Yasuhiro Akai

Hypoxia promotes fibrogenesis in vivo via HIF-1 stimulation of epithelial-to-mesenchymal transition

Hypoxia has been proposed as an important microenvironmental factor in the development of tissue fibrosis; however, the underlying mechanisms are not well defined. To examine the role of hypoxia-inducible factor-1 (HIF-1), a key mediator of cellular adaptation to hypoxia, in the development of fibrosis in mice, we inactivated Hif-1alpha in primary renal epithelial cells and in proximal tubules of kidneys subjected to unilateral ureteral obstruction (UUO) using Cre-loxP-mediated gene targeting. We found that Hif-1alpha enhanced epithelial-to-mesenchymal transition (EMT) in vitro and induced epithelial cell migration through upregulation of lysyl oxidase genes. Genetic ablation of epithelial Hif-1alpha inhibited the development of tubulointerstitial fibrosis in UUO kidneys, which was associated with decreased interstitial collagen deposition, decreased inflammatory cell infiltration, and a reduction in the number of fibroblast-specific protein-1-expressing (FSP-1-expressing) interstitial cells. Furthermore, we demonstrate that increased renal HIF-1alpha expression is associated with tubulointerstitial injury in patients with chronic kidney disease. Thus, we provide clinical and genetic evidence that activation of HIF-1 signaling in renal epithelial cells is associated with the development of chronic renal disease and may promote fibrogenesis by increasing expression of extracellular matrix-modifying factors and lysyl oxidase genes and by facilitating EMT.

Increased plasma concentrations of adrenomedullin correlate with relaxation of vascular tone in patients with septic shock

OBJECTIVE To investigate plasma concentrations of adrenomedullin in patients with septic shock and the potential association of these concentrations with relaxation of vascular tone. DESIGN Prospective, case series. SETTING Department of Emergency and Critical Care Medicine, Nara Medical University. PATIENTS Twelve patients who fulfilled the clinical criteria for severe sepsis or septic shock (as defined by the Members of the American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference Committee) and 13 healthy volunteers. INTERVENTIONS Arterial blood samples were obtained via a 20-gauge cannula inserted into each patients radial artery. MEASUREMENTS AND MAIN RESULTS After extraction and purification, plasma adrenomedullin was measured by radioimmunoassay. Systemic vascular resistance index, pulmonary vascular resistance, cardiac index, and stroke volume index were determined with a thermodilution catheter. The mean plasma concentration of adrenomedullin was markedly higher in patients than in controls (226.1 +/- 66.4 [SEM] vs. 5.05 +/- 0.21 fmol/mL, p < .01). Moreover, these concentrations correlated significantly with cardiac index, stroke volume index, and heart rate values, and correlated significantly with decreases in diastolic blood pressure, systemic vascular resistance index, and pulmonary vascular resistance index values. CONCLUSIONS Enhanced production of adrenomedullin in patients with septic shock may contribute to reduced vascular tone, hypotension, or both. More data are needed to clarify the role of adrenomedullin in the regulation of vascular tone in this patient population.

Epithelial-Mesenchymal Transition as a Potential Explanation for Podocyte Depletion in Diabetic Nephropathy

