Renal histopathological changes were evaluated by examining periodic acid-Schiff (PAS) staining in Protocol 2.

<p><b>A</b>, Representative images of PAS-stained renal sections (scale bar shows the values). <b>B</b>, The PAS-positive area within total glomerular area. <b>C</b>, Mean glomerular diameters expressed as fold changes to 10-week values of the DSS+NS group. ^#<i>P</i><0.05 <i>vs.</i> 10-week values of DSS + HS group. ^a<i>P</i><0.05 <i>vs.</i> DSS + NS, ^b<i>P</i><0.05 <i>vs.</i> DSS + HS, ^c<i>P</i><0.05 <i>vs.</i> DSS + HS → NS, ^d<i>P</i><0.05 <i>vs.</i> DSS + HS → NS + OLM or DSS + HS → NS + AZEL.</p

Genes expression from protocol 2 (fold changes).

<p>FSP-1; fibroblast-specific protein-1, Sgk-1; serum and glucocorticoid-regulated kinases-1, NHE-1; Na⁺/H⁺ exchanger isoform-1, DSS; Dahl salt-sensitive rats, NS; normal salt (0.5% NaCl), HS; High salt (4% NaCl), OLM; olmesartan, AZEL; azelnidipine, HYD; hydralazine. β-actin was used as an internal control and results are expressed as fold changes to 10-week values of the DSS + NS group. _#<i>P</i><0.05 <i>vs.</i> 10-week values of the DSS + HS group. _a<i>P</i><0.05 <i>vs.</i> DSS + NS, _b<i>P</i><0.05 <i>vs.</i> DSS + HS, _c<i>P</i><0.05 <i>vs.</i> DSS + HS → NS, _d<i>P</i><0.05 <i>vs.</i> DSS + HS → NS + OLM or DSS + HS → NS + AZEL.</p><p>Genes expression from protocol 2 (fold changes).</p

NADPH oxidase dependent superoxide anion production in renal tissue in Protocol 2.

<p><b>A</b>, Representative images of 4-hydroxy-2-nonenal (4-HNE) immunostaining (scale bar shows the values). <b>B</b>, Quantitative analysis of 4-HNE-positive area. <b>C</b>, NADPH oxidase activity in homogenized renal cortical tissues measured by lucigenin-enhanced chemiluminescence. Renal cortical tissue (<b>D</b>) gp91phox and (<b>E</b>) Nox-1 mRNA levels were determined by real time RT-PCR. β-actin was used as an internal control and results are expressed as fold changes to 10-week values of the DSS + NS group. ^#<i>P</i><0.05 <i>vs.</i> 10-week values of DSS + HS group. ^a<i>P</i><0.05 <i>vs.</i> DSS + NS, ^b<i>P</i><0.05 <i>vs.</i> DSS + HS, ^c<i>P</i><0.05 <i>vs.</i> DSS + HS → NS, ^d<i>P</i><0.05 <i>vs.</i> DSS + HS → NS + OLM or DSS + HS → NS + AZEL.</p

Regression of Glomerular and Tubulointerstitial Injuries by Dietary Salt Reduction with Combination Therapy of Angiotensin II Receptor Blocker and Calcium Channel Blocker in Dahl Salt-Sensitive Rats

<div><p>A growing body of evidence indicates that renal tissue injuries are reversible. We investigated whether dietary salt reduction with the combination therapy of angiotensin II type 1 receptor blocker (ARB) plus calcium channel blocker (CCB) reverses renal tissue injury in Dahl salt-sensitive (DSS) hypertensive rats. DSS rats were fed a high-salt diet (HS; 4% NaCl) for 4 weeks. Then, DSS rats were given one of the following for 10 weeks: HS diet; normal-salt diet (NS; 0.5% NaCl), NS + an ARB (olmesartan, 10 mg/kg/day), NS + a CCB (azelnidipine, 3 mg/kg/day), NS + olmesartan + azelnidipine or NS + hydralazine (50 mg/kg/day). Four weeks of treatment with HS diet induced hypertension, proteinuria, glomerular sclerosis and hypertrophy, glomerular podocyte injury, and tubulointerstitial fibrosis in DSS rats. A continued HS diet progressed hypertension, proteinuria and renal tissue injury, which was associated with inflammatory cell infiltration and increased proinflammatory cytokine mRNA levels, NADPH oxidase activity and NADPH oxidase-dependent superoxide production in the kidney. In contrast, switching to NS halted the progression of hypertension, renal glomerular and tubular injuries. Dietary salt reduction with ARB or with CCB treatment further reduced blood pressure and partially reversed renal tissues injury. Furthermore, dietary salt reduction with the combination of ARB plus CCB elicited a strong recovery from HS-induced renal tissue injury including the attenuation of inflammation and oxidative stress. These data support the hypothesis that dietary salt reduction with combination therapy of an ARB plus CCB restores glomerular and tubulointerstitial injury in DSS rats.</p></div

