Intact Proximal Tubules Research Articles

Comparison of Antioxidant Effect of Crocus Sativus and Vitamin E on Amakicin Induced Nephrotoxicity in Albino Rats

ABSTRACT
 Objectives: The present study evaluated the protective effect of Crocus Sativus against Amikacin-induced Nephrotoxicity.
 Methods: The prospective descriptive study was conducted at the department of orthopedic surgery, Dr. Ruth KM Pfau Adult laboratory albino rats of the Wistar strain were used in this experimental study. In this study, 75 albino rats were subdivided into 3 groups, including experimental groups B and C. The control group (A) received normal saline intraperitoneally, group (B) received Amikacin 30mg/kg body weight intraperitoneally and Group (C) received Crocus sativus 50 mg/kg body weight orally and vitamin E orally in a dose of 50mg/kg of body weight. Kidneys of rats obtained after dissection were processed, sectioned, and stained in PAS Hematoxylin and Gomoris Methenamine Silver stains. The present study was conducted at Dow University of Health Sciences in the Institute of Biomedical Sciences and Animal House of Dow University and Dow Diagnostic Research and Reference Laboratory. The duration of the study was Three months. Inclusion criteria were Albino rats of both sexes, kidneys of either side (right & left), rats with an approximate weight of 140 to 180 grams were included in the study. Exclusion criteria were overweight rats and pregnant rats.
 Results: A significant increase in kidney weight was observed in group B. A considerable addition was observed in proximal tubular in groups B and C. A considerable increase was also observed in the number of intact proximal tubule nuclei and the nuclear diameter of groups B & C.
 Conclusion: The study provides evidence that Crocus Sativus has protective effects in Amikacin-induced nephrotoxicity, comparable to widely used Antioxidant Vitamin E. The study recommends the addition of Crocus Sativus in dietary supplements after further evaluation in human subjects.

MO328ASYMPTOMATIC HYPERURICEMIA ACTS AS ANTIOXIDANT DURING ACUTE KIDNEY INJURY AND DISEASE*

Abstract Background and Aims Acute kidney injury (AKI) and disease (AKD) are major causes of morbidity and mortality worldwide. Hyperuricemia (HU) is common in patients with impaired kidney function. While there is no doubt that crystalline uric acid (UA) causes acute and chronic UA nephropathy, urolithiasis and kidney stone disease, the pathogenesis of asymptomatic HU in AKI/AKD is incompletely understood. In animal studies, elevated serum UA levels may lead to endothelial dysfunction, renin-angiotensin system activation and oxidative stress. However, such models do not mimic human HU. To overcome this issue, we established a model of AKI/AKD with clinically relevant serum UA levels and hypothesized that asymptomatic HU improves the outcomes after AKI/AKD by restoring metabolic activity and mitochondrial biogenesis in macrophages and tubular epithelial cells. Method Alb-creERT2;Glut9lox/lox and Glut9lox/lox control mice were injected with tamoxifen and placed on a chow diet enriched with inosine. Hyperuricemic mice (serum UA ≥7 mg/dL) and mice without HU (serum UA 4-5 mg/dL) underwent uninephrectomy followed by unilateral ischemia-reperfusion (IR) to induce AKI/AKD. Serum and kidneys were collected on day 3 and 14 after AKI/AKD, and kidney function, tubular injury, inflammation, mitochondrial dysfunction, metabolic activity (fatty acid oxidation) and macrophage infiltration were quantified using GFR measurement, immunohistochemistry, colorimetric assays, electron microscopy, RT-PCR and flow cytometry. Results We observed an increase in serum UA levels from 7 to 10 mg/dL in hyperuricemic mice on day 3 after IR-induced AKI/AKD that returned to 7 mg/dL after 14 days (Figure left). While there was no difference in GFR between hyperuricemic and mice without HU with AKI/AKD on day 3, we found an improved kidney function in hyperuricemic mice on day 14 (Figure middle). This was associated with significantly less tubular injury and inflammation as well as an increase in the number of infiltrating anti-inflammatory M2-like macrophages as compared to mice without HU. Intrarenal mRNA expression level of the pro-oxidant heme-oxygenase-1 was reduced in hyperuricemic mice. However, the expression of anti-oxidant enzymes (Nrf-1 and Sod) and metabolic genes associated with fatty acid oxidation (Cpt1, Pparg, and Pgc1b) significantly increased as compared to mice without HU 14 days after AKI/AKD. In addition, HU increased the number of phospho-Histone-3 and intact proximal tubules and restored tubular mitochondrial morphology as indicated by an increased mitochondrial aspect ratio (Figure right). Conclusion Our data imply that asymptomatic HU improves kidney outcomes after IR-induced AKI/AKD because HU attenuates tubular injury and inflammation. In addition, we found that HU enhances the metabolic activity and anti-inflammatory M2-like macrophage polarization as well as restores mitochondrial biogenesis in tubular epithelial cells, suggesting that HU acts as antioxidant by improving kidney recovery after AKI/AKD.