BACKGROUND Depletion of glomerular podocytes is an important feature of progressive diabetic nephropathy. Although the most plausible explanation for this podocyte depletion is detachment from the glomerular basement membrane after cellular apoptosis, the mechanism is unclear. Fibroblast-specific protein 1 (FSP1; encoded by the S100A4 gene) is a member of the S100 family of calcium-binding proteins and is constitutively expressed in the cytoplasm of tissue fibroblasts or epithelial cells converted into fibroblasts by means of epithelial-mesenchymal transition. STUDY DESIGN Retrospective cross-sectional analysis. SETTINGS & PARTICIPANTS 109 patients with type 2 diabetes mellitus, of whom 43 (39%) underwent kidney biopsy. PREDICTOR Clinical stage (4 categories) and histological grade (5 categories) of diabetic nephropathy. OUTCOME FSP1 expression in podocytes in urine and glomeruli in kidney biopsy specimens. MEASUREMENTS Immunohistochemistry, real-time polymerase chain reaction, and in situ hybridization. RESULTS 38 of 109 patients (35%) were normoalbuminuric, 16 (15%) had microalbuminuria, 8 (7%) had macroalbuminuria, and 47 (43%) had decreased kidney function. Approximately 95% of podocytes in urine sediment were not apoptotic, and 86% expressed FSP1. The number of FSP1-positive podocytes in urine sediment was significantly larger in patients with macroalbuminuria than in those with normoalbuminuria (P = 0.03). Intraglomerular expression of FSP1 occurred almost exclusively in podocytes from patients with diabetes, and the number of FSP1-positive podocytes was larger in glomeruli showing diffuse mesangiopathy than in those showing focal mesangiopathy (P = 0.01). The number also was larger in glomeruli with nodular lesions than in those without nodular lesions (P < 0.001). FSP1-positive podocytes selectively expressed Snail1 and integrin-linked kinase, a known trigger for epithelial-mesenchymal transition. LIMITATIONS Nonrepresentative study population. CONCLUSIONS These results suggest that the appearance of FSP1 in podocytes of patients with diabetes is associated with more severe clinical and pathological findings of diabetic nephropathy, perhaps because of induction of podocyte detachment through epithelial-mesenchymal transition-like phenomena.

Decreased renal α-Klotho expression in early diabetic nephropathy in humans and mice and its possible role in urinary calcium excretion

Hypercalciuria is one of the early manifestations of diabetic nephropathy. We explored here the role of α-Klotho, a protein expressed predominantly in distal convoluted tubules that has a role in calcium reabsorption. We studied 31 patients with early diabetic nephropathy and compared them with 31 patients with IgA nephropathy and 7 with minimal change disease. Renal α-Klotho expression was significantly lower and urinary calcium excretion (UCa/UCr) significantly higher in diabetic nephropathy than in IgA nephropathy or minimal change disease. Multiple regression analyses indicated that α-Klotho mRNA was inversely correlated with calcium excretion. We next measured these parameters in a mouse model of streptozotocin (STZ)-induced diabetic nephropathy, characterized by glomerular hyperfiltration, as seen in early diabetic nephropathy. We also confirmed a reduction of renal α-Klotho mRNA down to almost 50% and enhanced calcium excretion in mice with STZ-induced diabetic nephropathy in comparison with nondiabetic mice. Hypercalciuria was exacerbated in heterozygous α-Klotho knockout mice in comparison with wild-type mice, each with STZ-induced diabetic nephropathy. Thus, α-Klotho expression was decreased in distal convoluted tubules in diabetic nephropathy in humans and mice. Renal loss of α-Klotho may affect urinary calcium excretion in early diabetic nephropathy.

Prognostic value of increased plasma levels of brain natriuretic peptide in patients with septic shock.

ABSTRACT Our objective was to investigate the plasma levels of brain and atrial natriuretic peptides (BNP and ANP, respectively) in patients with septic shock/severe sepsis and to study the association of BNP and ANP levels with hemodynamic parameters, severity of the disease, and prognosis of those patients. This is a prospective case series study of 22 patients with septic shock, 11 patients with severe sepsis, and 20 healthy volunteers at the Department of Emergency and Critical Care Medicine, Nara Medical University Hospital, Japan. Blood collection was performed on admission and on days 1, 2, and 4. Plasma BNP and ANP levels were measured by radioimmunoassay. Right atrial pressure, mean pulmonary arterial pressure, pulmonary arterial wedge pressure, and left ventricular stroke work index were determined using a thermodilution catheter. Acute Physiological and Chronic Health Evaluation II scores were calculated. Plasma levels of BNP and ANP were markedly elevated in patients with septic shock/severe sepsis compared with controls (BNP, 7 ± 0.3 pg mL−1; ANP, 13 ± 1 pg mL−1). In patients with septic shock, both BNP and ANP peaked on day 2 (BNP, 987 ± 160 pg mL−1; ANP, 103 ± 17 pg mL−1). Plasma levels of BNP on day 2 in patients with septic shock significantly correlated with right atrial pressure (r = 0.744, P < 0.01), mean pulmonary arterial pressure (r = 0.670, P < 0.01), pulmonary arterial wedge pressure (r = 0.709, P < 0.01), left ventricular stroke work index (r = −0.552, P < 0.05), Acute Physiological and Chronic Health Evaluation II score (r = 0.581, P < 0.01), and poor prognosis (P < 0.05). The optimal cutoff point for predicting mortality in patients with septic shock was a BNP level of 650 pg mL−1 on day 2, in which sensitivity and specificity were 92% and 80%, respectively. Increased plasma levels of BNP may reflect not only the severity of myocardial depression but also the disease severity and could be of prognostic value in patients with septic shock.

