Waichi Sato

Diabetic Endothelial Nitric Oxide Synthase Knockout Mice Develop Advanced Diabetic Nephropathy

The pathogenesis of diabetic nephropathy remains poorly defined, and animal models that represent the human disease have been lacking. It was demonstrated recently that the severe endothelial dysfunction that accompanies a diabetic state may cause an uncoupling of the vascular endothelial growth factor (VEGF)-endothelial nitric oxide (eNO) axis, resulting in increased levels of VEGF and excessive endothelial cell proliferation. It was hypothesized further that VEGF-NO uncoupling could be a major contributory mechanism that leads to diabetic vasculopathy. For testing of this hypothesis, diabetes was induced in eNO synthase knockout mice (eNOS KO) and C57BL6 controls. Diabetic eNOS KO mice developed hypertension, albuminuria, and renal insufficiency with arteriolar hyalinosis, mesangial matrix expansion, mesangiolysis with microaneurysms, and Kimmelstiel-Wilson nodules. Glomerular and peritubular capillaries were increased with endothelial proliferation and VEGF expression. Diabetic eNOS KO mice showed increased mortality at 5 mo. All of the functional and histologic changes were improved with insulin therapy. Inhibition of eNO predisposes mice to classic diabetic nephropathy. The mechanism likely is due to VEGF-NO uncoupling with excessive endothelial cell proliferation coupled with altered autoregulation consequent to the development of preglomerular arteriolar disease. Endothelial dysfunction in human diabetes is common, secondary to effects of glucose, advanced glycation end products, C-reactive protein, uric acid, and oxidants. It was postulated that endothelial dysfunction should predict nephropathy and that correction of the dysfunction may prevent these important complications.

Midkine Is Involved in Neutrophil Infiltration into the Tubulointerstitium in Ischemic Renal Injury

Midkine (MK) is a multifunctional heparin-binding protein and promotes migration of neutrophils, macrophages, and neurons. In the normal mouse kidney, MK is expressed in the proximal tubules. After renal ischemic reperfusion injury, its expression in proximal tubules was increased. Immediate increase of MK expression was found when renal proximal tubular epithelial cells in culture were exposed to 5 mM H2O2. Histologically defined tubulointerstitial damage was less severe in MK-deficient (Mdk−/−) than in wild-type (Mdk+/+) mice at 2 and 7 days after ischemic reperfusion injury. Within 2 days after ischemic injury, inflammatory leukocytes, of which neutrophils were the major population, were recruited to the tubulointerstitium. The numbers of infiltrating neutrophils and also macrophages were lower in Mdk−/− than in Mdk+/+ mice. Induction of macrophage inflammatory protein-2 and macrophage chemotactic protein-1, chemokines for neutrophils and macrophages, respectively, were also suppressed in Mdk−/− mice. Furthermore, renal tubular epithelial cells in culture expressed macrophage inflammatory protein-2 in response to exogenous MK administration. These results suggested that MK enhances migration of inflammatory cells upon ischemic injury of the kidney directly and also through induction of chemokines, and contributes to the augmentation of ischemic tissue damage.

Uric Acid, the Metabolic Syndrome, and Renal Disease

Metabolic syndrome, characterized by truncal obesity, hypertriglyceridemia, elevated BP, and insulin resistance, is recognized increasingly as a major risk factor for kidney disease and also is a common feature of patients who are on dialysis. One feature that is common to patients with metabolic syndrome is an elevated uric acid. Although often considered to be secondary to hyperinsulinemia, recent evidence supports a primary role for uric acid in mediating this syndrome. Specifically, fructose, which rapidly can cause metabolic syndrome in rats, also raises uric acid, and lowering uric acid in fructose-fed rats prevents features of the metabolic syndrome. Uric acid also can accelerate renal disease in experimental animals and epidemiologically is associated with progressive renal disease in humans. It is proposed that fructose- and purine-rich foods that have in common the raising of uric acid may have a role in the epidemic of metabolic syndrome and renal disease that is occurring throughout the world.

Anti-monocyte chemoattractant protein-1 gene therapy attenuates renal injury induced by protein-overload proteinuria.