A Multicompartment Human Kidney Proximal Tubule-on-a-Chip Replicates Cell Polarization-Dependent Cisplatin Toxicity.

Drug-induced kidney injury is a major clinical problem and causes drug attrition in the pharmaceutical industry. To better predict drug-induced kidney injury, kidney in vitro cultures with enhanced physiologic relevance are developed. To mimic the proximal tubule, the main site of adverse drug reactions in the kidney, human-derived renal proximal tubule epithelial cells (HRPTECs) were injected in one of the channels of dual-channel Nortis chips and perfused for 7 days. Tubes of HRPTECs demonstrated expression of tight junction protein 1 (zona occludens-1), lotus lectin, and primary cilia with localization at the apical membrane, indicating an intact proximal tubule brush border. Gene expression of cisplatin efflux transporters multidrug and toxin extrusion transporter (MATE) 1 (SLC47A1) and MATE2-k (SLC47A2) and megalin endocytosis receptor increased 19.9 ± 5.0-, 23.2 ± 8.4-, and 106 ± 33-fold, respectively, in chip cultures compared with 2-dimensional cultures. Moreover, organic cation transporter 2 (OCT2) (SLC22A2) was localized exclusively on the basolateral membrane. When infused from the basolateral compartment, cisplatin (25 µM, 72 hours) induced toxicity, which was evident as reduced cell number and reduced barrier integrity compared with vehicle-treated chip cultures. Coexposure with the OCT2 inhibitor cimetidine (1 mM) abolished cisplatin toxicity. In contrast, infusion of cisplatin from the apical compartment did not induce toxicity, which was in line with polarized localization of cisplatin uptake transport proteins, including OCT2. In conclusion, we developed a dual channel human kidney proximal tubule-on-a-chip with a polarized epithelium, restricting cisplatin sensitivity to the basolateral membrane and suggesting improved physiologic relevance over single-compartment models. Its implementation in drug discovery holds promise to improve future in vitro drug-induced kidney injury studies. SIGNIFICANCE STATEMENT: Human-derived kidney proximal tubule cells retained characteristics of epithelial polarization in vitro when cultured in the kidney-on-a-chip, and the dual-channel construction allowed for drug exposure using the physiologically relevant compartment. Therefore, cell polarization-dependent cisplatin toxicity could be replicated for the first time in a kidney proximal tubule-on-a-chip. The use of this physiologically relevant model in drug discovery has potential to aid identification of safe novel drugs and contribute to reducing attrition rates due to drug-induced kidney injury.

Meprin β expression associated with decreased proximal tubule PKA Cβ levels in ischemia/reperfusion‐induced acute kidney injury

