Deficiency of the kidney tubular angiotensin II type1 receptor-associated protein ATRAP exacerbates streptozotocin-induced diabetic glomerular injury via reducing protective macrophage polarization.

Although activation of the renin-angiotensin system and of its glomerular components is implicated in the pathogenesis of diabetic nephropathy, the functional roles of the tubular renin-angiotensin system with AT1 receptor signaling in diabetic nephropathy are unclear. Tissue hyperactivity of the renin-angiotensin system is inhibited by the angiotensin II type 1 receptor-associated protein ATRAP, which negatively regulates receptor signaling. The highest expression of endogenous ATRAP occurs in the kidney, where it is mainly expressed by tubules but rarely in glomeruli. Here, we found that hyperactivation of angiotensin II type 1 receptor signaling in kidney tubules exacerbated diabetic glomerular injury in a mouse model of streptozotocin-induced diabetic nephropathy. These phenomena were accompanied by decreased expression of CD206, a marker of alternatively activated and tissue-reparative M2 macrophages, in the kidney tubulointerstitium. Additionally, adoptive transfer of M2- polarized macrophages into diabetic ATRAP-knockout mice ameliorated the glomerular injury. As a possible mechanism, the glomerular mRNA levels of tumor necrosis factor-α and oxidative stress components were increased in diabetic knockout mice compared to non-diabetic knockout mice, but these increases were ameliorated by adoptive transfer. Furthermore, proximal tubule-specific ATRAP downregulation reduced tubulointerstitial expression of CD206, the marker of M2 macrophages in diabetic mice. Thus, our findings indicate that tubular ATRAP-mediated functional modulation of angiotensin II type 1 receptor signaling modulates the accumulation of tubulointerstitial M2 macrophages, thus affecting glomerular manifestations of diabetic nephropathy via tubule-glomerular crosstalk.

Effects of Rikkunshito treatment on renal fibrosis/inflammation and body weight reduction in a unilateral ureteral obstruction model in mice.

Chronic kidney disease (CKD) progresses to end-stage renal failure via renal tubulointerstitial fibrosis. Malnutrition, inflammation, and arteriosclerosis interact to exacerbate the poor prognosis of CKD, and their effective management is thus essential. The traditional Japanese medicine Rikkunshito (RKT) exerts appetite-stimulating effects via ghrelin, which attenuates inflammation and fibrosis. We evaluated the therapeutic effect of RKT in unilateral ureter obstruction (UUO)-induced renal fibrosis/inflammation and body weight loss in mice. UUO and sham-operated mice were fed a standard diet or diet containing 3.0% RKT. Renal fibrosis was investigated by histopathology and macrophage infiltration was determined by immunohistochemistry. Expression levels of genes associated with fibrosis, inflammation, ghrelin, and mitochondrial function were determined by quantitative reverse transcription-polymerase chain reaction and western blot analyses. RKT treatment partially prevented UUO-induced weight loss but failed to attenuate renal fibrosis and inflammation. Renal expression of sirtuin 1, a ghrelin-downstream signalling molecule, and gene expression of peroxisome proliferator-activated receptor-γ coactivator 1α and Bcl-2/adenovirus E1B interacting protein 3 were unaffected by RKT. These results indicate that RKT inhibits weight loss but does not improve renal fibrosis or inflammation in a rapidly progressive renal fibrosis mouse model. RKT may have a protective effect on weight loss associated with CKD.

Angiotensin II type 1 receptor-associated protein deficiency attenuates sirtuin1 expression in an immortalised human renal proximal tubule cell line.

The proximal tubule is a particularly important site for ageing-related kidney damage. Sirtuin 1 (SIRT1), an NAD+ (nicotinamide adenine dinucleotide)-dependent deacetylase in the proximal tubule, may be involved in renal injury associated with ageing. However, the mechanisms of SIRT1 regulation remain to be elucidated. We recently reported that angiotensin II type 1 receptor (AT1R)-associated protein (ATRAP)-deficient mice displayed age-associated renal function decline and tubulointerstitial fibrosis. Our data showed that SIRT1 protein expression was reduced in ATRAP-deficient mice, although the relationship between ATRAP deficiency and age-associated renal fibrosis is still not fully understood. It is, therefore, necessary to investigate how ATRAP affects SIRT1 protein expression to resolve ageing-associated kidney dysfunction. Here, since ageing studies are inherently lengthy, we used an ex vivo model of the proximal tubule to determine the role of ATRAP in SIRT1 protein expression. We first generated a clonal immortalised human renal proximal tubule epithelial cell line (ciRPTEC) expressing AT1R and ATRAP. Using this cell line, we demonstrated that ATRAP knockdown reduced SIRT1 protein expression in the ciRPTEC but did not alter SIRT1 mRNA expression. Thus, ATRAP likely mediates SIRT1 protein abundance in ciRPTEC.

