Essential regulatory elements for NHE3 gene transcription in renal proximal tubule cells

The objectives of the present study were to identify the cis-elements of the promoter absolutely required for the efficient rat NHE3 gene transcription and to locate positive and negative regulatory elements in the 5’-flanking sequence (5’FS), which might modulate the gene expression in proximal tubules, and to compare this result to those reported for intestinal cell lines. We analyzed the promoter activity of different 5’FS segments of the rat NHE3 gene, in the OKP renal proximal tubule cell line by measuring the activity of the reporter gene luciferase. Because the segment spanning the first 157 bp of 5’FS was the most active it was studied in more detail by sequential deletions, point mutations, and gel shift assays. The essential elements for gene transcription are in the region -85 to -33, where we can identify consensual binding sites for Sp1 and EGR-1, which are relevant to NHE3 gene basal transcription. Although a low level of transcription is still possible when the first 25 bp of the 5’FS are used as promoter, efficient transcription only occurs with 44 bp of 5’FS. There are negative regulatory elements in the segments spanning -1196 to -889 and -467 to -152, and positive enhancers between -889 and -479 bp of 5’FS. Transcription factors in the OKP cell nuclear extract efficiently bound to DNA elements of rat NHE3 promoter as demonstrated by gel shift assays, suggesting a high level of similarity between transcription factors of both species, including Sp1 and EGR-1.

Gentamicin-induced preconditioning of proximal tubular LLC-PK1 cells stimulates nitric oxide production but not the synthesis of heat shock protein

Nephrotoxicity is the main side effect of antibiotics such as gentamicin. Preconditioning has been reported to protect against injuries as ischemia/reperfusion. The objective of the present study was to determine the effect of preconditioning with gentamicin on LLC-PK1 cells. Preconditioning was induced in LLC-PK1 cells by 24-h exposure to 2.0 mM gentamicin (G/IU). After 4 or 15 days of preconditioning, cells were again exposed to gentamicin (2.0 mM) and compared to untreated control or G/IU cells. Necrosis and apoptosis were assessed by acridine orange and HOESCHT 33346. Nitric oxide (NO) and endothelin-1 were assessed by the Griess method and available kit. Heat shock proteins were analyzed by Western blotting. After 15 days of preconditioning, LLC-PK1 cells exhibited a significant decrease in necrosis (23.5 ± 4.3 to 6.5 ± 0.3%) and apoptosis (23.5 ± 4.3 to 6.5 ± 2.1%) and an increase in cell proliferation compared to G/IU. NO (0.177 ± 0.05 to 0.368 ± 0.073 ìg/mg protein) and endothelin-1 (1.88 ± 0.47 to 2.75 ± 0.53 pg/mL) production significantly increased after 15 days of preconditioning compared toG/IU. No difference in inducible HSP 70, constitutive HSC 70 or HSP 90 synthesis in tubular cells was observed afterpreconditioning with gentamicin. The present data suggest that preconditioning with gentamicin has protective effects on proximal tubular cells, that involved NO synthesis but not reduction of endothelin-1 or production of HSP 70, HSC 70, or HSP 90. We conclude that preconditioning could be a useful tool to prevent the nephrotoxicity induced by gentamicin.

Co-localization and interaction of organic anion transporter 1 with caveolin-2 in rat kidney