Ischemia/ reperfusion (IR) is a leading cause of acute kidney injury (AKI). Meprins are metalloproteases that are expressed abundantly in the brush border membranes of renal proximal tubules (PTs). The levels of expression and the localization of meprins were previously shown to associate with IR‐induced AKI. Studies from our group showed that meprin B proteolytically cleaves three isoforms of the catalytic subunit of PKA, namely, PKA Cα1, Cβ1, and Cβ2. The cleavage by meprin B resulted in decreased kinase activity of these PKA C isoforms. Furthermore, the total levels of PKA C were significantly decreased in the meprin expressing PTs at 3h post‐IR when compared to distal tubules which are deficient in meprins. The objective of the current study was to determine which of the PKA C isoforms is impacted by meprin B and thus involved in the IR‐response mediated by meprin β. To this end, surgical procedures were used to induce unilateral IR (with contralateral nephrectomy) in 12‐week old wild‐type (WT) and meprin β knockout (βKO) mice on a C57BL/6 background. Kidney tissues were harvested at 6h post IR. Kidney proteins were extracted and fractionated into BBM‐, cytosolic‐, and nuclear‐enriched fractions. Western blot analysis was used to determine the levels of Cα and Cβ in the cytosolic‐ and nuclear‐enriched fractions. Immunofluorescence staining was used to determine the localization of PKA C (total), Cβ, and Cα. Statistical analysis utilized 2‐way ANOVA. The total levels of PKA C decreased in kidneys from WT mice subjected to IR but not in βKO counterparts. However, use of isoform‐specific antibodies demonstrated increases in the levels of Cβ but not Cα in the cytosolic‐ and nuclear‐enriched fractions. Two Cβ protein bands (41 and 37 kDa) were detected in cytosolic‐ and one band (41 kDa) in nuclear‐enriched fractions. The levels of the 41 kDa Cβ protein species were higher in lysates from WT kidneys at 6h post‐IR (P=0.0001). In contrast, the 37 kDa species, which was detected in nuclear‐enriched fractions only, decreased in βKO (P=0.002) but not in WT kidneys subjected to IR. A closer histological analysis of kidney sections showed that the level of damage to the brush borders was not uniform in all proximal tubules. More importantly, immunohistochemical analysis showed that the PKA Cβ levels decreased in damaged/injured meprin β expressing PTs but appeared to increase in intact PTs. These changes were not observed in PTs from meprin βKO kidneys. Individual interstitial cells (presumed to be resident macrophages) also stained positively for Cβ in WT but not in βKO kidney sections. Taken together, the data suggest that the PKA Cβ isoform is involved in the response to IR‐induced renal injury. The Cβ levels are known to be abundant in lymphoid tissue but our data indicate expression in immune cells resident in kidney tissue as well as epithelial tubular cells in response to IR‐induced AKI. More studies are needed to determine the response genes mediated by PKA Cβ signaling in IR to increase our understanding of how meprins modulate kidney injury in mice.Support or Funding InformationThis study was supported by the National Institute of Health/National Institutes of General Medical Sciences (NIGMS) Grant #s SC3GM102049 and SC1GM118271

Effect of AQP1 knock out on mouse exercise tolerance

The water channel Aquaporin 1 (AQP1) is widely expressed throughout the body. Our lab demonstrated that AQP1 also can act as a CO2 channel when heterologously expressed in oocytes or when studied in the intact proximal tubule. We hypothesized that AQP1 plays a physiologically important role in O2 transport. Because AQP1 is present at high levels in erythrocytes and the pulmonary capillary endothelium, we compared voluntary wheel running over a 24‐h period in AQP1‐null vs wild‐type mice under conditions of hypoxia (ambient [O2] = 16%), normoxia (21%), and hyperoxia (40%). For wild‐type mice, the distances run were 16%: 9.2 ± 0.5 km (n = 28), 21%: 10.7 ± 0.5 km (n = 37), and 40%: 12.1 ± 0.6 km (n = 21). For AQP1‐null mice, the distances were 16%: 4.7 ± 0.5 km (n = 8), 21%: 6.2 ± 0.6 km (n = 13), and 40%: 6.9 ± 0.6 km (n = 12). We performed a linear‐regression analysis of distance run as a function of AQP1 status and [O2], treating [O2] categorically in reference to 21% O2. The AQP1 knockout reduced the distance run by 4.7 ± 0.5 km (p < 0.001), adjusting for [O2]. Compared to 21% O2, reducing [O2] to 16% reduced the distance run by 1.6 ± 0.6 km (p = 0.01), whereas increasing [O2] to 40% increased the distance run by 1.2 ± 0.6 km (p = 0.04), adjusting for AQP1 status. Thus, AQP1‐null mice have a major defect in voluntary exercise tolerance, consistent with the hypothesis that AQP1 plays an important physiological role in O2 transport across plasma membranes.