Improved home BP profile with dapagliflozin is associated with amelioration of albuminuria in Japanese patients with diabetic nephropathy: the Yokohama add-on inhibitory efficacy of dapagliflozin on albuminuria in Japanese patients with type 2 diabetes study (Y-AIDA study).

BACKGROUND: The Y-AIDA study was designed to investigate the renal- and home blood pressure (BP)-modulating effects of add-on dapagliflozin treatment in Japanese individuals with type 2 diabetes mellitus (T2DM) and albuminuria. METHODS: We conducted a prospective, multicenter, single-arm study. Eighty-six patients with T2DM, HbA1c 7.0-10.0%, estimated glomerular filtration rate (eGFR) ≥ 45 mL/min/1.73 m2, and urine albumin-to-creatinine ratio (UACR) ≥ 30 mg/g creatinine (gCr) were enrolled, and 85 of these patients were administered add-on dapagliflozin for 24 weeks. The primary and key secondary endpoints were change from baseline in the natural logarithm of UACR over 24 weeks and change in home BP profile at week 24. RESULTS: Baseline median UACR was 181.5 mg/gCr (interquartile range 47.85, 638.0). Baseline morning, evening, and nocturnal home systolic/diastolic BP was 137.6/82.7 mmHg, 136.1/79.3 mmHg, and 125.4/74.1 mmHg, respectively. After 24 weeks, the logarithm of UACR decreased by 0.37 ± 0.73 (P < 0.001). In addition, changes in morning, evening, and nocturnal home BP from baseline were as follows: morning systolic/diastolic BP - 8.32 ± 11.42/- 4.18 ± 5.91 mmHg (both P < 0.001), evening systolic/diastolic BP - 9.57 ± 12.08/- 4.48 ± 6.45 mmHg (both P < 0.001), and nocturnal systolic/diastolic BP - 2.38 ± 7.82/- 1.17 ± 5.39 mmHg (P = 0.0079 for systolic BP, P = 0.0415 for diastolic BP). Furthermore, the reduction in UACR after 24 weeks significantly correlated with an improvement in home BP profile, but not with changes in other variables, including office BP. Multivariate linear regression analysis also revealed that the change in morning home systolic BP was a significant contributor to the change in log-UACR. CONCLUSIONS: In Japanese patients with T2DM and diabetic nephropathy, dapagliflozin significantly improved albuminuria levels and the home BP profile. Improved morning home systolic BP was associated with albuminuria reduction. Trial registration The study is registered at the UMIN Clinical Trials Registry (UMIN000018930; http://www.umin.ac.jp/ctr/index-j.htm ). The study was conducted from July 1, 2015 to August 1, 2018.

Early Enhanced Leucine-Rich α-2-Glycoprotein-1 Expression in Glomerular Endothelial Cells of Type 2 Diabetic Nephropathy Model Mice.

Abnormal angiogenesis plays a major role in the development of early stage diabetic nephropathy. Vascular endothelial growth factor (VEGF) is a classical proangiogenic factor that regulates abnormal glomerular angiogenesis linked to glomerular hypertrophy in the early stage of diabetic nephropathy. Leucine-rich α-2-glycoprotein-1 (LRG1) was recently reported as a novel proangiogenic factor that is expressed in endothelial cells and promotes angiogenesis by modulating the transforming growth factor-ß signaling pathway. However, the pathophysiology of LRG1 in diabetic nephropathy remains largely unknown. In the present study, we investigated intrarenal expression of the novel proangiogenic factor LRG1 in diabetic db/db mice by immunohistochemistry and a laser capture microdissection method during the development of diabetic nephropathy. We hypothesized that glomerular LRG1 expression is increased earlier than VEGF expression under conditions of pathological angiogenesis in the early stage of diabetic nephropathy. Thus, we compared glomerular expression of VEGF and LRG1 in diabetic db/db mice at 16 and 24 weeks of age. At 16 weeks, diabetic db/db mice exhibited glomerular hypertrophy with abnormal angiogenesis characterized by endothelial cell proliferation, which was concomitant with an increase in LRG1 expression of glomerular endothelial cells. However, glomerular VEGF expression was not increased at this early stage. At 24 weeks, the features of early diabetic nephropathy in db/db mice had developed further, along with further enhanced glomerular LRG1 expression. At this late stage, glomerular VEGF and fibrosis-related-gene expression was also significantly increased compared with nondiabetic db/m mice. These results suggest that LRG1 plays a pivotal role in the initial development of diabetic nephropathy by promoting abnormal angiogenesis, thereby suggesting that LRG1 is a potential preemptive therapeutic target of diabetic nephropathy.

