated the role of adiponectin for kidney disease in a subtotal nephrectomized mouse model. Methods and Results—Subtotal (5/6) nephrectomy was performed in adiponectin-knockout (APN-KO) and wild-type (WT) mice. The procedure resulted in significant accumulation of adiponectin in glomeruli and interstitium in the remnant kidney. Urinary albumin excretion, glomerular hypertrophy, and tubulointerstitial fibrosis were significantly worse in APN-KO mice compared with WT mice. Intraglomerular macrophage infiltration and mRNA levels of vascular cell adhesion molecule (VCAM)-1, MCP-1, tumor necrosis factor (TNF)-&agr;, transforming growth factor (TGF)-&bgr;1, collagen type I/III, and NADPH oxidase components were significantly increased in KO mice compared with WT mice. Treatment of APN-KO mice with adenovirus-mediated adiponectin resulted in amelioration of albuminuria, glomerular hypertrophy, and tubulointerstitial fibrosis and reduced the elevated levels of VCAM-1, MCP-1, TNF-&agr;, TGF-&bgr;1, collagen type I/III, and NADPH oxidase components mRNAs to the same levels as those in WT mice. Conclusions—Adiponectin accumulates to the injured kidney, and prevents glomerular and tubulointerstitial injury through modulating inflammation and oxidative stress.

Suppression of macrophage functions impairs skeletal muscle regeneration with severe fibrosis

When damaged, skeletal muscle regenerates. In the early phases of regeneration, inflammatory cells such as neutrophils/granulocytes and macrophages infiltrate damaged muscle tissue. To reveal the roles of macrophages during skeletal muscle regeneration, we injected an antibody, AFS98 that blocks the binding of M-CSF to its receptor into normal mice that received muscle damages. Anti-M-CSF receptor administration suppressed macrophage but not neutrophil infiltration. Histological study indicated that suppression of macrophages function leads to the incomplete muscle regeneration. In addition FACS and immunohistochemical study showed that the acute lack of macrophages delayed proliferation and differentiation of muscle satellite cells in vivo. Furthermore, mice injected with the anti-M-CSF receptor antibody exhibited not only adipogenesis, but also significant collagen deposition, i.e., fibrosis and continuous high expression of connective tissue growth factor. Finally we indicate that these fibrosis markers were strongly enriched in CD90(+) cells that do not include myogenic cells. These results indicate that macrophages directly affect satellite cell proliferation and that a macrophage deficiency severely impairs skeletal muscle regeneration and causes fibrosis.

Exploring RNA interference as a therapeutic strategy for renal disease.

The short synthetic interfering RNA duplexes (siRNAs) can selectively suppress gene expression in somatic mammalian cells without nonselective toxic effects of double-stranded RNA (dsRNA). However, a selective in vivo delivery of siRNA transfer has not been reported in kidney. Here, we investigated whether injection of synthetic siRNAs via renal artery followed by electroporation could be effective and therapeutic in silencing specific gene in glomerulus. We investigated the effect of siRNA in rat cultured mesangial cells (MCs) and showed that siRNA sequence-specific suppression of transgene expression was over a 1000-fold more potent than that by antisense oligodeoxynucleotide (ASODN). Transfection of siRNA targeting luciferase into rat kidneys significantly inhibited expression of a cotransfected luciferase expression vector in vivo. The delivery of siRNA targeting enhanced green fluorescent protein (EGFP) in the transgenic ‘green’ rat reduced endogenous EGFP expression, mainly in glomerular MCs. Furthermore, RNAi targeting against TGF-β1 significantly suppressed TGF-β1 mRNA and protein expression, thereby ameliorated the progression of matrix expansion in experimental glomerulonephritis. In addition, vector-based RNAi also inhibited TGF-β1 expression in vitro and in vivo. In conclusion, siRNA-directed TGF-β1 silencing may be of therapeutic value in the prevention and treatment of fibrotic diseases.