Avian renal proximal tubule epithelium urate secretion is mediated by Mrp4

Birds are uricotelic and, like humans, maintain high plasma urate concentrations (approximately 300 microM). The majority of their urate waste, as in humans, is eliminated by renal proximal tubular secretion; however, the mechanism of urate transport across the brush-border membrane of the intact proximal tubule epithelium during secretion is uncertain. The dominance of secretory urate transport in the bird provides a convenient model for examining this process. The present study shows that short hairpin RNA interference (shRNAi) effectively knocked down gene expression of multidrug resistance protein 4 (Mrp4; 25% of control) in primary monolayer cultures of isolated chicken proximal tubule epithelial cells (cPTCs). Control and Mrp4-shRNAi-treated cPTCs were mounted in Ussing chambers and unidirectional transepithelial fluxes of urate were measured. To detect nonspecific effects, transepithelial electrical resistance (TER) and sodium-dependent glucose transport (Iglu) were monitored throughout experiments. Knocking down Mrp4 expression resulted in a reduction of transepithelial urate secretion to 35% of control with no effects on TER or Iglu. Although electrical gradient-driven urate transport in isolated brush-border membrane vesicles was confirmed, potassium-induced depolarization of the plasma membrane in intact cPTCs failed to inhibit active transepithelial urate secretion. However, electrical gradient-dependent vesicular urate transport was inhibited by the MRP4 inhibitor MK-571 also known to inhibit active transepithelial urate transport by cPTCs. Based on these data, direct measure of active transepithelial urate secretion in functional avian proximal tubule epithelium indicates that Mrp4 is the dominant apical membrane exit pathway from cell to lumen.

Roles of ERK and cPLA2 in the Angiotensin II-Mediated Biphasic Regulation of Na+-HCO3 − Transport

Regulation of renal proximal transport by angiotensin II (Ang II) is biphasic: low concentrations (picomolar to nanomolar) stimulate reabsorption, but higher concentrations (nanomolar to micromolar) inhibit reabsorption. Traditionally, the stimulatory effect has been attributed to activation of protein kinase C and/or a decrease in intracellular cAMP, whereas the inhibitory action has been attributed to the activation of phospholipase A2 (PLA2) and the subsequent release of arachidonic acid. The Ang II receptor subtype responsible for these effects and the intracellular signaling pathways involved are not completely understood. We isolated proximal tubules from wild-type, Ang II type 1A receptor (AT1A)-deficient, and group IVA cytosolic phospholipase A2 (cPLA2alpha)-deficient mice, and compared their responses to Ang II. In wild-type mice, we found that the stimulatory and inhibitory effects of Ang II on Na+-HCO3(-) cotransporter activity are both AT1-mediated but that ERK activation only plays a role in the former. The stimulatory effect of Ang II was also observed in AT1A-deficient mice, suggesting that this occurs through AT1B. In contrast, the inhibitory effects of Ang II appeared to be mediated by cPLA2alpha activation because high-concentration Ang II stimulated Na+-HCO3(-) cotransporter activity when cPLA2alpha activity was abrogated by pharmacological means or genetic knockout. Consistent with this observation, we found that activation of the cPLA2alpha/P450 pathway suppressed ERK activation. We conclude that Ang II activates ERK and cPLA2alpha in a concentration-dependent manner via AT1, and that the balance between ERK and cPLA2alpha activities determines the ultimate response to Ang II in intact proximal tubules.

Dipeptidyl peptidase IV inhibition downregulates Na+-H+exchanger NHE3 in rat renal proximal tubule

In the microvillar microdomain of the kidney brush border, sodium hydrogen exchanger type 3 (NHE3) exists in physical complexes with the serine protease dipeptidyl peptidase IV (DPPIV). The purpose of this study was to explore the functional relationship between NHE3 and DPPIV in the intact proximal tubule in vivo. To this end, male Wistar rats were treated with an injection of the reversible DPPIV inhibitor Lys [Z(NO2)]-pyrrolidide (I40; 60 mg.kg(-1).day(-1) ip) for 7 days. Rats injected with equal amounts of the noninhibitory compound Lys[Z(NO2)]-OH served as controls. Na(+) - H(+) exchange activity in isolated microvillar membrane vesicles was 45 +/- 5% decreased in rats treated with I40. Membrane fractionation studies using isopycnic centrifugation revealed that I40 provoked redistribution of NHE3 along with a small fraction of DPPIV from the apical enriched microvillar membranes to the intermicrovillar microdomain of the brush border. I40 significantly increased urine output (67 +/- 9%; P < 0.01), fractional sodium excretion (63 +/- 7%; P < 0.01), as well as lithium clearance (81 +/- 9%; P < 0.01), an index of end-proximal tubule delivery. Although not significant, a tendency toward decreased blood pressure and plasma pH/HCO(3)(-) was noted in I40-treated rats. These findings indicate that inhibition of DPPIV catalytic activity is associated with inhibition of NHE3-mediated NaHCO3 reabsorption in rat renal proximal tubule. Inhibition of apical Na(+) - H(+) exchange is due to reduced abundance of NHE3 protein in the microvillar microdomain of the kidney brush border. Moreover, this study demonstrates a physiologically significant interaction between NHE3 and DPPIV in the intact proximal tubule in vivo.

