Shunya Uchida

Regulation of cortical collecting duct function: Effect of endothelin☆

We recently showed that endothelin-1 (ET-1) increases cell Ca2+ in the mouse cortical collecting duct. To clarify the cellular action and target cell of ET-1, electrophysiologic techniques and cell Ca2+ measurement were applied to rabbit cortical collecting ducts perfused in vitro. When 10(-8) mol/L ET-1 was added to the bath, a transient increase followed by a sustained increase in cell Ca2+ was observed. A sustained increase in cell Ca2+ lasted 10 to 20 minutes and was associated with a decrease in lumen-negative transepithelial voltage. To confirm the target cell type of ET-1, confocal laser microscopy was used. An increase in cell Ca2+ was observed in the same cell, which also showed an increase in cell Ca2+ in response to arginine vasopressin (AVP), which indicated that the principal cell has ET-1 receptors in the basolateral membrane. When ET-1 was applied to the bath, total cellular membrane resistance (Ri) decreased initially and then gradually increased because of inhibition of the luminal Na+ channel. An initial decrease in Ri was considered an influx of Ca2+ from the basolateral membrane. To further determine the source of an increase in cell Ca2+, the effect of ET-1 was tested in the absence of external Ca2+ and in the presence of a Ca2+ channel blocker in the bath. Cell Ca2+ did not respond to ET-1 in the absence of external Ca2+, a condition in which an AVP-stimulated increase in cell Ca2+ was preserved.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of chronic metabolic acidosis on ammonia production fromL-glutamine in microdissected rat nephron segments

To evaluate the role of each nephron segment in renal ammoniagenesis, distribution of renal ammoniagenic activity along the nephron in control and acidotic rats was examined. We used our original aerobic incubation system and ammonia produced from glutamine in 7 defined segments of microdissected nephron was measured using the enzymatic cycling method.When ammonia production in the control was compared in each nephron segment, the highest specific activity of ammoniagenesis per mm tubular length and that per μg protein were observed in the proximal straight tubule (PST) and the thick ascending limb of Henles loop, respectively. Chronic metabolic acidosis increased ammonia production per mm tubular length markedly in the proximal convoluted tubule (PCT) (+171%), moderately in the medullary collecting tubule (+123%) and PST (+77%), and slightly in the distal convoluted tubule (+52%), revealing that the highest activity of ammoniagenesis was located in PCT and PST in acidosis.These data indicate that proximal tubules have major roles in renal ammoniagenesis both in the control and in acidosis. From the early observation of glutaminase I isoenzyme distribution along the nephron, our data suggest that not only phosphate-dependent glutaminase but also phosphate-independent glutaminase may have important roles in renal ammoniagenesis.

Detection of endothelin-1 mRNA by RT-PCR in isolated rat renal tubules

A combination of reverse transcription and polymerase chain reaction was utilized to detect baseline endothelin-1 (ET-1) mRNA expressed in renal tubules dissected from rats. 5 and 3 primers were constructed according to human ET-1 genomic DNA. Rat ET-1 mRNA was found to be expressed only in cortical through medullary collecting ducts in addition to glomeruli. Sites for tubular synthesis of ET-1 corroborate well with major ET-1 binding sites along the nephron, indicating autocrine/paracrine role of ET-1 in the renal tubules and supporting a prevailing concept on such function of ET-1 in many differing tissues.

Localization and properties of NAD+-dependent 15-hydroxyprostaglandin dehydrogenase activity in the rat kidney

