Proton gradient formation in early endosomes from proximal tubules

AbstractHeavy endosomes were isolated from proximal tubules using a combination of magnesium precipitation and wheat-germ agglutinin negative selection techniques. Two small GTPases (Rab4 and Rab5) known to be specifically present in early endosomes were identified in our preparations. Endosomal acidification was followed fluorimetrically using acridine orange. In presence of chloride ions and ATP, the formation of a proton gradient (ΔpH) was observed. This process is due to the activity of an electrogenic V-type ATPase present in the endosomal membrane since specific inhibitors bafilomycin and folimycin effectively prevented or eliminated endosomal acidification. In presence of chloride ions (Km = 30 mM) the formation of the proton gradient was optimal. Inhibitors of chloride channel activity such as DIDS and NPPB reduced acidification. The presence of sodium ions stimulated the dissipation of the proton gradient. This effect of sodium was abolished by amiloride derivative (MIA) but only when loaded into endosomes, indicating the presence of a physiologically oriented Na+/H+-exchanger in the endosomal membrane. Monensin restored the gradient dissipation. Thus three proteins (V-type ATPase, Cl−-channel, Na+/H+-exchanger) present in early endosomas isolated from proximal tubules may regulate the formation, maintenance and dissipation of the proton gradient

Effect of sodium bicarbonate supplementation on the renin-angiotensin system in patients with chronic kidney disease and acidosis:a randomized clinical trial

Background Acidosis-induced kidney injury is mediated by the intrarenal renin-angiotensin system, for which urinary renin is a potential marker. Therefore, we hypothesized that sodium bicarbonate supplementation reduces urinary renin excretion in patients with chronic kidney disease (CKD) and metabolic acidosis. Methods Patients with CKD stage G4 and plasma bicarbonate 15-24 mmol/l were randomized to receive sodium bicarbonate (3 x 1000 mg/day, similar to 0.5 mEq/kg), sodium chloride (2 x 1,00 mg/day), or no treatment for 4 weeks (n = 15/arm). The effects on urinary renin excretion (primary outcome), other plasma and urine parameters of the renin-angiotensin system, endothelin-1, and proteinuria were analyzed. Results Forty-five patients were included (62 +/- 15 years, eGFR 21 +/- 5 ml/min/1.73m(2), plasma bicarbonate 21.7 +/- 3.3 mmol/l). Sodium bicarbonate supplementation increased plasma bicarbonate (20.8 to 23.8 mmol/l) and reduced urinary ammonium excretion (15 to 8 mmol/day, both P <0.05). Furthermore, a trend towards lower plasma aldosterone (291 to 204 ng/L, P = 0.07) and potassium (5.1 to 4.8 mmol/l, P = 0.06) was observed in patients receiving sodium bicarbonate. Sodium bicarbonate did not significantly change the urinary excretion of renin, angiotensinogen, aldosterone, endothelin-1, albumin, or alpha 1-microglobulin. Sodium chloride supplementation reduced plasma renin (166 to 122 ng/L), and increased the urinary excretions of angiotensinogen, albumin, and alpha 1-microglobulin (all P <0.05). Conclusions Despite correction of acidosis and reduction in urinary ammonium excretion, sodium bicarbonate supplementation did not improve urinary markers of the renin-angiotensin system, endothelin-1, or proteinuria. Possible explanations include bicarbonate dose, short treatment time, or the inability of urinary renin to reflect intrarenal renin-angiotensin system activity

Dietary salt modifies the blood pressure response to renin-angiotensin inhibition in experimental chronic kidney disease

