Motokazu Matsuura

Expression Levels of Renal Organic Anion Transporters (OATs) and Their Correlation with Anionic Drug Excretion in Patients with Renal Diseases

AbstractPurpose. Because the urinary excretion of drugs is often decreased in renal diseases, dosage regimens are adjusted to avoid adverse drug reactions. The aim of present study was to clarify the alteration in the levels of renal drug transporters and their correlation with the urinary drug excretion in renal diseases patients. Methods. We quantified the mRNA levels of human organic anion transporters (hOATs) by real-time polymerase chain reaction and examined the excretion of the anionic drug, cefazolin, in renal disease patients. Moreover, transport of cefazolin by hOAT1 and hOAT3 were examined using HEK293 transfectants. Results. Among four hOATs, the level of hOAT1 mRNA was significantly lower in the kidney of patients with renal diseases than in the normal controls. The elimination constant of cefazolin showed a significant correlation with the values of phenolsulfonphthalein test and mRNA levels of hOAT3. The uptake study using HEK293 transfectants revealed that cefazolin and phenolsulfonphthalein were transported by hOAT3. Conclusions. These results suggest that hOAT3 plays an important role for anionic drug secretion in patients with renal diseases and that the expression levels of drug transporters may be related to the alteration of renal drug secretion.

Expression of Smad1 is directly associated with mesangial matrix expansion in rat diabetic nephropathy.

Diabetic nephropathy is the leading cause of end-stage renal disease, and glomerular mesangial matrix expansion is the hallmark in diabetic nephropathy. However, the precise mechanism for the development of mesangial matrix expansion has remained unknown. The key component involved in mesangial matrix expansion is type IV collagen (Col4). Recently, we have reported that Smad1 transcriptionally regulates expression of Col4 under diabetic conditions in vitro. Here we show that this direct regulator of Col4 also plays a crucial role for mesangial matrix expansion in vivo. Streptozotocin-induced diabetic rats are the model of incipient diabetic nephropathy, and showed various levels of mesangial matrix expansion at 24 weeks. The glomerular expression of Smad1 was significantly increased in diabetic rats with more mesangial matrix expansion by Western blot and immunohistochemical analysis. Furthermore, the glomerular expression of Smad1 was closely correlated with the glomerular expression of Col4 and smooth muscle alpha actin (α-SMA), while albuminuria or glomerular filtration rate was not correlated with mesangial matrix expansion. We also found that urinary excretion of Smad1 was closely associated with the severity of mesangial matrix expansion. In cultured mesangial cells expression of Smad1 upregulated the transcriptional activity of key molecules in mesangial matrix expansion, such as Col4 and α-SMA. These data indicate the critical involvement of Smad1 in mesangial matrix expansion in the early phase of diabetic nephropathy. Our data imply that urinary Smad1 might be a representative diagnostic marker for mesangial matrix expansion in diabetic nephropathy.

Activation of STAT3/Smad1 is a key signaling pathway for progression to glomerulosclerosis in experimental glomerulonephritis.

Mesangial cell proliferation is a significant event in the development of progressive glomerular injuries. However, the issue of how cell proliferation is involved in the development of glomerulosclerosis is unclear. Recently, we showed that the overexpression of type IV collagen (Col IV), a major component of mesangial extracellular matrix, is transcriptionally regulated by Smad1 in diabetic glomerulosclerosis. In this study, we have demonstrated the effect of the administration of an anti-platelet-derived growth factor (PDGF) β-receptor antibody (APB5) blocking activation by the PDGF-B chain on rat glomerulonephritis and have examined the signaling pathways that regulate both glomerular cell proliferation and glomerulosclerosis in vivo and in vitro. Experimental mesangial proliferative glomerulonephritis (Thy1 GN) was induced by a single intravenous injection of anti-rat Thy-1.1 monoclonal antibody. In Thy1 GN, mesangial cell proliferation and the expression of Col IV peaked at day 6. Immunohistochemical staining for the expression of Smad1, phospho-Smad1 (pSmad1), and phospho-STAT3 (pSTAT3) revealed that the peak for glomerular Smad1 expression occurred at day 6, consistent with the peak for mesangial proliferation. The expression of pSmad1 was up-regulated at day 1, and the peak for glomerular pSmad1 expression occurred at day 4 of the disease. When treated with APB5, both mesangial proliferation and sclerosis were reduced significantly. The expression of Smad1, pSmad1, and pSTAT3 was also significantly reduced by the administration of APB5. PDGF induced both mesangial cell replication and Col IV synthesis in association with an increased expression of pSTAT3 and pSmad1 on cultured mesangial cells. In addition, APB5 reduced mesangial cell proliferation in association with decreased pSmad1, pSTAT3, and Col IV protein expressions in vitro. The introduction of dominant negative STAT3 significantly decreased the expression of Col IV in cultured mesangial cells. These data suggest that the activation of STAT3 and Smad1 participates in the developing process of glomerulosclerosis in experimental glomerulonephritis.

