Taichi Murakami

Activation of Bone Morphogenetic Protein 4 Signaling Leads to Glomerulosclerosis That Mimics Diabetic Nephropathy

Diabetic nephropathy (DN) is the most common cause of chronic kidney disease. We have previously reported that Smad1 transcriptionally regulates the expression of extracellular matrix (ECM) proteins in DN. However, little is known about the regulatory mechanisms that induce and activate Smad1. Here, bone morphogenetic protein 4 (Bmp4) was found to up-regulate the expression of Smad1 in mesangial cells and subsequently to phosphorylate Smad1 downstream of the advanced glycation end product-receptor for advanced glycation end product signaling pathway. Moreover, Bmp4 utilized Alk3 and affected the activation of Smad1 and Col4 expressions in mesangial cells. In the diabetic mouse, Bmp4 was remarkably activated in the glomeruli, and the mesangial area was expanded. To elucidate the direct function of Bmp4 action in the kidneys, we generated transgenic mice inducible for the expression of Bmp4. Tamoxifen treatment dramatically induced the expression of Bmp4, especially in the glomeruli of the mice. Notably, in the nondiabetic condition, the mice exhibited not only an expansion of the mesangial area and thickening of the basement membrane but also remarkable albuminuria, which are consistent with the distinct glomerular injuries in DN. ECM protein overexpression and activation of Smad1 in the glomeruli were also observed in the mice. The mesangial expansion in the mice was significantly correlated with albuminuria. Furthermore, the heterozygous Bmp4 knock-out mice inhibited the glomerular injuries compared with wild type mice in diabetic conditions. Here, we show that BMP4 may act as an upstream regulatory molecule for the process of ECM accumulation in DN and thereby reveals a new aspect of the molecular mechanisms involved in DN.

SOX9 protein induces a chondrogenic phenotype of mesangial cells and contributes to advanced diabetic nephropathy.

Diabetic nephropathy (DN) is the most important chronic kidney disease. We previously reported that Smad1 transcriptionally regulates the expression of extracellular matrix in DN. Phenotypic change in mesangial cells (MCs) is a key pathologic event in the progression of DN. The aim of this study is to investigate a novel mechanism underlying chondrogenic phenotypic change in MCs that results in the development of DN. MCs showed chondrogenic potential in a micromass culture, and BMP4 induced the expression of chondrocyte markers (SRY-related HMG Box 9 (SOX9) and type II collagen (COL2)). Advanced glycation end products induced the expression of chondrocyte marker proteins downstream from the BMP4-Smad1 signaling pathway in MCs. In addition, hypoxia also induced the expression of BMP4, hypoxia-inducible factor-1α (HIF-1α), and chondrocyte markers. Overexpression of SOX9 caused ectopic expression of proteoglycans and COL2 in MCs. Furthermore, forced expression of Smad1 induced chondrocyte markers as well. Dorsomorphin inhibited these inductions. Glomerular expressions of HIF-1α, BMP4, and chondrocyte markers were observed in diabetic nephropathy mice. These positive stainings were observed in mesangial sclerotic lesions. SOX9 was partially colocalized with HIF-1α and BMP4 in diabetic glomeruli. BMP4 knock-in transgenic mice showed not only similar pathological lesions to DN, but also the induction of chondrocyte markers in the sclerotic lesions. Here we demonstrate that HIF-1α and BMP4 induce SOX9 expression and subsequent chondrogenic phenotype change in DN. The results suggested that the transdifferentiation of MCs into chondrocyte-like cells in chronic hypoxic stress may result in irreversible structural change in DN.

Scleraxis modulates bone morphogenetic protein 4 (BMP4)-Smad1 protein-smooth muscle α-actin (SMA) signal transduction in diabetic nephropathy.