The oligonucleotide primer sequences for real-time RT-PCR.

<p>TGF-β; transforming growth factor β, MCP-1; monocyte chemoattractant protein 1, PAI-1; plasminogen activator inhibitor 1, α-SMA; alpha smooth muscle actin, FSP-1; fibroblast-specific protein-1.</p><p>The oligonucleotide primer sequences for real-time RT-PCR.</p

Cellular infiltration and proinflammatory cytokine mRNA levels in Protocol 2.

<p><b>A</b>, The number of mononuclear cells in the interstitial space of the kidney was quantified. Renal cortical tissues (<b>B</b>) monocyte chemoattractant protein 1 (MCP-1), and (<b>C</b>) plasminogen activator inhibitor 1 (PAI-1) mRNA levels were determined by real time RT-PCR. β-actin was used as an internal control and results are expressed as fold changes to 10-week values of the DSS + NS group. ^#<i>P</i><0.05 <i>vs.</i> 10-week values of DSS + HS group. ^a<i>P</i><0.05 <i>vs.</i> DSS + NS, ^b<i>P</i><0.05 <i>vs.</i> DSS + HS, ^c<i>P</i><0.05 <i>vs.</i> DSS + HS → NS, ^d<i>P</i><0.05 <i>vs.</i> DSS + HS → NS + OLM or DSS + HS → NS + AZEL.</p

Glomerular podocyte injury was detected by immunohistochemistry for Desmin in Protocol 2.

<p><b>A</b>, Representative micrographs of Desmin-stained renal sections (scale bar shows the values). <b>B</b>, Quantitative analysis of Desmin-positive area. <b>C</b>, mRNA expression of nephrin, and <b>D</b>, podocin were quantitatively analyzed by real time RT-PCR. β-actin was used as an internal control and results are expressed as fold changes to 10-week values of the DSS + NS group. ^#<i>P</i><0.05 <i>vs.</i> 10-week values of the DSS + HS group. ^a<i>P</i><0.05 <i>vs.</i> DSS + NS, ^b<i>P</i><0.05 <i>vs.</i> DSS + HS, ^c<i>P</i><0.05 <i>vs.</i> DSS + HS → NS, ^d<i>P</i><0.05 <i>vs.</i> DSS + HS → NS + OLM or DSS + HS → NS + AZEL.</p

Systolic blood pressure (SBP) profile measured by tail-cuff plethysmography in Protocol2.

<p><b>A</b>, SBP. Four weeks after high-salt (4%) loading (from 6 to 10 weeks of age), dietary salt reduction and/or drug treatment were given for 10 weeks (from 10 to 20 weeks of age). <b>B</b>, Delta changes in SBP were calculated by deducting 10-week values from 20-week values of the respective group. NS; normal salt, HS; high salt, OLM; olmesartan, AZEL; azelnidipine, HYD; hydralazine. *<i>P</i><0.05 vs. baseline values of each respective group. ^#<i>P</i><0.05 <i>vs.</i> 10-week values of each respective group or of DSS + HS group. ^a<i>P</i><0.05 <i>vs.</i> DSS + NS, ^b<i>P</i><0.05 <i>vs.</i> DSS + HS, ^c<i>P</i><0.05 <i>vs.</i> DSS + HS → NS, ^d<i>P</i><0.05 <i>vs.</i> DSS + HS → NS + OLM or DSS + HS → NS + AZEL.</p

The Influence of a Single Nucleotide Polymorphism within <em>CNDP1</em> on Susceptibility to Diabetic Nephropathy in Japanese Women with Type 2 Diabetes