It has been postulated that protein filtered through glomeruli activates tubular epithelial cells, which secrete vasoactive and inflammatory substances including chemokines, leading to tubulointerstitial renal injury. The present study was designed to investigate the role of monocyte chemoattractant protein-1 (MCP-1) in this process and to evaluate the effectiveness of a kidney-targeted gene transfer technique using hydrodynamic pressure. Naked plasmid encoding 7ND (an MCP-1 antagonist) or a control plasmid was introduced into the left kidney of rats. Three days after gene transfer (day 0), intraperitoneal administration of bovine serum albumin (10 mg/g body wt per day) was started and continued for 14 or 21 d. RT-PCR showed that 7ND mRNA was expressed only in the gene-transfected kidney. Immunostaining showed that 7ND protein was localized in the interstitial cells. Macrophage infiltration was significantly reduced in the left kidney of rats treated with 7ND on days 14 and 21. In the right kidney, such effects were not observed. 7ND also attenuated tubular damage and decreased the number of apoptotic cells. Computer-assisted analysis revealed that the areas positively stained for alpha-smooth muscle actin (alpha SMA), fibronectin-EDA, type I collagen, and collagen fibrils were significantly reduced in the 7ND-treated kidney on day 21. Furthermore, 7ND gene therapy significantly reduced MCP-1 and TGF-beta 1 mRNA expression. These results demonstrate that MCP-1 plays an important role in the development of tubulointerstitial inflammation, tubular damage, and fibrosis induced by proteinuria. The fact that 7ND gene therapy had little effect on the contralateral kidney indicates that 7ND acted locally. This strategy may have a potential usefulness as a gene therapy against tubulointerstitial renal injury.

High‐fat and high‐sucrose (western) diet induces steatohepatitis that is dependent on fructokinase

Fructose intake from added sugars has been implicated as a cause of nonalcoholic fatty liver disease. Here we tested the hypothesis that fructose may interact with a high‐fat diet to induce fatty liver, and to determine if this was dependent on a key enzyme in fructose metabolism, fructokinase. Wild‐type or fructokinase knockout mice were fed a low‐fat (11%), high‐fat (36%), or high‐fat (36%) and high‐sucrose (30%) diet for 15 weeks. Both wild‐type and fructokinase knockout mice developed obesity with mild hepatic steatosis and no evidence of hepatic inflammation on a high‐fat diet compared to a low‐fat diet. In contrast, wild‐type mice fed a high‐fat and high‐sucrose diet developed more severe hepatic steatosis with low‐grade inflammation and fibrosis, as noted by increased CD68, tumor necrosis factor alpha, monocyte chemoattractant protein‐1, alpha‐smooth muscle actin, and collagen I and TIMP1 expression. These changes were prevented in the fructokinase knockout mice. Conclusion: An additive effect of high‐fat and high‐sucrose diet on the development of hepatic steatosis exists. Further, the combination of sucrose with high‐fat diet may induce steatohepatitis. The protection in fructokinase knockout mice suggests a key role for fructose (from sucrose) in this development of steatohepatitis. These studies emphasize the important role of fructose in the development of fatty liver and nonalcoholic steatohepatitis. (Hepatology 2013;58:1632–1643)

Metabolic profiling reveals new serum biomarkers for differentiating diabetic nephropathy

Capillary electrophoresis coupled with time-of-flight mass spectrometry was used to explore new serum biomarkers with high sensitivity and specificity for diabetic nephropathy (DN) diagnosis, through comprehensive analysis of serum metabolites with 78 diabetic patients. Multivariate analyses were used for identification of marker candidates and development of discriminative models. Of the 289 profiled metabolites, orthogonal partial least-squares discriminant analysis identified 19 metabolites that could distinguish between DN with macroalbuminuria and diabetic patients without albuminuria. These identified metabolites included creatinine, aspartic acid, γ-butyrobetaine, citrulline, symmetric dimethylarginine (SDMA), kynurenine, azelaic acid, and galactaric acid. Significant correlations between all these metabolites and urinary albumin-to-creatinine ratios (p < 0.009, Spearman’s rank test) were observed. When five metabolites (including γ-butyrobetaine, SDMA, azelaic acid and two unknowns) were selected from 19 metabolites and applied for multiple logistic regression model, AUC value for diagnosing DN was 0.927 using the whole dataset, and 0.880 in a cross-validation test. In addition, when four known metabolites (aspartic acid, SDMA, azelaic acid and galactaric acid) were applied, the resulting AUC was still high at 0.844 with the whole dataset and 0.792 with cross-validation. Combination of serum metabolomics with multivariate analyses enabled accurate discrimination of DN patients. The results suggest that capillary electrophoresis-mass spectrometry based metabolome analysis could be used for DN diagnosis.

Endothelial injury due to eNOS deficiency accelerates the progression of chronic renal disease in the mouse

The vascular endothelium expresses endothelial nitric oxide synthase (eNOS) that generates nitric oxide (NO) to help maintain vascular integrity due to its anti-inflammatory, antiproliferative, and antithrombogenic effects. Pharmacological blockade of NO production has been shown to exacerbate renal injury in chronic renal disease and induces endothelial cell loss. However, pharmacological inhibition of NO nonspecifically blocks other types of NOS and therefore does not define the specific role of eNOS in kidney disease. We hypothesized that a lack of endothelial eNOS can induce a loss of glomerular and peritubular capillary endothelium and exacerbate renal injury in progressive renal disease. We tested out this hypothesis using remnant kidney (RK) in eNOS knockout (eNOS KO) mice. Systolic blood pressure was significantly higher, and renal function was worse in RK-eNOS KO mice compared with those in RK-C57BL6 mice. eNOS deficiency resulted in more severe glomerulosclerosis, mesangiolysis, and tubular damage. Glomerular and tubular macrophage infiltration and collagen deposition were also greater in RK-eNOS KO mice. Renal injuries in the RK-eNOS KO mice were accompanied by a greater loss of endothelial cells that was shown to be due to both a decrease in endothelial cell proliferation and an increase in apoptosis. A lack of eNOS accelerates both glomerular and tubulointerstitial injury with a loss of glomerular capillaries and peritubular capillaries. Impaired endothelial function is likely a direct risk factor for renal disease.