Angiotensin receptor-binding molecule in leukocytes in association with the systemic and leukocyte inflammatory profile.

BACKGROUND AND AIMS: The components of the renin-angiotensin system in leukocytes is involved in the pathophysiology of non-communicable diseases (NCDs), including hypertension, atherosclerosis and chronic kidney disease. Angiotensin II type 1 receptor (AT1R)-associated protein (ATRAP) is an AT1R-specific binding protein, and is able to inhibit the pathological activation of AT1R signaling in certain animal models of NCDs. The aim of the present study was to investigate the expression and regulation of ATRAP in leukocytes. METHODS: Human leukocyte ATRAP mRNA was measured with droplet digital polymerase chain reaction system, and analyzed in relation to the clinical variables. We also examined the leukocyte cytokines mRNA in bone-marrow ATRAP-deficient and wild-type chimeric mice after injection of low-dose lipopolysaccharide. RESULTS: The ATRAP mRNA was abundantly expressed in leukocytes, predominantly granulocytes and monocytes, of healthy subjects. In 86 outpatients with NCDs, leukocyte ATRAP mRNA levels correlated positively with granulocyte and monocyte counts and serum C-reactive protein levels. These positive relationships remained significant even after adjustment. Furthermore, the leukocyte ATRAP mRNA was significantly associated with the interleukin-1ß, tumor necrosis factor-α and monocyte chemotactic protein-1 mRNA levels in leukocytes of NCDs patients. In addition, the leukocyte interleukin-1ß mRNA level was significantly upregulated in bone marrow ATRAP-deficient chimeric mice in comparison to wild-type chimeric mice after injection of lipopolysaccharide. CONCLUSIONS: These results suggest that leukocyte ATRAP is an emerging marker capable of reflecting the systemic and leukocyte inflammatory profile, and plays a role as an anti-inflammatory factor in the pathophysiology of NCDs.

Identification of peptides present in sour milk whey that ameliorate scopolamine-induced memory impairment in mice.

Cognitive impairment is treated with cholinesterase inhibitors that slow cognitive decline but cause significant adverse effects. Functional foods that improve memory without such effects would therefore be valuable. We reported that unidentified components of sour milk whey produced by fermentations using Lactobacillus helveticus and Saccharomyces cerevisiae improved memory in a mouse model of scopolamine-induced memory impairment. Here, we show that casein-derived peptides were the most active components of orally administered fractions of this milk product. Of five peptides tested, ß-casein (residues 73-91) was the most effective for ameliorating scopolamine-induced cognitive deficits, as indicated by a significantly higher percentage of alternations of mice orally administered 0.05 nmol/kg peptide (58.0 ± 9.3%) versus vehicle (51.0 ± 5.8%). This orally active peptide may improve cognitive function of patients with dementia.

Adipocyte-Specific Enhancement of Angiotensin II Type 1 Receptor-Associated Protein Ameliorates Diet-Induced Visceral Obesity and Insulin Resistance.

BACKGROUND: The renin-angiotensin system has a pivotal role in the pathophysiology of visceral obesity. Angiotensin II type 1 receptor (AT1R) is a major player in the signal transduction of the renin-angiotensin system, and the overactivation of this signaling contributes to the progression of visceral obesity. We have shown that the AT1R-associated protein (ATRAP) promotes AT1R internalization from the cell surface into cytoplasm along with the suppression of overactivation of tissue AT1R signaling. In this study, we examined whether the enhancement of adipose ATRAP expression could efficiently prevent diet-induced visceral obesity and insulin resistance. METHODS AND RESULTS: We generated adipocyte-specific ATRAP transgenic mice using a 5.4-kb adiponectin promoter, and transgenic mice and littermate control mice were fed either a low- or high-fat diet for 10 weeks. Although the physiological phenotypes of the transgenic and control mice fed a low-fat diet were comparable, the transgenic mice exhibited significant protection against high-fat diet-induced adiposity, adipocyte hypertrophy, and insulin resistance concomitant with an attenuation of adipose inflammation, macrophage infiltration, and adipokine dysregulation. In addition, when mice were fed a high-fat diet, the adipose expression of glucose transporter type 4 was significantly elevated and the level of adipose phospho-p38 mitogen-activated protein kinase was significantly attenuated in the transgenic mice compared with control mice. CONCLUSIONS: Results presented in this study suggested that the enhancement in adipose ATRAP plays a protective role against the development of diet-induced visceral obesity and insulin resistance through improvement of adipose inflammation and function via the suppression of overactivation of adipose AT1R signaling. Consequently, adipose tissue ATRAP is suggested to be an effective therapeutic target for the treatment of visceral obesity.