<div><h3>Background</h3><p>Several linkage analyses have mapped a susceptibility locus for diabetic nephropathy to chromosome 18q22–23, and polymorphisms within the carnosine dipeptidase 1 gene (<em>CNDP1)</em>, located on 18q22.3, have been shown to be associated with diabetic nephropathy in European subjects with type 2 diabetes. However, the association of this locus with diabetic nephropathy has not been evaluated in the Japanese population. In this study, we examined the association of polymorphisms within the <em>CNDP1/CNDP 2</em> locus with diabetic nephropathy in Japanese subjects with type 2 diabetes.</p> <h3>Methodology/Principal Findings</h3><p>We genotyped a leucine repeat polymorphism (D18S880) that is within <em>CNDP1</em> along with 29 single nucleotide polymorphisms (SNPs) in the <em>CNDP1</em>/<em>CNDP2</em> locus for 2,740 Japanese subjects with type 2 diabetes (1,205 nephropathy cases with overt nephropathy or with end-stage renal disease [ESRD], and 1,535 controls with normoalbuminuria). The association of each polymorphism with diabetic nephropathy was analysed by performing logistic regression analysis. We did not observe any association between D18S880 and diabetic nephropathy in Japanese subjects with type 2 diabetes. None of the 29 SNPs within the <em>CNDP1/CNDP2</em> locus were associated with diabetic nephropathy, but a subsequent sex-stratified analysis revealed that 1 SNP in <em>CNDP1</em> was nominally associated with diabetic nephropathy in women (rs12604675-A; <em>p</em> = 0.005, odds ratio [OR] = 1.76, 95% confidence interval [CI], 1.19−2.61). Rs12604675 was associated with overt proteinuria (<em>p</em> = 0.002, OR = 2.18, 95% CI, 1.32−3.60), but not with ESRD in Japanese women with type 2 diabetes.</p> <h3>Conclusions/Significance</h3><p>Rs12604675-A in <em>CNDP1</em> may confer susceptibility to overt proteinuria in Japanese women with type 2 diabetes.</p> </div

Predictive Significance of Kidney Myeloid-Related Protein 8 Expression in Patients with Obesity- or Type 2 Diabetes-Associated Kidney Diseases

<div><p>Background and Objective</p><p>We have reported that toll-like receptor 4 (TLR4) and one of its endogenous ligands, myeloid-related protein 8 (MRP8 or S100A8), play an important role in the progression of diabetic nephropathy in mice. The aim of this study was to evaluate significance of kidney MRP8 expression in patients with obesity- or type 2 diabetes-associated kidney diseases.</p><p>Methods</p><p>In diabetic, obese or control subjects, MRP8 mRNA and protein expression levels in renal biopsy samples were determined by real-time RT-PCR and immunohistochemistry (n = 28 and 65, respectively), and their associations with baseline and prognostic parameters were analyzed. Effects of MRP8 upon pro-inflammatory gene expressions were examined using macrophages.</p><p>Results</p><p>Kidney MRP8 gene and protein expression levels were elevated in obese or diabetic groups compared to control group. Among all subjects, by univariate linear regression analysis, glomerular MRP8-positive cell count and tubulointerstitial MRP8-positive area at baseline were both, respectively, correlated not only with various known risk factors for diabetic nephropathy (such as systolic blood pressure, proteinuria and serum creatinine) but also with extent of glomerulosclerosis and tubulointerstitial fibrosis. Independent factors predicting urinary protein levels a year later were examined by multivariate analysis, and they included glomerular MRP8-positive cell count (β = 0.59, P<0.001), proteinuria (β = 0.37, P = 0.002) and systolic blood pressure (β = 0.21, P = 0.04) at baseline, after adjustment for known risk factors. MRP8 protein expression was observed in CD68-positive macrophages and atrophic tubules. In cultured mouse macrophages, MRP8 protein induced proinflammatory cytokine expressions and also triggered auto-induction of MRP8 in a TLR4-dependent manner.</p><p>Conclusions</p><p>Glomerular MRP8 expression appears to be associated with progression of proteinuria in obese or type 2 diabetic patients, possibly by inducing inflammatory changes in macrophages through TLR4 signaling.</p></div