Possible role of adenosine in macula densa control of glomerular hemodynamics

Possible role of adenosine in macula densa control of glomerular hemodynamics.BackgroundThe macula densa (MD), a plaque of specialized tubular epithelial cells, senses changes in tubular NaCl concentration and sends a signal(s) that controls the resistance of the glomerular afferent arteriole (Af-Art). This mechanism, called tubuloglomerular feedback (TGF), is thought to be important in the homeostasis of body fluids and electrolytes. Our aim was to determine the range of NaCl concentrations in tubular fluid at the MD that would elicit the Af-Art response. In addition, we examined the possible involvement of adenosine in transmitting the signal from the MD to the Af-Art.MethodsRabbit Af-Arts and attached MD were simultaneously microperfused in vitro, keeping pressure in the Af-Art at 60mm Hg.ResultsIncreasing the Na+/Cl- concentration of the MD perfusate from 26/7 to 41/22 mEq/L decreased the luminal diameter of the terminal Af-Art segment by 10 ± 4% (N = 9; P < 0.01). The response was maximal at 55/36 mEq/L (18 ± 6%), so that further elevation of NaCl concentration had no additional effect (20 ± 6% at 84/65 mEq/L). When FK838 (10-6 mol/L), a specific adenosine A1 receptor antagonist, was added to both Af-Art perfusate and bath, Af-Art constriction was completely abolished. The maximum response was 20 ± 3% before FK838 and 0.6 ± 1% afterward (N = 12). Adding adenosine at 10-8 mol/L to both bath and perfusate significantly augmented Af-Art constriction induced by increased NaCl at the MD (P < 0.01); however, adding 10-8 to 10-6 mol/L adenosine to the MD perfusate had no effect regardless of the NaCl concentration at the MD.ConclusionsThese results demonstrate that MD control of Af-Art resistance is induced by relatively low NaCl concentrations at the MD, and that activation of the adenosine A1 receptor in the vascular and interstitial space (but not the tubular lumen) may be essential for signal transmission from the MD to the Af-Art

Role of neuronal nitric oxide synthase in the macula densa

Role of neuronal nitric oxide synthase in the macula densa.BackgroundThere is evidence that macula densa nitric oxide (NO) inhibits tubuloglomerular feedback (TGF). However, TGF response is not altered in mice deficient in neuronal nitric oxide synthase (nNOS) (-/-). Furthermore, nNOS expression in the macula densa is inversely related to salt intake, yet micropuncture studies have shown that NOS inhibition potentiates TGF in rats on high sodium intake but not in rats on a low-salt diet. These inconsistencies may be due to confounding systemic factors, such as changes in circulating renin. To further clarify the role of macula densa nNOS in TGF response, independent of systemic factors, we tested the hypothesis that (1) TGF response is inversely related to sodium intake, and (2) during low sodium intake, NO produced by macula densa nNOS tonically controls the basal diameter of the afferent arteriole (Af-Art).MethodsAf-Arts and attached macula densas were simultaneously microperfused in vitro. TGF response was determined by measuring Af-Art diameter before and after increasing NaCl in the macula densa perfusate. TGF response was studied in wild-type (+/+) and nNOS knockout mice (-/-), as well as in juxtaglomerular apparatuses (JGAs) from rabbits fed a low-, normal-, or high-NaCl diet.ResultsTGF responses were similar in nNOS +/+ and -/- mice. However, in nNOS +/+ mice, 7-nitroindazole (7-NI) perfused into the macula densa significantly potentiated the TGF response (P = 0.001), while in nNOS -/- mice, this potentiation was absent. In rabbits on three different sodium diets, TGF responses were similar and were potentiated equally by 7-NI. However, in JGAs from rabbits on a low-NaCl diet, adding 7-NI to the macula densa while perfusing it with low-NaCl fluid caused Af-Art vasoconstriction, decreasing the diameter by 14% (from 21.7 ± 1.3 to 18.6 ± 1.5 μm; P < 0.001). This effect was not observed in JGAs from rabbits fed a normal- (19.0 ± 0.5 vs. 19.3 ± 0.8 μm after 7-NI) or high-NaCl diet (18.6 ± 0.7 vs. 18.4 ± 0.7 μm).ConclusionsFirst, in this in vitro preparation, chronic changes in macula densa nNOS do not play a major role in the regulation of TGF. Compensatory mechanisms may develop during chronic alteration of nNOS that keep TGF relatively constant. Second, nNOS regulates TGF response acutely. Third, the results obtained in the +/+ and -/- mice also confirm that the effect of 7-NI is due to inhibition of macula densa nNOS. Finally, during low sodium intake (without induction of TGF), the regulation of basal Af-Art resistance by macula densa nNOS suggests that NO in the macula densa helps maintain renal blood flow during the high renin secretion caused by low sodium intake