Localization of NAD+-dependent (type I) 15-hydroxyprostaglandin dehydrogenase (15PGDH) in the rat kidney was examined using an ultramicro assay of the enzyme activity based on the enzymatic cycling method. The enzyme activities during first 3 weeks of age were 30- to 40-fold higher than the adult and rapidly decreased by 4th week. 15PGDH activities measured with either PGE2 or PGF2α as a substrate were five times higher in slices from midcortical or juxtamedullary layers than in slices from the superficial cortex of 3 week-old rat kidney. Little activity was found in inner medulla and papilla. When the enzyme activity was assayed using isolated nephron segments dissected from collagenase treated slices of 3 week-old rat kidneys, the activity was localized only in the proximal convoluted and straight tubules with either PGs (PGE2: 1.75±0.25 in PCT, 7.70±1.19 in PST, and PGF2α: 1.63±0.39, 6.18±1.52 pmoles NADH/mm/40 min). The kinetic analysis for renal 15PGDH of 3 week-old rats revealed thatKm for PGE2 (8.4 μM) was lower than that for PGF2α (22.6 μM) with constant NAD+, whileVmax for both was similar. In contrast, bothKm andVmax for NAD+ were identical with either PGs. These data suggest that the rate-limiting factor of type I 15PGDH is the concentration of prostaglandins in the kidney rather than the concentration of NAD+. It is thought that the enzyme may be physiologically significant in inactivating the prostaglandins reaching the proxial tubule in order to ensure that the classical prostaglandins (PGE2 and PGF2α) produced in the medullary interstitium and collecting tubules can effectively regulate the distal nephron function.

Polarized Secretion of Endothelin-1 and Big Et-1 in Mdck Cells Is Inhibited by Cell Na+ Flux and Disrupted by Nh4cl

We recently found that TGF-beta increases ET-1 secretion in MDCK, a renal tubular cell line. The secretion of ET-1, by a confluent monolayer of MDCK cells grown on a filter, to the basolateral side of the epithelium was two times greater than that to the apical side. However, TGF-beta-increased ET-1 and big ET-1 secretion were exclusively released to the basolateral side. We investigated the mechanism of polarized secretion of ET-1 and big ET-1 in MDCK cells. After 24 h of incubation with 80 pM TGF-beta, basolateral secretion of both ET-1 and big ET-1 increased two to threefold without a significant increase on the apical side. TGF-beta-stimulated ET-1 and big ET-1 secretion in the basolateral bath were inhibited when 10 microM amiloride (Na+ channel blocker) or 1 microM ouabain (Na+, K(+)-ATPase inhibitor) was added for 3 h. Polarized secretion of ET-1 and big ET-1 was not affected. In contrast, 10 mM NH4Cl or 0.2 mM chloroquine (both lysosomal function inhibitors) reduced TGF-beta-stimulated ET-1 secretion in the basolateral bath whereas big ET-1 secretion in the apical bath increased two times. Thus, the polarity of big ET-1 secretion was reversed. In addition, the conversion rate from big ET-1 to ET-1 was significantly decreased from 80 to 55% by inhibiting lysosomal function. Prepro-ET-1 mRNAs 3 h after these perturbations were virtually unaltered. Our data show that ET-1 and big ET-1 secretion may be regulated by cell Na+ flux and that lysosomal functions may play some roles in the conversion of big ET-1 to mature ET-1 and in polarized secretion of big ET-1. Translational or posttranslational regulation may play an additional role in ET-1 secretion as well as the mRNA level.

Mechanisms of Endothelin-1 mRNA and Peptides Induction by TGF-β and TPA in MDCK Cells

Mechanisms of prepro-ET-1 mRNA expression and mature endothelin-1 (ET-1) peptide secretion in MDCK cells (dog collecting duct origin) were investigated. MDCK cells constitutively expressed prepro-ET-1 mRNA (approximately 2.3 kb). TGF-beta time-dependently increased prepro-ET-1 mRNA levels between 30 min and 6 h. Induction of the mRNA 6 h following TGF-beta addition was dose-dependent with a half-maximal concentration of 10 pM. The half-life of prepro-ET-1 mRNA was 15 min in controls when the cells were treated with 10 micrograms/ml of actinomycin D, whereas it was extended to 30-45 min by TGF-beta treatment. Prepro-ET-1 mRNA was rapidly (15-30 min) induced by 0.5 microM TPA, one of the phorbol esters, but downregulated below baseline after 1 h. Our data show that MDCK cells constitutively secrete ET-1 and increase its production in response to TGF-beta. An axis of TGF-beta-ET-1-collecting duct may play an important role in regulation of electrolyte transport and cell growth of the renal tubules.