Does membrane trafficking play a role in regulating the sodium/hydrogen exchanger isoform 3 in the proximal tubule?

The proximal tubule sodium/hydrogen exchanger continuously reabsorbs the bulk of the filtered sodium, controlling salt delivery to the distal nephron which is critical for tubuloglomerular feedback autoregulation and for fine control of salt excretion in the distal nephron. This review focuses on recent studies of the mechanisms of regulation of sodium transport in the proximal tubule, and addresses whether results from studies in proximal tubule cell lines are applicable to the proximal tubule in situ. Recent in-vivo studies provided evidence that sodium/hydrogen exchanger isoform 3 can move into and out of the apical microvilli accompanied by parallel changes in renal sodium transport: the exchanger is retracted from the microvilli in response to hypertension, parathyroid hormone or dopamine treatment and moved into the microvilli in response to sympathetic nervous system stimulation, puromycin aminonucleoside induced nephritic syndrome, and insulin treatment. Studies in cultured opossum kidney proximal tubule cells provided evidence for clathrin coated vesicle mediated, dynamin dependent, cytoskeleton dependent internalization of sodium/hydrogen exchanger isoform 3 from the surface to an endosomal pool in response to dopamine or parathyroid hormone. In the intact proximal tubule there is evidence for a two-step internalization process: (1) from villi to the intermicrovillar cleft region and (2) to a higher density membrane pool that may be either below the microvilli or deep in intermicrovillar clefts. Recent studies have described a significant inactive pool of the exchanger in the intermicrovillar region in vivo that may serve as a storage and recruitable pool. The molecular mechanisms responsible for increasing or decreasing sodium transport in the proximal tubule appear to include redistribution of sodium/hydrogen exchanger isoform 3 to or from the microvillar region. Detailed studies in cultured proximal tubule cell lines provide evidence for endocytosis and exocytosis of the exchanger dependent on cytoskeleton and clathrin coated vesicles. In vivo, the apical membrane is differentiated into discrete villar and intermicrovillar membrane domains and the intermicrovillar domain, not observed in cultured cells, may serve as a recruitable storage pool for sodium/hydrogen exchanger isoform 3.

Clonal Analysis of Immortalized Renal Proximal Tubule Cells: Na +/Glucose Cotransport System Levels Are Maintained Despite a Decline in Transport Function

Primary rabbit kidney proximal tubule (RPT) cells (S.D. Chung et al., 1982, J. Cell Biol. 95, 118–126) were transfected with the vector pRSV-T, which contains SV40 early region genes. After the third passage (when normal cells had stopped dividing), individual colonies formed in cultures transfected with pRSV-T. Clonal isolates (RPT-I cells) could be obtained in a simple and reproducible manner. Southern analysis of clone RPT-I8 indicated the presence of SV40 early region genes. Nuclear SV40 T was detected. After 23 passages, and subcloning, RPT-I8 (and subclones) was found to express renal proximal tubule markers to a similar extent, indicating that the phenotype was stable. Nevertheless, the activities of the Na +/glucose cotransport system, γ-glutamyl transpeptidase and alkaline phosphatase, were reduced as compared with primary cultures. Western analysis indicated that the level of Na +/glucose cotransporters was maintained in RPT-I8 cells, when compared with intact proximal tubules and primary cultures. Thus, the reduction in α-MG uptake in RPT-I8 cells may be attributed to other types of cellular alterations, including changes in energy metabolism. Indeed, growth in glucose-free medium was not observed in RPT-I8 cell cultures, suggesting that unlike primary RPT cells (J. C. Chung et al., 1992, J. Cell. Physiol. 150, 243–250), the gluconeogenic pathway was not intact.