Chronic kidney disease contributes to hypertension, but the mechanisms are incompletely understood. To address this, we applied the 5/6th nephrectomy rat model to characterize hypertension and the response to dietary salt and renin-Angiotensin inhibition. 5/6th nephrectomy caused low-renin, salt-sensitive hypertension with hyperkalemia and unsuppressed aldosterone. Compared with sham rats, 5/6th nephrectomized rats had lower Na/H exchanger isoform 3, Na-K-2Cl cotransporter, Na-Cl cotransporter, a-epithelial Na channel (ENaC), and Kir4.1 levels but higher serum and glucocorticoid-regulated kinase 1, prostasin, c-ENaC, and Kir5.1 levels. These differences correlated with plasma renin, aldosterone, and/or K. On a normal-salt diet, adrenalectomy (0 ± 9 mmHg) and spironolactone (11 ± 10 mmHg) prevented a progressive rise in blood pressure (10 ± 8 mmHg), and this was enhanced in combination with losartan (41 ± 12 and 43 ± 9 mmHg). A high-salt diet caused skin Na and water accumulation and aggravated hypertension that could only be attenuated by spironolactone (16 ± 7 mmHg) and in which the additive effect of losartan was lost. Spironolactone also increased natriuresis, reduced skin water accumulation, and restored vasorelaxation. In summary, in the 5/6th nephrectomy rat chronic kidney disease model, salt-sensitive hypertension develops with a selective increase in c-ENaC and despite appropriate transporter adaptations to low renin and hyperkalemia. With a normal-salt diet, hypertension in 5/6th nephrectomy depends on angiotensin II and aldosterone, whereas a high-salt diet causes more severe hypertension mediated through the mineralocorticoid receptor. NEW and NOTEWORTHY Chronic kidney disease (CKD) causes salt-sensitive hypertension, but the interactions between dietary salt and the renin-Angiotensin system are incompletely understood. In rats with CKD on a normal-salt diet targeting aldosterone, the mineralocorticoid receptor (MR) and especially angiotensin II reduced blood pressure. On a high-salt diet, however, only MR blockade attenuated hypertension. These results reiterate the importance of dietary salt restriction to maintain renin-Angiotensin system inhibitor efficacy and specify the MR as a target in CKD

A plasma creatinine- and urea-based equation to estimate glomerular filtration rate in rats

Monitoring renal function is a vital part of kidney research involving rats. The laborious measurement of glomerular filtration rate (GFR) with administration of exogenous filtration markers does not easily allow serial measurements. Using an in-house database of inulin clearances, we developed and validated a plasma creatinine- and plasma urea-based equation to estimate GFR in a large cohort of male rats [development cohort n = 325, R2 = 0.816, percentage of predictions that fell within 30% of the true value (P30) = 76%] that had high accuracy in the validation cohort (n = 116 rats, R2 = 0.935, P30 = 79%). The equation was less accurate in rats with nonsteady-state creatinine, in which the equation should therefore not be used. In conclusion, applying this equation facilitates easy and repeatable estimates of GFR in rats. NEW & NOTEWORTHY This is the first equation, that we know of, which estimates glomerular filtration rate in rats based on a single measurement of body weight, plasma creatinine, and plasma urea

New chondrosarcoma cell lines and mouse models to study the link between chondrogenesis and chemoresistance

Chondrosarcomas are cartilage-forming, poorly vascularized tumors. They represent the second malignant primary bone tumor of adults after osteosarcoma, but in contrast to osteosarcoma they are resistant to chemotherapy and radiotherapy, surgical excision remaining the only therapeutic option. Few cell lines and animal models are available, and the mechanisms behind their chemoresistance remain largely unknown. Our goal was to establish new cell lines and animal cancer models from human chondrosarcoma biopsies to study their chemoresistance. Between 2007 and 2012, 10 chondrosarcoma biopsies were collected and used for cell culture and transplantation into nude mice. Only one transplanted biopsy and one injected cell line has engrafted successfully leading to conventional central high-grade chondrosarcoma similar to the original biopsies. In culture, two new stable cell lines were obtained, one from a dedifferentiated and one from a grade III conventional central chondrosarcoma biopsy. Their genetic characterization revealed triploid karyotypes, mutations in IDH1, IDH2, and TP53, deletion in CDKN2A and/or MDM2 amplification. These cell lines expressed mesenchymal membrane markers (CD44, 73, 90, 105) and were able to produce a hyaline cartilaginous matrix when cultured in chondrogenic three-dimensional (3D) pellets. Using a high-throughput quantitative RT-PCR approach, we observed that cell lines cultured in monolayer had lost expression of several genes implicated in cartilage development (COL2A1, COMP, ACAN) but restored their expression in 3D cultures. Chondrosarcoma cells in monolayer were sensitive to several conventional chemotherapeutic agents but became resistant to low doses of mafosfamide or doxorubicin when cultured in 3D pellets, in parallel with an altered nucleic accumulation of the drug. Our results indicate that the cartilaginous matrix produced by chondrosarcoma cells may impair diffusion of several drugs and thus contribute to chemoresistance. Therefore, 3D chondrogenic cell pellets constitute a more relevant model to study chondrosarcoma chemoresistance and may be a valuable alternative to animal experimentations