Endothelin Action and Production in Renal Tubular Cells

Recent studies suggest that the renal tubule is not only a target of endothelin-1 (ET-1) but is also a possible source of endothelin. Thus, we attempted to detect specific preproET-1 messenger RNA (mRNA) as well as immunoreactive ET-1 secretion in renal epithelial cell lines. Madin-Darby canine kidney (MDCK) cells expressed a single 2.3 kb preproET-1 mRNA. In medium conditioned by MDCK for 24 hours there was a significant amount of immunoreactive ET-1 detected by sandwich type immunoassay. Both mRNA expression and mature ET-1 secretion were stimulated by an addition of transforming growth factor (TGF)-β to the basolateral side in a time-dependent manner, but the increase in peptide secretion lagged behind ET-1 mRNA expression by several hours. The basal secretion of ET-1 was polarized, with 2.3-fold greater secretion into the basolateral side over the apical side, while the difference was augmented to 7.5-fold by TGF-β. In contrast, LLCPK1 (pig kidney) cells secreted little ET-1 associated with a lower level of ET-1 mRNA expression. Regarding the effect of ET-1, it elicited [Ca2+]i transients in LLC-PK1 but not in MDCK. These data clearly show that renal tubules can express preproET-1 mRNA, synthesize mature ET-1, and secrete it preferentially to the basolateral side of the tubule cells, all of which are enhanced by TGF-β. The data suggest that ET- l produced by the renal tubule may function as a paracrine hormone in regulating tubular functions.

Time to Target Uric Acid to Retard Chronic Kidney Disease Progression

Uric acid (UA) remains a risk factor for the progression of chronic kidney disease (CKD). Most observational studies showed a slight elevation in the serum UA level and this independently predicts the incidence and development of CKD. The recent meta-analysis, however, did not reach the conclusion that urate-lowering therapy with allopurinol retards the progression of CKD. The target level of serum UA if treated is another issue of debate. Our recent analysis by propensity score analysis has shown that the serum UA should be targeted below 6.0 mg/dL to inhibit the progression towards end-stage renal disease. Underlying mechanisms whereby an increase in serum UA induces kidney injury have been elucidated in animal models. Hyperuricemic models can lead to systemic hypertension, arteriolosclerosis including afferent arteriolopathy as well as albuminuria probably due to the activation of oxidative stress. Discoveries of urate transporters have elucidated the novel mechanism of UA transport in the kidney and intestine. The intestinal ABCG2 may play a compensatory role in light of decreased renal clearance of UA in CKD model rats, the trigger of which is not a uremic toxin but serum UA itself. Insulin directly upregulates URAT1 and downregulates ABCG2 in the kidney tubules, suggesting a possible link between UA and metabolic syndrome. This review summarizes the recent knowledge on the causal effect of serum UA on the kidney injury.

Urinary Exosomal Aquaporin-2 Expression and the Efficacy of Tolvaptan in Advanced CKD Patients with Congestive Heart Failure: An Interim Report

Background: The efficacy of tolvaptan, a selective antagonist for vasopressin V2 receptor, on water diuresis has been reported in congestive heart failure (CHF) patients with chronic kidney disease (CKD). However, the mechanism of tolvaptan resistance is not well understood. In this study the effect of tolvaptan on urinary expression of exosomal aquaporin-2 (AQP-2) was examined in relation to effectiveness of tolvaptan. nMethods: We examined 13 CHF patients with advanced CKD (M:F=11:2, age 49 to 81, serum creatinine 1.7 to 10.4 mg/dL, eGFR 3.5 to 69 ml/min/1.73 m2, median 9.2) who were not under control with conventional treatment including furosemide therapy (40 to 80 mg orally). Tolvaptan of 7.5 mg/day was instituted and blood and urine parameters were measured as well as urinary exosomal AQP-2 and Na-K-2Cl cotransporter (NKCC2). Responders were defined as increase in urine volume greater than 500 ml/day after the treatment. nResults: Eight patients were diagnosed as responders who increased urine volume together with decrease in body weight within several days (p<0.05 vs. before treatment). Baseline data did not show the significant difference between both groups. Serum levels of creatinine and Na did not change over 1 week in either responders or non-responders. All responders showed a significant reduction in urinary exosomal AQP-2 after the treatment whereas non-responders showed rather increase in its expression. In contrast, urinary exosomal NKCC2 expression was unaltered in any case. nConclusions: Tolvaptan exerts its diuretic action in more than half the CHF patients complicated with advanced CKD. The responsiveness corroborates well with urinary exosomal AQP2 expression. The predictor for resistance to tolvaptan needs further examination.