Hesr1 and Hesr3 are essential to generate undifferentiated quiescent satellite cells and to maintain satellite cell numbers

Satellite cells, which are skeletal muscle stem cells, divide to provide new myonuclei to growing muscle fibers during postnatal development, and then are maintained in an undifferentiated quiescent state in adult skeletal muscle. This state is considered to be essential for the maintenance of satellite cells, but their molecular regulation is unknown. We show that Hesr1 (Hey1) and Hesr3 (Heyl) (which are known Notch target genes) are expressed simultaneously in skeletal muscle only in satellite cells. In Hesr1 and Hesr3 single-knockout mice, no obvious abnormalities of satellite cells or muscle regenerative potentials are observed. However, the generation of undifferentiated quiescent satellite cells is impaired during postnatal development in Hesr1/3 double-knockout mice. As a result, myogenic (MyoD and myogenin) and proliferative (Ki67) proteins are expressed in adult satellite cells. Consistent with the in vivo results, Hesr1/3-null myoblasts generate very few Pax7+ MyoD– undifferentiated cells in vitro. Furthermore, the satellite cell number gradually decreases in Hesr1/3 double-knockout mice even after it has stabilized in control mice, and an age-dependent regeneration defect is observed. In vivo results suggest that premature differentiation, but not cell death, is the reason for the reduced number of satellite cells in Hesr1/3 double-knockout mice. These results indicate that Hesr1 and Hesr3 are essential for the generation of adult satellite cells and for the maintenance of skeletal muscle homeostasis.

Fully phosphorylated fetuin-A forms a mineral complex in the serum of rats with adenine-induced renal failure

The serum glycoprotein fetuin-A is an important inhibitor of extra-osseous calcification, but correlations between serum fetuin-A levels and the extent of vascular calcification are controversial. In this study, we used a rat model of adenine-induced renal failure with secondary hyperparathyroidism that exhibits all characteristic features of patients with chronic kidney disease. These rats had medial vascular calcification along with reduced levels of both serum and hepatic fetuin-A. Treatment with an inhibitor of ectopic calcification, alendronate, decreased bone turnover and eliminated completely the vascular calcification in this rat model, but there was no change in the levels of hepatic and serum fetuin-A. Centrifugation of the serum of untreated rats with renal failure gave a small precipitate composed of fetuin-A, calcium, magnesium, and phosphate; this complex, absent from normal rat serum, was not found in the serum of alendronate-treated rats with renal failure. Rat serum contained three types of phosphorylated fetuin-A, as well as unphosphorylated forms, but only the fully phosphorylated fetuin-A was present in the mineral complex. The amount of this complex reflected the risk of mineral precipitation. Our results suggest that the measurement of serum fetuin-mineral complex rather than fetuin-A alone might provide a better indication of extra-osseous calcification propensity.

Activation of the Signal Transducer and Activator of Transcription Signaling Pathway in Renal Proximal Tubular Cells by Albumin

Renal proximal tubular cells activated by reabsorption of protein are thought to play significant roles in the progression of kidney diseases. It was hypothesized that the signal transducer and activator of transcription (STAT) proteins may be activated by proteinuria in proximal tubular cells. To test this hypothesis, murine proximal tubular cells were treated with albumin (30 mg/ml medium) for various lengths of time. The results showed that albumin could activate Stat1 and Stat5 within 15 min in proximal tubular cells. The activation of STATs was mediated mostly by Jak2 and required no protein synthesis. In addition, activation of Stat1 occurred even after neutralization of IFN-gamma. The activation of STATs was inhibited by N-acetyl-L-cysteine, a precursor of glutathione and a reactive oxygen species (ROS) scavenger, and fluorescence-activated cell sorter analysis showed upregulation of intracellular ROS after albumin overloading, suggesting that albumin per se could generate ROS in proximal tubular cells. The activation of STATs occurred by way of the ROS generating system, and especially through the membrane-bound NADPH oxidase system. Reduced activities of glutathione peroxidase and catalase could also be responsible for the accumulation of intracellular ROS. Hence, not only the ROS generating system, but also the ROS scavenging system may contribute to the induction of ROS by albumin. These findings support the hypothesis that proximal tubular cells are activated and generate ROS by reabsorption of abundant urinary proteins filtered through the glomerular capillaries, and as a consequence, various IFN-gamma-inducible proteins are synthesized through IFN-gamma-independent activation of STAT signaling.