半月体性腎炎におけるbeta2 integrin 関与の病型間の相違について

An immunohistochemical study was performed to investigate the involvement of beta2 integrins in the glomerular infiltration of leukocytes in different types of human crescentic glomerulonephritis (CrGN). On 51 ethanol-fixed paraffin embedded renal biopsy specimens (34 cases of pauci-immune type and 17 cases of immune-mediated type), the following antigens were evaluated by immunoperoxidase method ; the glomerular (intra-capillary and extra-capillary) expression of ICAM-1, deposition of FRA and infiltration of leukocytes bearing beta2 integrins (LFA-1, CR3 and CR4). ICAM-1 was expressed both in extra-capillary and intra-capillary areas in very early stage of crescent formation and was rapidly decreased with the progression of the stage of crescent. Similarly, infiltration of leukocytes bearing beta2 integrins were observed both in extra-capillary and intra-capillary areas in early stage of crescent formation and were rapidly decreased with the progression of the stage of crescent. In pauci-immune type, the extra-capillary expression of ICAM-1 was significantly correlated with extra-capillary infiltration of LFA-1+cells (γ=0.800, p<0.0001) and CR3+cells (γ=0.791, p<0.0001). The extra-capillary deposition of FRA was also significantly correlated with extra-capillary infiltration of CR3+cells (γ=0.685, p<0.001) and CR4+cells (γ=0. 741, p<0.0001). In both types, the intra-capillary expression of ICAM-1 was significantly correlated with intra-capillary infiltration of LFA-1+cells (γ=0.618, p<0.01 and γ=0.754, p<0.05 respectively). These results suggested that ICAM-1/LFA-1 and ICAM-1/CR3 interaction might be involved in both the extra- and intra-capillary infiltration of leukocytes in pauci-immune CrGN. CR3 and CR4 might also function as fibrin/fibrinogen receptors in extra-capillary lesions in this type. On the other hand, only ICAM-1/LFA-1 interaction might be involved only in the intra-capillary infiltration of leukocytes in immune mediated CrGN

Lipoprotein glomerulopathy: Significance of lipoprotein and ultrastructural features

Lipoprotein glomerulopathy: Significance of lipoprotein and ultrastructural features.BackgroundLipoprotein glomerulopathy (LPG) is a unique disease characterized by intraglomerular lipoprotein thrombi and type III hyperlipoproteinemia. Recently, we have demonstrated that LPG is associated with inherited apolipoprotein E (apoE) variants including apoE Sendai. On the other hand, electron microscopy shows that intraglomerular lipoprotein thrombi consist of lipid granules of various sizes. To elucidate the relationship between the peculiar histology and abnormal lipid metabolism related to apoE Sendai, we studied lipoprotein profiles and ultrastructural features.MethodsThe subjects were 11 patients with LPG. Four patients were nephrotic, and two others became nephrotic within six months following the biopsy. Eight patients underwent apoE gene analysis and showed apoE Sendai. The other three were presumed to have apoE Sendai because this mutation was demonstrated in their kindreds. Under electron microscopy, diameters of more than 1000 lipid granules were measured in several glomeruli, and a mean value was calculated in each case. Lipoprotein profiles were analyzed by the ultracentrifugation methods.ResultsThe mean diameter of intraglomerular lipid granules correlated inversely with the levels of plasma triglyceride (TG; rs = -0.73, P < 0.05), TG (rs = -0.77, P < 0.01) and cholesterol (Chol; rs = -0.75, P < 0.05) in very low-density lipoprotein (VLDL) fraction and TG in high-density lipoprotein (HDL) fraction (rs = -0.75, P < 0.05). The inverse correlation was also seen between the mean lipid diameter and TG/Chol ratios in whole plasma (rs = -0.80, P < 0.01) and in HDL (rs = -0.80, P < 0.01). In addition, the cases showing smaller lipid granules and higher TG/Chol ratios in plasma and in HDL were nephrotic or became nephrotic within six months.ConclusionThese results suggest that the size of lipid granules in LPG may become smaller under the influence of hypertriglyceridemia and particularly elevated plasma VLDL and HDL-TG, which may lead to heavy proteinuria