Urinary Smad1 is a novel marker to predict later onset of mesangial matrix expansion in diabetic nephropathy

OBJECTIVE—We reported that Smad1 is a key transcriptional factor for mesangial matrix expansion in diabetic nephropathy. In this study, we examined whether urinary Smad1 in an early phase of diabetes can predict later development of glomerulosclerosis in diabetic nephropathy and how an angiotensin II type 1 receptor blocker (ARB) can modulate structural changes and urinary markers. RESEARCH DESIGN AND METHODS—Smad1 and albumin in the urine were examined 4 weeks after injection of streptozotocin in 48 rats or 6 weeks of diabetes in db/db mice. Their renal pathology was analyzed after 20 weeks in rats or 12 weeks in mice. Among 48 diabetic rats 7 rats were treated with olmesartan for 20 weeks. RESULTS—Urinary Smad1 of diabetic rats at 4 weeks was nicely correlated with mesangial matrix expansion at 24 weeks (r = 0.70, P < 0.001), while albuminuria showed a weaker association (r = 0.31, P = 0.043). Olmesartan treatment significantly ameliorated glomerulosclerosis and dramatically decreased urinary Smad1 (from 3.9 ± 2.9 to 0.3 ± 0.3 ng/mg creatinine, P < 0.05). In db/db mice, urinary Smad1 at 6 weeks was also significantly correlated with mesangial expansion at 18 weeks. In contrast, there was no change in urinary Smad1 in control diabetic rats or mice. CONCLUSIONS—The increase of urinary Smad1 in the early stages of diabetes is correlated with later development of glomerulosclerosis in two rodent models. These data indicate that urinary Smad1 could be a novel predictor for later onset of morphological changes and can be used to monitor the effect of ARBs in diabetic nephropathy.

Pharmacokinetic Significance of Renal OAT3 (SLC22A8) for Anionic Drug Elimination in Patients with Mesangial Proliferative Glomerulonephritis

PurposeOur previous studies showed that the mRNA level of human organic anion transporter (hOAT) 3 in the kidney was correlated with the rate of elimination of an anionic antibiotic cefazolin. However, the correlation coefficient was not so high. In the present study, therefore, we enrolled more patients to examine whether additional factors were responsible for the correlation.MethodshOAT mRNA levels in renal biopsy specimens were quantified using the real-time polymerase chain reaction method. The elimination rates for the free fraction of cefazolin were determined in patients with various renal diseases.ResultsIn the present study, the coefficient of correlation between the hOAT3 mRNA level and the elimination rates for the free fraction of cefazolin was not so high in the patients overall as in our previous study (r = 0.536). However, following the classification of renal diseases, a better correlation was obtained in patients with mesangial proliferative glomerulonephritis (r = 0.723). In contrast, multiple regression analyses including gender, age, and liver function did not result in any improvements in the correlation coefficients.ConclusionsThese results suggest that the hOAT3 mRNA level is a significant marker of pharmacokinetics with which to predict the rate of elimination of cefazolin in patients with mesangial proliferative glomerulonephritis.

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.

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.

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.

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.