Metallothionein isoform 3 and proximal tubule vectorial active transport

Metallothionein isoform 3 (MT-3) is expressed in the proximal tubule cells of the human kidney. The goal of the present study was to further characterize the basal expression of MT-3 in the proximal tubule and to determine if MT-3 participates in the maintenance of proximal tubule cell function. Expression of MT-3 mRNA was determined in the intact proximal tubule using microdissection and reverse transcription-polymerase chain reaction (RT-PCR). Basal expression of MT-3 mRNA and protein was determined in cultured human proximal tubule (HPT) cells and an immortalized proximal tubular cell line, HK-2 cells, using RT-PCR and immunoblotting. The MT-3 gene was stably transfected into the HK-2 cell line using the pcDNA3.1/Hygro (+) vector. MT-3 mRNA was detected in the proximal tubule of the in situ kidney with relative expression in excess to that of the beta-actin housekeeping gene. The mortal HPT cells were shown to express both MT-3 mRNA and protein and to form domes, while immortal HK-2 cells were shown to have no expression of MT-3 mRNA and protein nor to form domes. The stable transfection of MT-3 in HK-2 restored MT-3 expression and dome formation to the HK-2 cells. MT-3 mRNA is present in the human proximal tubule, and MT-3 expression is involved in the transport function of a human renal cell line that retains properties of the proximal tubule.

Intracellular pH regulatory mechanism in a human renal proximal cell line (HKC-8): evidence for Na+/H+ exchanger, CI-/HCO3- exchanger and Na+-HCO3- cotransporter.

In the present study we investigated whether an immortalized human renal proximal cell line, HKC-8, expresses a recently cloned Na+-HCO3- cotransporter (NBC-1) and, if so, which isoform (kNBC-1 from kidney or pNBC-1 from pancreas) is expressed in this cell line. Cell pH (pHi) measurements using a pH-sensitive fluorescence probe in the absence of HCO3-/CO2 revealed the presence of a Na+/H+ exchanger that required high concentrations of amiloride for full inhibition. In the presence of HCO3-/CO2 another pHi recovery process, dependent on Na+ but independent of Cl-, was identified. This process was electrogenic and was inhibited by 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulphonic acid (DIDS), being consistent with the Na+-HCO3- cotransporter. In addition, the pHi responses to Cl- removal were compatible with the presence of a Na+-independent Cl-/HCO3- exchanger that was also inhibited by DIDS. Reverse transcriptase polymerase chain reaction (RT-PCR) using primers designed for specific and common regions detected mRNAs of both kNBC-1 and pNBC-1 and Western blot analysis confirmed the expression of NBC-1 protein. These results indicate that HKC-8 has transport activities similar to intact proximal tubules and also suggest that both kNBC-1 and pNBC-1 may contribute to the Na+-HCO3- cotransport activity in this cell line.

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Epithelial transport of anthelmintic ivermectin in a novel model of isolated proximal kidney tubules.

The mechanism of excretion of the anthelmintic drug ivermectin was investigated in a novel experimental model of functionally intact proximal tubules isolated from a teleost fish (Fundulus heteroclitus). Secretion into the lumens of freshly isolated proximal tubules was studied by means of confocal laser scanning microscopy and digital image analysis using ivermectin and fluorescent labelled ivermectin (BODIPY-ivermectin; BI) as substrates. The tubular cells rapidly accumulated BI from the medium and attained steady state within 25 minutes. Luminal fluorescence in the steady state was 5-7 times higher as compared to intracellular fluorescence. The secretion of BI into the tubular lumens was inhibited in a dose dependent manner by unlabelled ivermectin and inhibitors of the renal excretory membrane pump p-glycoprotein, namely SDZ PSC-833 and verapamil, but not by leukotriene C4, a substrate of the renal export protein mrp2. Accumulation inside the tubular cells was not affected by the added inhibitors. Ivermectin inhibited the renal secretion of the fluorescent cyclosporin derivative NBDL-CS, a substrate of p-glycoprotein, but not the secretion of the mrp2-substrate fluorescein-methotrexate, nor the secretion of fluorescein, a substrate of the classical renal organic anion transporter. The data are consistent with BI and ivermectin interacting in teleost kidney tubules exclusively with p-glycoprotein, but not with one of the other known excretory transport systems. In addition, the studies demonstrate that freshly isolated functionally intact kidney tubules from killifish are a useful tool to differentiate the substrate specificity of renal transport systems with respect to drug elimination.