Increased Excretion of Urinary Transforming Growth Factor Beta 1 in Patients with Diabetic Nephropathy

The accumulation of extracellular matrix in the glomeruli of human and experimental models of diabetic nephropathy is associated with disease progression. Transforming growth factor beta 1 (TGF-β1), which is a multifunctional peptide growth factor, plays a key role in the synthesis of extracellular matrix protein in vitro, and the expression of TGF-β1 is elevated in human and rat diabetic nephropathy. In this study, we measured the urinary TGF-β1 excretion in 57 patients with non-insulin-dependent diabetes mellitus and in 20 healthy volunteers to examine whether the determination of urinary TGF-β1 excretion would facilitate the evaluation of the degree of mesangial expansion in patients with diabetic nephropathy. Both active and total TGF-β1 levels in 24-hour urine samples collected from patients with diabetes mellitus and normal controls were measured using an enzyme-linked immunosorbent assay. We observed a higher excretion of urinary TGF-β1 in patients with diabetes mellitus than in normal controls. In addition, the urinary TGF-β1 excretion was elevated in patients with severe mesangial expansion. These results suggest that urinary TGF-β1 may represent one parameter that can be used to evaluate the progression of diabetic nephropathy.

Intraglomerular expression of transforming growth factor-beta 1 (TGF-β1) mRNA in patients with glomerulonephritis: quantitative analysis by competitive polymerase chain reaction

TGF‐β1 is involved in the pathogenesis of glomerular sclerosis. We studied the intraglomerular expression of TGF‐β1 mRNA in patients with glomerulonephritis using competitive polymerase chain reaction (PCR). This method is sensitive enough to quantify cDNA copies of mRNA present in small amounts of samples. Renal biopsy specimens were obtained from 42 patients with various kinds of glomerulonephritis. Ten glomeruli were dissected from renal biopsy specimens. Normal glomeruli were also obtained from the resected kidneys of eight patients with renal cell cancer. Total RNA was extracted from the glomeruli and reverse transcribed into cDNA with reverse transcriptase. To prepare samples containing identical amounts of β‐actin cDNA (8 pg), we performed competitive PCR by co‐amplifying mutant templates of β‐actin with a unique EcoRI site. Next, to measure TGF‐β1 cDNA, we performed competitive PCR by co‐amplifying mutant templates of TGF‐β1. We observed a higher glomerular expression of TGF‐β1 mRNA in cases of mesangial proliferative glomerulonephritis having a moderate increase in mesangial matrix, diabetic nephropathy and diffuse proliferative lupus nephritis, compared with normal glomeruli. Results suggest that the intraglomerular synthesis of TGF‐β1 may be involved in the progression of glomerulonephritis in humans.