An angiotensin II type 1 receptor binding molecule has a critical role in hypertension in a chronic kidney disease model.

Angiotensin II type 1 receptor-associated protein (ATRAP) promotes AT1R internalization along with suppression of hyperactivation of tissue AT1R signaling. Here, we provide evidence that renal ATRAP plays a critical role in suppressing hypertension in a mouse remnant kidney model of chronic kidney disease. The effect of 5/6 nephrectomy on endogenous ATRAP expression was examined in the kidney of C57BL/6 and 129/Sv mice. While 129/Sv mice with a remnant kidney showed decreased renal ATRAP expression and developed hypertension, C57BL/6 mice exhibited increased renal ATRAP expression and resistance to progressive hypertension. Consequently, we hypothesized that downregulation of renal ATRAP expression is involved in pathogenesis of hypertension in the remnant kidney model of chronic kidney disease. Interestingly, 5/6 nephrectomy in ATRAP-knockout mice on the hypertension-resistant C57BL/6 background caused hypertension with increased plasma volume. Moreover, in knockout compared to wild-type C57BL/6 mice after 5/6 nephrectomy, renal expression of the epithelial sodium channel α-subunit and tumor necrosis factor-α was significantly enhanced, concomitant with increased plasma membrane angiotensin II type 1 receptor in the kidneys. Thus, renal ATRAP downregulation is involved in the onset and progression of blood pressure elevation caused by renal mass reduction, and implicates ATRAP as a therapeutic target for hypertension in chronic kidney disease.

Effects of the Angiotensin receptor blocker olmesartan on adipocyte hypertrophy and function in mice with metabolic disorders.

In the present study, we examined the therapeutic effects of olmesartan, an angiotensin II (Ang II) type 1 receptor (AT1R)-specific blocker, in genetically obese diabetic KKAy mice, a model of human metabolic disorders with visceral obesity, with a focus on an olmesartan effect on the adipose tissue. Olmesartan treatment (3 mg/kg per day) for 4 weeks significantly lowered systolic blood pressure but did not affect body weight during the study period in KKAy mice. However, there were three interesting findings possibly related to the pleiotropic effects of olmesartan on adipose tissue in KKAy mice: (1) an inhibitory effect on adipocyte hypertrophy, (2) a suppressive effect on IL-6 gene expression, and (3) an ameliorating effect on oxidative stress. On the other hand, olmesartan exerted no evident influence on the adipose tissue expression of AT1R-associated protein (ATRAP), which is a molecule interacting with AT1R so as to inhibit pathological AT1R activation and is suggested to be an emerging molecular target in metabolic disorders with visceral obesity. Collectively, these results suggest that the blood pressure lowering effect of olmesartan in KKAy mice is associated with an improvement in adipocyte, including suppression of adipocyte hypertrophy and inhibition of the adipose IL-6-oxidative stress axis. Further study is needed to clarify the functional role of adipose ATRAP in the pleiotropic effects of olmesartan.

Milk-derived peptides, Val-Pro-Pro and Ile-Pro-Pro, attenuate atherosclerosis development in apolipoprotein e-deficient mice: a preliminary study.

Milk-derived peptides, Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP), have angiotensin I-converting enzyme inhibitory activities and blood pressure-lowering effects. We examined the effects of these peptides on the development of atherosclerosis in apolipoprotein E-deficient [apoE(-/-)] mice. For 31 weeks, six-week-old male apoE(-/-) mice received a diet that included one of the following: fermented milk containing both VPP and IPP; casein hydrolysate containing both of these peptides; synthesized VPP; synthesized IPP; enalapril; captopril; or control diet. At the end of feeding, blood biochemistry, aortic atherogenesis, and gene expression by DNA microarray analysis were evaluated. There were no significant changes in the plasma lipid levels and 8-isoprostane, a marker of oxidative stress. The area ratio of intima to media in the aortic arch was significantly lower in the fermented milk, casein hydrolysate, synthesized VPP, enalapril, and captopril groups than in the control group. As is common with diets containing VPP and/or IPP, we observed reductions in mRNA expression of inflammatory cytokines, such as interleukin (IL)-6 and IL-1ß, oxidized low-density lipoprotein receptor, and transcription regulators. These results suggest that a continuous intake of VPP and IPP might be beneficial for preventing atherosclerosis caused by hypercholesterolemia.