Difference in H 2O 2 toxicity between intact renal tubules and cultured proximal tubular cells

The present study was undertaken to examine the response to H 2O 2 and t-butylhydroperoxide ( t-BHP) in various in vitro model systems of renal proximal tubules: rabbit renal cortical slices, freshly isolated rabbit proximal tubules, rabbit primary cultured proximal tubular cells, and opossum kidney (OK) cells. t-BHP increased lactate dehydrogenase release and lipid peroxidation in a concentration-dependent manner over the concentration range of 0.2 to 3 mM in cortical slices, whereas H 2O 2 caused a similar concentration-dependent increase in both parameters at 5–100 mM. The sensitivity of isolated tubules to both peroxides was similar to that of cortical slices. In primary cultured cells and OK cells, however, the cytotoxicity of H 2O 2 was identical to that of t-BHP. The cytotoxicity of t-BHP was not different among all the systems examined. The specific activity of catalase in cortical slices was similar to that of isolated tubules, but it was much higher than that of primary cultured cells or opossum kidney cells. Glutathione (GSH) peroxidase activity was not different among all the systems examined. The expression of catalase mRNA in cortical slices and isolated tubules was higher than that in primary cultured cells, whereas those of superoxide dismutase, glutathione peroxidase, or β-actin were not different among the systems. These results indicate that intact proximal tubules are more resistant to H 2O 2 than are cultured proximal tubular cells, and the resistance is due to a higher specific activity of catalase resulting from the increased expression of its mRNA.

Cross-Talk between the Na&lt;sup&gt;+&lt;/sup&gt;-K&lt;sup&gt;+&lt;/sup&gt;-ATPase and the H&lt;sup&gt;+&lt;/sup&gt;-ATPase in Proximal Tubules in Suspension

The cellular energy required for the activity of the Na(+)-K(+)-ATPase and of the H(+)-ATPase was estimated in intact proximal tubules in suspension. Both the fall in oxygen consumption (directly measured) and NADH oxidation (as estimated from exogenous substrate metabolism) were measured before and following application of ouabain (1 mM) to inhibit the sodium pump, following bafilomycin (0.1 mM) to inhibit the proton pump or following a combination of these inhibitors. The data demonstrate that the sodium pump utilizes 43% and the proton pump 19% of the phosphorylating NADH turnover of canine proximal tubules studied in vitro. However, a significant and stoichiometric stimulation of one pump was observed upon inhibition of the other. The NADH turnover related to the sodium pump increased from 308 to 402 (delta = 94) mumol.g-1 wet weight.h-1 following bafilomycin application and that of the proton pump from 136 to 230 (delta = 94) following ouabain application. This stimulation was largely abolished by inhibition of the Na+/H+ exchange occurring in either direction by amiloride or methylisobutylamiloride. It is concluded that a cross-talk occurs between the basolateral sodium pumps and the proton pumps located on the brush border membrane and/or on endosomes in proximal tubules. This cross-talk appears to be mediated by Na+/H+ exchange suggesting that both the proton pump and the Na+/H+ exchanger may contribute in a cooperative fashion to the proximal secretion of protons.

Water and urea diffusive permeabilities in isolated proximal tubule cells

We measured the water (3H2O) and urea ([14C]urea) diffusive permeabilities (Pd) in intact proximal tubule cells (PTC). Isolated rabbit PTC were packed in polyethylene tubes to measure the diffusion coefficients for 3H2O and [14C]urea at 14 degrees C. Pd values for water and urea were estimated from the diffusion coefficients in intact cells, in extracellular and in intracellular media using a model of parallel and series diffusion in the packed PTC. Pd was 65 +/- 8 and 18 +/- 3 microns/s for water and urea, respectively. We examined the effect of different pharmacological agents on the Pd values for water and for urea. p-Chloromercuribenzenesulfonate at 2 mM markedly inhibited the Pd of water and urea. Phloretin (1 mM) inhibited the Pd for urea, whereas it increased Pd for water. KMnO4 (30 microM) markedly inhibited urea Pd but did not alter water Pd. The values for energy of activation of Pd for water and for urea were 2.9 +/- 2 and 7.3 +/- 4 kcal/mol, respectively. These results show that water and urea do not share the same pathway across the PTC membrane.