LOW BMI IS THE RISK OF CARDIO-VASCULAR MORTALITY WITHOUT PROGRESSION OF CKD

The paradoxical risk of BMI on mortality is known in CKD as well in dialysis populations, but studies of CVD risk in CKD including underweight is limited. We hypothesized lean CKD increase the CVD risk, contributing different factors from obese. 2,676 CKD patients recruited from 11 outpatients’ hospitals. BMI and estimated GFR (eGFR) were calculated, and change of eGFR and CVD mortality during 2 years were collected. Patients were divided by BMI under cut off value of normal, thus 7% grouped in lean subjects (BMI <18.5). Systolic blood pressure (sBP), albumin, hemoglobin, age and prevalence of diabetes were lower in lean BMI group compared to other subjects. However CVD history, urinary protein, baseline eGFR and smoking didn't differ between the groups. The lean BMI increased significantly the risk of CVD mortality, in spite of low prevalence of comorbidities and young age in unadjusted model (HR 2.38, 95%CI 1.49-5.21, p<0.01). This significance remained after adjusted for CVD risk factors, such as primary disease of CKD, age, sex, smoking, albumin, cholesterol, sBP and eGFR. On the other hand, BMI was not associated with the decline rate of eGFR. We concluded that BMI less than 18.5 was an independent predictor of CVD, and that BMI did not effect on CKD progression rate in Japanese CKD

High glucose level and angiotensin II type 1 receptor stimulation synergistically amplify oxidative stress in renal mesangial cells

Abstract Oxidative stress in renal mesangial cell causes diabetic glomerular changes. High glucose levels and angiotensin II (Ang II) are known to stimulate superoxide production in renal mesangial cells. However, it has been unclear whether Ang II stimulation and pre-conditioning with high glucose affects the same pathway of superoxide production in renal mesangial cells or not. In this study, we examined the levels of oxidative stress under Ang II stimulation in renal mesangial cells preincubated for six hours at various glucose levels. Intracellular levels of reactive oxidative species (ROS) were measured using dihydroethidium or 5′,6′-chloromethyl- 2′,7′ dichlorodihydro-fluorescein diacetate, which facilitates the detection of intracellular ROS under real-time fluorescent microscope. Ang II-induced elevated intracellular ROS levels were detected only when the cells were pre-incubated with high levels of glucose (13.5 mM, 27.8 mM), but was not detected under normal glucose condition (5.5 mM). Production of Ang II-induced intracellular ROS was higher under pre-treatment with 27.8 mM glucose compared to pretreatment with 13.5 mM glucose level. This ROS production in mesangial cells was induced within several minutes of the initiation of Ang II stimulation under high glucose levels. The production of intracellular ROS was significantly reduced in the presence of angiotensin II type1-receptor (AT1R) antagonist, whereas it was augmented in the presence of angiotensin II type2-receptor antagonist. In conclusion, Ang II-induced oxidative stress was augmented by high glucose levels and ROS levels were further alleviated in the presence of AT1R antagonists

Characterization of a mouse cortical collecting duct cell line

Characterization of a mouse cortical collecting duct cell line. A cortical collecting duct (CCD) cell line has been developed from a mouse transgenic for the early region of simian virus 40, Tg(SV40E)Bri/7. CCDs were microdissected and placed on collagen gels. Monolayers were subsequently subcultured onto permeable collagen membranes and maintained in serum-supplemented medium. One line, designated M-1, retained many characteristics of the CCD, including a typical epithelial appearance and CCD-specific antigens. M-1 cells, when grown in monolayers on permeable supports, exhibited a high transepi-thelial resistance (885.7 ± 109.6 ohms/cm2) and developed a lumen negative transepithelial potential difference (PD) of -45.7 ± 3.5mV. The associated short-circuit current (SCC) averaged 71.8 ± 10.3 µA/cm2, and was reduced by 95% by luminal application of amiloride. The cultured cells responded to arginine vasopressin (AVP) with a significant increase in SCC. M-1 cells generated significant transepithelial solute gradients. After 24 hours incubation, the composition of the luminal (L) and basolateral (B) media (in mM) was: [Na+], L = 106.7 ± 0.9 and B = 127.4 ± 0.4; [K+], L = 8.6 ± 0.6 and B = 2.1 ± 0.3; [Cl], L = 68.6 ± 5.8 and B = 101.8 ± 6.6; [HCO3], L = 15.5 ± 1.5 and B = 8.6 ± 1.2; while pH was 7.16 ± 0.03 at the luminal and 6.94 ± 0.03 at the basolateral side. The formation of these concentration gradients indicates that the CCD cultures absorb Na+ and Cl- and secrete K+. Lactate accumulated predominantly at the basolateral side (L = 7.1 ± 0.44 and B = 17.5 ± 0.52 mM); osmotic concentration was 272 ± 1.4 at the luminal and 290 ±3.0 mOsm/kg at the basolateral side. These data demonstrate that the M-1 cell line retains many phenotypic properties of the CCD epithelium and thus should prove useful as a model in studying mechanisms of ion transport in this segment