The organic anion transporters (OAT) have recently been identified. Although the some transport properties of OATs in the kidney have been verified, the regulatory mechanisms for OAT's functions are still not fully understood. The rat OAT1 (rOAT1) transports a number of negatively charged organic compounds between the cells and their extracellular milieu. Caveolin (Cav) also plays a role in membrane transport. Therefore, we investigated the protein-protein interactions between rOAT1 and caveolin-2. In the rat kidney, the expressions of rOAT1 mRNA and protein were observed in both the cortex and the outer medulla. With respect to Cav-2, the expressions of mRNA and protein were observed in all portions of the kidney (cortex < outer medulla = inner medulla). The results of Western blot analysis using the isolated caveolae-enriched membrane fractions or the immunoprecipitates by respective antibodies from the rat kidney showed that rOAT1 and Cav-2 co-localized in the same fractions and they formed complexes each other. These results were confirmed by performing confocal microscopy with immunocytochemistry using the primary cultured renal proximal tubular cells. When the synthesized cRNA of rOAT1 along with the antisense oligodeoxynucleotides of Xenopus Cav-2 were co-injected into Xenopus oocytes, the [14C]p-aminohippurate and [3H]methotrexate uptake was slightly, but significantly decreased. The similar results were also observed in rOAT1 over-expressed Chinese hamster ovary cells. These findings suggest that rOAT1 and caveolin-2 are co-expressed in the plasma membrane and rOAT1's function for organic compound transport is upregulated by Cav-2 in the normal physiological condition.

Structure of the Bufo arenarum kidney: renal corpuscle, neck segment and proximal tubule

The structural organization of the renal corpuscle (RC), ciliated neck segment (NS) and the proximal tubule (PT) were studied in the toad, Bufo arenarum, by means of light and transmission electron microscopy. The ciliated neck segment and the proximal tubule are located in the dorsolateral zone of the kidney, while the distal tubules are located in a ventromedial zone. RC are found between these two zones. The glomerular filter apparatus consists of the podocyte epithelium, a basement membrane, a subendothelial space and an endothelium. The podocyte emits cytoplasmatic processes extending over the surface of the glomerular capillaries. These processes divide into further processes ending in expansions known as pediceles. The basement membrane consists of a lamina rara externa and a rather thin lamina densa, while the subendothelial space contains collagen fibers and slender cytoplasmic processes of the mesangial cells. NS are composed of ciliated cells with a characteristic location of the mitochondria. The PT consists of prismatic cells with a dense luminal brush border of long microvilli and numerous apical vesicles. The basal cell membrane is increased by small infoldings. One characteristic structure of the cytoplasm is the presence of lipid droplets. The cytological structure of PT cells can be considered as an adaptation for the reabsorption of organic materials

Effect of D-glucose and L-glutamate on the kinetics of bicarbonate reabsorption in rat proximal tubules

The effect of D-glucose or L-glutamate on the kinetics of bicarbonate reabsorption in the early (EPT) and middle proximal tubule (MPT) was studied in vivo in Munich-Wistar rats by microperfusion techniques. The presence of 20 mM D-glucose in the lumen increased acidification half-time (t/2) (from 2.54 + or - 0.09s to 3.11 + or - 0.17 s in EPT and from 4.75 + or - 0.20 s to 6.04 + or - 0.49 s in MPT). Bicarbonate reabsorption 9JHCO-3) decreased as a consequence of this change (from 3.80 + or - 0.17 to 2.46 + or - 0.20 nmol cm-2s-1 in EPT and from 2.30 + or - 0.10 to 1.64 + or - 0.10 nmol cm-2s-1 in MPT). In this situation the basolateral membrane potential difference (BLMPD) in the MPT decereased from -41.6 + or - 2.47 to -29.7 + or - 2.45 mV and returned to control values after perfusion with D-glucose. The addition of 20 mM L-glutamate to the luminal perfusion caused an opposite effect, i.e., a decrease in t/2 (1.54 + or - 0.21 s in EPT and 3.25 + or - 0.26 s in MPT) and a consequent increase in JHCO-3 in both segments (5.09 + or - 0.58 nmol cm-2s-1 in EPT and 3.92 + or - 0.30 nmol cm-2s-1 in MPT). The BLMPD of MPT increased during L-glutamate perfusion (-39 + or - 2.48 mV in control and -52.0 + or - 2.72 mV with L-glutamate) and returned to control values after perfusion. The results observed may be the consequence of coupled Na+ - substrate transport altering the BLMPD with modifies the electrical driving force for coupled Na+ -HCO-3 efflux across the basolateral membrane. The alteration of this process may in turn affect intracellular pH, which is an important modulator of luminal Na+./H+ enchange. This possibility is supported by the observed depolarization of BLMPD by D-glucose (electroneutral molecule), and hyperpolarization by glutamate anion