Intracellular compartmentation of organic anions within renal cells

Epifluorescence microscopy and video-image analysis were used to measure the distribution of the monovalent organic anion fluorescein (FL) within the cells of three organic anion-secreting renal epithelia: crab urinary bladder (a proximal tubule analogue), opossum kidney (OK) cells in culture, and intact teleost proximal tubules. In all three preparations the intracellular FL distribution was nonuniform. Two distinct intracellular compartments were detected, one being diffuse and cytoplasmic and the other punctate. With low FL concentrations in the medium (1 microM and below) dye accumulation in the punctate compartment exceeded that of the cytoplasm. In crab bladder epithelium FL uptake into both compartments was inhibited by external probenecid, p-aminohippurate (PAH), and LiCl and stimulated by 10-50 microM external glutarate, suggesting that the punctate compartment loaded by a two-step mechanism: transport into the cytoplasm at the basolateral membrane, followed by accumulation at specific intracellular sites. Experiments in which FL was microinjected into OK cells directly demonstrated movement of FL from the cytoplasmic to the punctate compartment. Accumulation in the latter was specific, i.e., inhibitable by coinjected PAH and probenecid, and energy dependent. Together, these findings indicate that during secretion organic anions are sequestered within renal cells. The role of sequestration in overall transport remains to be determined.

PAH/alpha-KG countertransport stimulates PAH uptake and net secretion in isolated rabbit renal tubules.

Possible stimulation of both basolateral uptake and net transepithelial secretion of p-aminohippurate (PAH) by PAH/alpha-ketoglutarate (alpha-KG) countertransport was examined in intact perfused and nonperfused rabbit proximal S2 renal tubules. Preloading tubules with alpha-KG (100 microM) for 30 min increased [14C]-PAH rate of uptake by nonperfused tubules and rate of net secretion by perfused tubules approximately three- to sixfold. During stimulation of net secretion, intracellular [14C]PAH concentration increased to about the same extent as net secretion. Presence of Li+ (2 mM) or absence of Na+ (inhibitors of Na(+)-dicarboxylate cotransport) in bathing medium during alpha-KG preloading eliminated stimulation of PAH transport. Addition of unlabeled alpha-KG (1 mM) to bathing medium stimulated efflux of [14C]PAH across the basolateral membrane of tubules with oil-filled lumina, further supporting the concept of PAH/alpha-KG countertransport. Preloading with succinate (100 microM) also stimulated rates of [14C]PAH uptake by nonperfused tubules and net secretion by perfused tubules, but stimulation was only approximately 1.5-fold. Moreover, preloading with methyl succinate, a slowly metabolized derivative of succinate, did not stimulate [14C]PAH uptake by nonperfused tubules or net secretion by perfused tubules. Thus it seems most likely that succinate preloading stimulates PAH transport via metabolism, possibly by conversion to alpha-KG, not by direct countertransport for PAH. This study indicates for the first time in intact mammalian proximal S2 renal tubules that PAH/alpha-KG countertransport can stimulate net PAH secretion by generating an increased intracellular PAH concentration.

The renal sodium-calcium exchanger.

The functional expression of the renal sodium-calcium exchanger has been amply documented in studies on renal cortical basolateral membranes. In perfused renal tubules, other investigators have shown sodium-calcium exchange activity in the proximal convolution of the rat and in the distal convolution, the connecting tubule, and the collecting tubule of the rabbit. In rat proximal tubules, we found that the sodium-calcium exchanger is an important determinant of cytosolic calcium homeostasis, since inhibition of sodium-dependent calcium efflux mode caused a large accumulation of tubular calcium. In membranes from rat proximal tubules sodium-calcium activity was high, and in intact proximal tubules, the tubular sodium-calcium exchanger exhibited a high affinity for cytosolic calcium and had a substantial transport capacity, which may be absolute requirements for the maintenance of stable cytosolic calcium in proximal tubules.