Transcriptional Regulation of Nephrin Gene by Peroxisome Proliferator–Activated Receptor-γ Agonist: Molecular Mechanism of the Antiproteinuric Effect of Pioglitazone

The renoprotective potential of the peroxisome proliferator-activated receptor-gamma (PPAR-gamma) agonist pioglitazone was explored in an immune model of progressive nephropathy, passive Heymann nephritis (PHN), compared with that of an angiotensin II receptor antagonist, taken as standard therapy for renoprotection. PHN rats received orally vehicle, pioglitazone (10 mg/kg twice daily), or candesartan (1 mg/kg twice daily) from months 2 to 8. Pioglitazone reduced proteinuria as effectively as candesartan and limited renal functional and structural changes. Kidneys from untreated PHN rats showed lower nephrin mRNA and protein than controls, both restored by pioglitazone. The effect was seen both early and late during the course of the disease. Whether the antiproteinuric effect of pioglitazone could be due to its effect on nephrin gene transcription also was investigated. HK-2 cells were transfected with plasmids that harbor the luciferase gene under portions (2-kb or 325-bp) of human nephrin gene promoter that contain putative peroxisome proliferator-responsive elements (PPRE) and incubated with pioglitazone (10 muM). Transcriptional activity of luciferase gene was highly increased by pioglitazone, with the strongest expression achieved with the 325-bp fragment. Increase in luciferase activity was prevented by bisphenol A diglycidyl ether, a PPAR-gamma synthetic antagonist. Electrophoretic mobility shift assay experiments showed a direct interaction of PPAR/retinoid X receptor heterodimers to PPRE present in the enhancer region of the nephrin promoter. In conclusion, pioglitazone exerts an antiproteinuric effect in immune-mediated glomerulonephritis as angiotensin II receptor antagonist does. Enhancement of nephrin gene transcription through specific PPRE in its promoter discloses a novel mechanism of renoprotection for PPAR-gamma agonists.

Retinoids Regulate the Repairing Process of the Podocytes in Puromycin Aminonucleoside-induced Nephrotic Rats

The foot processes forming the slit diaphragm are disrupted in diseases associated with proteinuria. Although they are often repairable, regulators for the repairing process remain unknown. By extrapolating from the fact that vitamin A is essential for the nephrogenesis, this study examined whether or not injured podocytes in the middle of the repairing process require retinaldehyde dehydrogenase type 2 (RALDH2), one of the key enzymes to produce all-trans-retinoic acid (ATRA). RALDH2 was dramatically upregulated in podocytes of puromycin aminonucleoside-induced nephrosis (PAN nephrosis) rats. On day 5 of PAN nephrosis, RALDH2 showed the remarkable induction, whereas glomerular expression levels of nephrin and midkine, one of the ATRA target genes, were downregulated. Daily administration of ATRA ameliorated proteinuria, which was accompanied by the improvement in the effacement of the foot processes and by the induction of nephrin and midkine. In contrast, recovery from PAN nephrosis was delayed in rats fed with a vitamin A-deficient diet. Consistently, the promoter region of human nephrin gene (NPHS1) contained three putative retinoic acid response elements (RARE) and showed the enhancer activity in response to ATRA in a dose-dependent manner. This transcriptional activation was regulated through the receptors for retinoids because BMS-189453, an antagonist to the retinoid receptors, counteracted it in a dose-dependent manner. In conclusion, active metabolites of vitamin A, especially ATRA produced by RALDH2 play relevant roles during the repairing process of injured podocytes. The results obtained from PAN nephrosis rats might be applicable to human renal diseases.