Serum levels of VEGF and basic FGF in the subacute phase of myocardial infarction

We examined serial changes in serum levels of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) measured by ELISAs in 45 patients with acute myocardial infarction (AMI) who received heparin intravenously for 3 to 5 days after the onset and in 30 control subjects with an old myocardial infarction. To evaluate the effect of heparin on these serum levels, heparin was administered intravenously in 10 patients with AMI on day 21. Blood samples were obtained from all AMI patients on days 1, 2, 3, 7, 14, 21, and 28 and from 10 AMI patients before and 1 h after heparin administration. Serum VEGF level was significantly reduced after heparin administration (P<0.001). Serum samples from day 1 to 3 were therefore excluded from the subsequent analysis. Serum VEGF level in AMI patients was significantly higher on day 7 than in the control subjects (P<0.0001), and then decreased over time (P<0.0001). The serum VEGF level on day 7 was independently associated with the peak serum CK level (P<0.05). The serum bFGF level did not differ significantly between the AMI patients and the control subjects. In conclusion, the serum VEGF level may be selectively elevated during the healing process after AMI.

Prognostic Value of Urinary Interleukin 6 in Patients with IgA Nephropathy: An 8-Year Follow-Up Study

We evaluated the significance of urinary IL-6 levels in the prediction of long-term renal outcome in patients with IgA nephropathy (IgAN). Fifty-nine patients with biopsy-proven IgAN were enrolled in this study. All patients had a creatinine clearance (Ccr) greater than 80 ml/min and normal serum creatinine concentration (Scr) on enrollment and were followed for 8.07 ± 1.72 years. Twenty- four-hour urine samples were obtained and urinary IL-6 was measured by ELISA. Fifteen patients exhibited a worsening of renal function (progressor). Renal function of the remaining 44 patients was stable during follow-up (non-progressor). The urinary IL-6 levels of progressors on enrollment were significantly higher than those of non-progressors (3.8 ± 3.8 and 1.8 ± 1.5 ng/day, respectively, p = 0.0138). We calculated the risk ratio for the progression to renal failure. Patients with a urinary IL-6 level greater than 2.5 ng/day at diagnosis had a 7.8-fold higher risk for the progression of the disease (95% CI 1.31–46.47, p = 0.024) compared with those whose urinary IL-6 level was less than 1.0 ng/day. In conclusion, urinary IL-6 levels could be used as a predictor of long-term renal outcome in patients with IgAN. Patients with a urinary IL-6 level greater than 2.5 ng/day at diagnosis may have a worse prognosis.

Reduction of Circulating Soluble Fms-Like Tyrosine Kinase-1 Plays a Significant Role in Renal Dysfunction–Associated Aggravation of Atherosclerosis

Background— Renal dysfunction is commonly accompanied by a worsening of atherosclerosis; however, the underlying molecular mechanism is not fully understood. We examined the role played by soluble fms-like tyrosine kinase-1 (sFlt-1), an endogenous antagonist of the proatherogenic cytokine placental growth factor (PlGF), in the worsening of atherosclerosis in patients with renal dysfunction and in an animal model of renal failure. Methods and Results— In this study, 329 patients who received cardiac catheterization and 76 patients who underwent renal biopsy were enrolled. Both plasma sFlt-1 levels and renal sFlt-1 mRNA expression were positively correlated with estimated glomerular filtration rate (P<0.01). The PlGF/sFlt-1 ratio was negatively correlated with estimated glomerular filtration rate (P<0.01), whereas plasma PlGF levels were not affected by it. The PlGF/sFlt-1 ratio was significantly higher in patients with multivessel coronary artery disease than in patients with single-vessel or no coronary artery disease. The reduction of circulating sFlt-1 and renal sFlt-1 mRNA levels was confirmed in five-sixths (5/6)–nephrectomized apolipoprotein E–deficient mice that developed experimental renal dysfunction. Atherosclerotic plaque area and macrophage infiltration into the plaque were significantly higher in 5/6–nephrectomized apolipoprotein E–deficient mice than in control mice, but replacement therapy with recombinant sFlt-1 significantly reduced both plaque formation and macrophage infiltration. Conclusions— The present study demonstrates that a reduction in the circulating levels of sFlt-1 is associated with the worsening of atherosclerosis that accompanies renal dysfunction.