Background: Activated mesangial cells exhibit SMA and contribute to the progression of diabetic nephropathy. Results: Scleraxis negatively regulated the AGE-induced expression and secretion of BMP4. Conclusion: Scleraxis and Id1 are involved in the BMP4-SMA pathway and modulate phenotypic changes. Significance: Deeper insight into the impact of regulatory mechanism of scleraxis-BMP4-Smad1 signal activation might help to prevent diabetic glomerular damage. Activation of mesangial cells (MCs), which is characterized by induction of smooth muscle α-actin (SMA) expression, contributes to a key event in various renal diseases; however, the mechanisms controlling MC differentiation are still largely undefined. Activated Smad1 induced SMA in a dose-dependent manner in MCs. As a direct regulating molecule for SMA, we identified and characterized scleraxis (Scx) as a new phenotype modulator in advanced glycation end product (AGE)-exposed MCs. Scx physically associated with E12 and bound the E-box in the promoter of SMA and negatively regulated the AGE-induced SMA expression. Scx induced expression and secretion of bone morphogenetic protein 4 (BMP4), thereby controlling the Smad1 activation in AGE-treated MCs. In diabetic mice, Scx was concomitantly expressed with SMA in the glomeruli. Inhibitor of differentiation 1 (Id1) was further induced by extended treatment with AGE, thereby dislodging Scx from the SMA promoter. These data suggest that Scx and Id1 are involved in the BMP4-Smad1-SMA signal transduction pathway besides the TGFβ1-Smad1-SMA signaling pathway and modulate phenotypic changes in MCs in 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.

An Autopsy Case of Mitochondrial Myopathy, Encephalopathy, Lactic Acidosis, and Stroke-Like Episodes (MELAS) with Intestinal Bleeding in Chronic Renal Failure

Abstract A 50-year-old man who underwent hemodialysis (HD) at local outpatient HD center due to end-stage renal disease (ESRD) was transferred to our hospital because of pneumonia. He had severe emaciation and past history of congestive heart failure. Presenting symptoms almost consistently involved difficulty in hearing and recurrent attacks of migraine-like headaches. He was diagnosed with dilated cardiomyopathy, showing diastolic mechanical dyssynchrony by tissue Doppler echocardiography. On the day of death, he had hematemesis and hemorrhagic shock. Autopsy revealed perforation of duodenum, and genetic analysis using mitochondrial DNA from cardiac muscle and iliopsoas muscle revealed a 3243A > G mutation in the mitochondrial tRNALeu(UUR) gene, which is related to mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS). Multiple organ failure due to the mutation of mitochondrial DNA with gastrointestinal bleeding is not a common.

Transcription factor 7-like 2 (TCF7L2) regulates activin receptor-like kinase 1 (ALK1)/Smad1 pathway for development of diabetic nephropathy.

Smad1 has previously been shown to play a key role in the development of diabetic nephropathy (DN), by increasing synthesis of extracellular matrix. However, the regulatory mechanism of Smad1 in DN is still unclear. This study aims to elucidate molecular interactions between activin receptor-like kinase 1 (ALK1)/Smad1 signaling pathway and transcription factor 7-like 2 (TCF7L2) in the progression of DN in vitro and in vivo. The expressions of TCF7L2 and ALK1 were induced by advanced glycation end products (AGEs) in parallel with Smad1, phosphorylated Smad1 (pSmad1), and alpha-smooth muscle actin (α-SMA) through TGF-β1 in cultured mesangial cells. Constitutively active ALK1 increased pSmad1 and α-SMA expressions. The binding of TCF7L2 to ALK1 promoter was confirmed by chromatin immunoprecipitation assay. Furthermore, TCF7L2 induced promoter activity of ALK1. AGEs and TGF-β1 induced a marked increase in TCF7L2 expression in parallel with ALK1. Overexpression of TCF7L2 increased the expressions of ALK1 and Smad1. Inversely, TCF7L2 knockdown by siRNA suppressed α-SMA expression as well as ALK1 and Smad1. The iNOS transgenic mice (iNOS-Tgm), which developed diabetic glomerulosclerosis resembling human diabetic nephropathy, exhibited markedly increased expressions of ALK1, TCF7L2, Smad1, pSmad1, and α-SMA in glomeruli in association with mesangial matrix expansion. These results provide a new evidence that the TCF7L2/ALK1/Smad1 pathway plays a key role in the development of DN.

Re-evaluation of Pre-pump Arterial Pressure to Avoid Inadequate Dialysis and Hemolysis: Importance of Prepump Arterial Pressure Monitoring in Hemodialysis Patients.