The growth factor midkine regulates the renin-angiotensin system in mice

The renin-angiotensin system plays a pivotal role in regulating blood pressure and is involved in the pathogenesis of kidney disorders and other diseases. Here, we report that the growth factor midkine is what we believe to be a novel regulator of the renin-angiotensin system. The hypertension induced in mice by 5/6 nephrectomy was accompanied by renal damage and elevated plasma angiotensin II levels and was ameliorated by an angiotensin-converting enzyme (ACE) inhibitor and an angiotensin receptor blocker. Notably, ACE activity in the lung, midkine expression in the lung, and midkine levels in the plasma were all increased after 5/6 nephrectomy. Exposure to midkine protein enhanced ACE expression in primary cultured human lung microvascular endothelial cells. Furthermore, hypertension was not induced and renal damage was less severe in midkine-deficient mice. Supplemental administration of midkine protein to midkine-deficient mice restored ACE expression in the lung and hypertension after 5/6 nephrectomy. Oxidative stress might be involved in midkine expression, since expression of NADH/NADPH oxidase-1, -2, and -4 was induced in the lung after 5/6 nephrectomy. Indeed, the antioxidative reagent tempol reduced midkine expression and plasma angiotensin II levels and consequently ameliorated hypertension. These results suggest that midkine regulates the renin-angiotensin system and mediates the kidney-lung interaction after 5/6 nephrectomy.

The E-Selectin Ligand Basigin/CD147 Is Responsible for Neutrophil Recruitment in Renal Ischemia/Reperfusion

E-selectin and its ligands are essential for extravasation of leukocytes in inflammation. Here, we report that basigin (Bsg)/CD147 is a ligand for E-selectin that promotes renal inflammation in ischemia/reperfusion. Compared with wild-type mice, Bsg-deficient (Bsg(-/-)) mice demonstrated striking suppression of neutrophil infiltration in the kidney after renal ischemia/reperfusion. Although E-selectin expression increased similarly between the two genotypes, Bsg(-/-) mice exhibited less renal damage, suggesting that Bsg on neutrophils contribute to renal injury in this model. Neutrophils expressed Bsg with N-linked polylactosamine chains and Bsg(-)(/)(-) neutrophils showed reduced binding to E-selectin. Bsg isolated from HL-60 cells bound to E-selectin, and tunicamycin treatment to abolish N-linked glycans from Bsg abrogated this binding. Furthermore, Bsg(-)(/)(-) neutrophils exhibited reduced E-selectin-dependent adherence to human umbilical vein endothelial cells in vitro. Injection of labeled neutrophils into mice showed that Bsg(-)(/)(-) neutrophils were less readily recruited to the kidney after renal ischemia/reperfusion than Bsg(+/+) neutrophils, regardless of the recipients genotype. Taken together, these results indicate that Bsg is a physiologic ligand for E-selectin that plays a critical role in the renal damage induced by ischemia/reperfusion.

Midkine is involved in tubulointerstitial inflammation associated with diabetic nephropathy

The concept that inflammation plays a crucial role in the pathogenesis of diabetic nephropathy has been recently emerging, although the principal pathology of diabetic nephropathy comprises glomerular sclerosis and associated changes in nephrons. Here, we identified the growth factor midkine (MK) as a novel key molecule involved in inflammation associated with Streptozotocin-induced diabetic nephropathy. The tubulointerstitial damage, as assessed as morphological changes, osteopontin expression, collagen I deposition and macrophage infiltration, were strikingly less in MK-deficient (Mdk−/−) mice than in Mdk+/+ mice. Monocyte chemoattractant protein (MCP)-1 expression, but not that of intercellular adhesion molecule-1, was also lower in Mdk−/− mice. High glucose upregulated MK expression in primary-cultured tubular epithelial cells, and induced MCP-1 to a larger extent in Mdk+/+ cells than in Mdk−/− cells. Correspondingly, the combination of exogenous MK and high glucose enhanced MCP-1 expression in Mdk−/− cells. Furthermore, high glucose and oxidant stress enhanced MK expression in macrophages. Consistent with the findings in the mouse model, MK expression was detected in the glomeruli, tubular epithelium and interstitium of kidneys from patients with diabetic nephropathy. Our data indicate that MK plays a critical role in the tubulointerstitial inflammation associated with diabetic nephropathy through activation of the MCP-1 pathway.