Effect of bafilomycin on proximal bicarbonate absorption in the rat

To evaluate the relative importance of the V-type H(+)-ATPase in proximal bicarbonate reabsorption in vivo, proximal tubules of male and female Wistar rats (180 to 260 g) were perfused with bicarbonate-Ringer solution with and without the addition of 2 microM bafilomycin A1. Bafilomycin significantly increased stationary pH from 6.75 +/- 0.05 (N = 39) to 6.86 +/- 0.03 (N = 82), the stationary concentration of bicarbonate from 5.24 +/- 0.62 to 6.33 +/- 0.46 mM and the half-time of acidification from 3.72 +/- 0.22 to 4.65 +/- 0.25 s, and significantly decreased net bicarbonate reabsorption from 3.17 +/- 0.21 to 2.55 +/- 0.15 nmol s-1 cm-2, that is, by 20 per cent . Since bafilomycin is considered to be a specific inhibitor for V-type H(+)-ATPase, these data establish 1) the existence of this type of transport in the rat proximal tubule and 2) that approximately a fifth of the total proximal bicarbonate reabsorption is due to this mechanism of transport

Clearance de lítio como método da avaliaçäo do manuseio tubular de sódio em ratos acordados, estudados em gaiolas metabólicas / Lithium clearance like evaluation method of tubular handling of sodium in awake rats, studieds in metabolic cages

Os autores estudaram o clearance de lítio (CLi), em ratos acordados näo restritos, em gaiolas metabólicas individuais. Os resultados do presente trabalho foram obtidos utilizando-se de fórmulas matemáticas definidas a partir dos conceitos básicos do manuseio tubular renal de lítio (Li), dentre os quais: 1) a absorçäo de Li no túbulo proximal é proporcional àquela de água e sódio (Na); 2) näo ocorre absorçäo ou secreçäo de Li em segmentos do nefro distais ao túbulo proximal; nessas condiçöes, o CLi seria um marcador da reabsorçäo proximal de sódio e água. Esses resultados mostraram uma rebsorçäo de Na pelo túbulo proximal de 77,1 ñ 1,04%, comparável aos valores aceitos classicamente na literatura. O valor verificado para a reabsorçäo distal (ou "pós-proximal") de sódio foi de 99,5 ñ 0,03%. Para sensibilizar o CLi como marcador tubular proximal, foi empregada a amilorida para bloquear eventual reabsorçäo distal de lítio. Os resultados do grupo I (controle) foram comparados àqueles do grupo que recebeu amilorida (Amd - grupo II) e näo mostraram diferenças significativas na reabsorçäo proximal de sódio entre os dois grupos (controle 77,1 ñ 1,04% vs. amilorida 75,5 ñ 1,70%). Porém, o efeito natriurético observado no grupo II fica evidente quando se compara a reabsorçäo distal deste íon, com a do grupo I (amilorida 98,2 ñ 0,11%vs. controle 99,5 ñ 0,03%, p < 0,001). Este último dado mostra a açäo farmacológica da droga nos segmentos distais do nefro, sem contudo alterar o CLi, validando esta técnica para o estudo de funçäo tubular proximal neste modelo

Effect of propranolol and nifedipine on in vitro fluid absorption by the isolated perfused proximal convoluted tubule of the rabbit