Prepump arterial pressure (PreAP) is monitored to avoid generating excessive negative pressure. The National Kidney Foundation K/DOQI clinical practice guidelines for vascular access recommend that PreAP should not fall below -250 mm Hg because excessive negative PreAP can lead to a decrease in the delivery of blood flow, inadequate dialysis, and hemolysis. Nonetheless, these recommendations are consistently disregarded in clinical practice and pressure sensors are often removed from the dialysis circuit. Thus far, delivered blood flow has been reported to decrease at values more negative than -150 mm Hg of PreAP. These values have been analyzed by an ultrasonic flowmeter and not directly measured. Furthermore, no known group has evaluated whether PreAP-induced hemolysis occurs at a particular threshold. Therefore, the aim of this study was to clarify the importance of PreAP in the prediction of inadequate dialysis and hemolysis. By using different diameter needles, human blood samples from healthy volunteers were circulated in a closed dialysis circuit. The relationship between PreAP and delivered blood flow or PreAP and hemolysis was investigated. We also investigated the optimal value for PreAP using several empirical monitoring methods, such as a pressure pillow. Our investigation indicated that PreAP is a critical factor in the determination of delivered blood flow and hemolysis, both of which occured at pressure values more negative than -150 mm Hg. With the exception of direct pressure monitoring, commonly used monitoring methods for PreAP were determined to be ineffective. We propose that the use of a vacuum monitor would permit regular measurement of PreAP.

Protein inhibitor of activated STAT, PIASy regulates α-smooth muscle actin expression by interacting with E12 in mesangial cells.

Phenotypic transformation of mesangial cells (MCs) is implicated in the development of glomerular disease; however, the mechanisms underlying their altered genetic program is still unclear. α-smooth muscle actin (α-SMA) is known to be a crucial marker for phenotypic transformation of MCs. Recently, E-boxes and the class I basic helix-loop-helix proteins, such as E12 have been shown to regulateα-SMA expression. Therefore, we tried to identify a novel E12 binding protein in MCs and to examine its role in glomerulonephritis. We found that PIASy, one of the protein inhibitors of activated STAT family protein, interacted with E12 by yeast two-hybrid screens and coimmunopreciptation assays. Overexpression of E12 significantly enhanced theα-SMA promoter activity, and the increase was blocked by co-transfection of PIASy, but not by a PIASy RING mutant. In vivo sumoylation assays revealed that PIASy was a SUMO E3 ligase for E12. Furthermore, transforming growth factor-β (TGF-β) treatment induced expression of both PIASy and E12, consistent with α-SMA expression. Moreover, reduced expression of PIASy protein by siRNA specific for PIASy resulted in increased TGF-β-mediated α-SMA expression. In vivo, PIASy and E12 were dramatically upregulated along with α-SMA and TGF-β in the proliferative phase of Thy1 glomerulonephritis. Furthermore, an association between PIASy and E12 proteins was observed at day 6 by IP-western blotting, but not at day 0. These results suggest that TGF-β up-regulates PIASy expression in MCs to down-regulateα-SMA gene transcription by the interaction with E12.

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.

A case of rapid amelioration of hepatitis C virus-associated cryoglobulinemic membranoproliferative glomerulonephritis treated by interferon-free directly acting antivirals for HCV in the absence of immunosuppressant

Mixed cryoglobulinemic syndrome, which is a systemic vasculitis characterized by the immune complex deposition in small- and medium-sized arteries and most often due to chronic hepatitis C virus (HCV) infection, sometimes clinically manifests as refractory glomerulonephritis or nephritic syndrome. Patients with mixed cryoglobulinemic nephropathy who have a rapidly progressive glomerulonephritis should receive immunosuppressive therapy. After disease stabilization, patients should receive concurrent therapy for the underlying HCV infection. The standard therapy of a chronic HCV infection is IFN monotherapy or IFN combined with ribavirin; however, after the introduction of direct-acting antivirals (DAAs), the standard therapy for patients with HCV genotype 1 has dramatically changed. We report a case of HCV-associated cryoglobulinemic membranoproliferative glomerulonephritis (MPGN) successfully treated by daclatasvir and asunaprevir, which are IFN-free DAAs for HCV, in combination with angiotensin II receptor blocker without immunosuppressive therapy. The patient developed severe nephrotic syndrome with progressive kidney dysfunction. Blood examination revealed a high copy number of HCV-RNA (6.4 log IU/mL, type 1), cryoglobulinemia, paraproteinemia of IgM-κ, and hypocomplementemia. Histological analysis showed MPGN type 1. These findings were compatible with those observed in HCV-associated cryoglobulinemic MPGN. This case offers original evidence for the application of newer generation of IFN-free DAAs in the treatment of HCV-associated cryoglobulinemic nephropathy.