The inhibition of fluid absorption (Jv) by the antiarrhythmic and antihypertensive drugs propranolol and nifedipine, which increase cytosolic Ca*+ concentration, was studied using the isolated rabbid proximal convoluted tubule perfused in vitro. Proximal convuluted tubules were perfused and bathed with a modified Krebs-Henseleit solution containing bovine serum albumin. Jv was measured after a 30-min control period, after 40 min with eithe 0.1 mM propranolol or 1.0 mM mifedipine on the peritubular side and after a 40-min recovery period. Both drugs inhibited Jv (58% propranolol, and 21% nifedipine) The 40-min recovery period was sufficient to reserve the effect of nifedipine, but propranolol-treated tubules (N=6) only reached 78% of the control Jv value. These results demonstrate that antiarrhythmic and anthypertensive drugs are powerful inhbitors of net fluid absorption by exerting a direct effect on proximal or distal tubule cells, thus acting like "local diuretics"

Mechanism of potassium transport across proximal tubule epithelium in the rat

The mechanism of proximal tubule potassium reabsorption was studied by stopped-flow microperfusion and determination of potassium activities by ion-sensitive resin microelectrodes. The proximal tubule was unable to establish transepithelial potassium gradients. perfusion with 20 mM K+ turned the lumen 3 mV more negative, an effect abolished by Ba2+. the half-time for K+ activities to reach their stationary level after perfusion with 1 mMK+ was significantly increased by Ba2+ from 4.25 ñ 0.14sto 11.0 ñ 1.71s, and aftr perfusion with 20 mMK+, from 5.43 ñ 0.20 to 12.53 ñ 0.90s. These data indicate that a significant fraction of potassium is transferred across proximal tubule spithelium by a transcellular, K+-channel-dependent route

Effect of glucose on the kinetics of bicarbonate reabsorption in the early and middle proximal tubule

The kinetics of bicarbonate reabsorption in the early (EPT) and middle proximal tubule (MPT) was studied in Munich Wistar rats by means of microperfusion techniques. The early proximal tubule (EPT) had a higher capacity of reabsorbing bicarbonate than the middle segment. The calculated bicarbonate flow (JHCO3) was 4.47 nmol s**-1 cm**-2 int the EPT and 2.30 nmol s**-1 cm**-2 in the MPT and the half time of injected bicarbonate (t/2) was less in the EPT (2.24 s) than in the MPT (5.20 s). The presence of 20 mM glucose in the lumen led to a reduction in the velocity of tubular acidification in the EPT (3.11 s) and caused a decrease in JHCO3 in both segments (2.46 nmol s**-1 cm**-2 in the EPT and 1.64 nmols s**-1 cm**-2 in the MPT). These alterations may be the result either of lumen-to-cell sodium gradient dissipation or electrical changes induced by sodium-glucose cotransport that may depolarize the basolateral membrane leading to intracellular alkalinization. This effect may then impair the Na +/H+ exchanger, thus decreasing bicarbonate reabsorption

Filtered load of buffer and renal H-ion secretion: mechanism of proximal tubule load dependence

Cuando la carga filtrada de "buffers" como bicarbonato o fosfato es incrementada por elevación del FG o de la concentración plasmática de "buffer", la reabsorción global de bicarbonato o la formación de acidez titulable son estimuladas. Lo mismo ocurre con la reabsorción proximal de bicarbonato durante la perfusión con concentraciones crecientes de este ion o cuando se eleva el flujo de fluido. En los últimos años, hemos estudiado los mecanismos de esta dependencia funcional. Observamos que el ritmo de reabsorción de bicarbonato es siempre proporcional a la concentración luminal de "buffer" cuando se inyecta una columna estacionaria de fluido en la luz tubular. La secreción de H**+ es tambiénn proporcional a nivels luminales de otros "buffers" distintos al bicarbonato. Empleando la técnica de pH-stat adaptada a túbulos renales, demostramos que la secreción proxim de H**+ depende del pH luminal y es independiente del sistema de "buffer" utilizado. Un análisis cinético de estos datos demuestra una relación no linear entre pH luminal y secreción de H**+, compatible con transporte mediado por "carrier"