Search

To determine the renal localization of oligopeptide transporters, Northern blot analyses were performed and polyclonal antisera were generated against PEPT1 and PEPT2, the two cloned rat H+/peptide transporters. Under high-stringency conditions, a 3.0-kb mRNA transcript of rat PEPT1 was expressed primarily in superficial cortex, whereas a 3.5-kb mRNA transcript of PEPT2 was expressed primarily in deep cortex/outer stripe of outer medulla. PEPT1 antisera detected a specific band on immunoblots of renal and intestinal brush-border membrane vesicles (BBMV) with an apparent mobility of approximately 90 kDa. PEPT2 antisera detected a specific broad band of approximately 85 kDa in renal but not in intestinal BBMV. PEPT1 immunolocalization experiments showed detection of a brush border antigen in S1 segments of the proximal tubule and in the brush border of villi from all segments of the small intestine. In contrast, PEPT2 immunolocalization was primarily confined to the brush border of S3 segments of the proximal tubule. All other nephron segments in rat were negative for PEPT1 and PEPT2 staining. Overall, our results conclusively demonstrate that although PEPT1 is expressed in early regions of the proximal tubule (pars convoluta), PEPT2 is specific for the latter regions of proximal tubule (pars recta).

Angiotensin IV, [[des-Asp1,Arg2]ANG II or ANG-(3-8)], has been shown to preferentially bind to a novel angiotensin binding site (AT4 receptor). The cellular location and function of this receptor in the rat kidney is unknown. Autoradiography localized AT4 receptors to the cell body and apical membrane of convoluted and straight proximal tubules in the cortex and outer stripe of the outer medulla. ANG IV (0.1 pM-1 microM) elicited a concentration-dependent decrease in transcellular Na+ transport (as measured by proximal tubule O2 consumption rates) in fresh suspensions of control or nystatin-stimulated (bypasses rate-limiting step of apical Na+ entry) rat proximal tubules. The inhibitory effect of 1 pM ANG IV was unaltered by either 1 microM losartan (AT1-receptor antagonist) or 1 microM PD-123319 (AT2-receptor antagonist) and yet was abolished by 1 microM divalinal-ANG IV (AT4-receptor antagonist) or ouabain pretreatment. These results demonstrate that the kidney AT4-receptor system is localized to the proximal tubule and suggests that one potential biological role of this system is in the regulation of Na+ transport by inhibiting a ouabain-sensitive component of Na(+)-K(+)-adenosinetriphosphatase activity in the rat.

Localization of a recently described and cloned Na-Pi cotransport system from flounder was investigated by reverse transcription-polymerase chain reaction (RT-PCR) of microdissected tubules and by immunocytochemistry of kidney of winter flounder. Histological examination showed a small glomerulus, an extremely short proximal tubule PI with a selective affinity to Lens culinaris agglutinin from lentils, and an extensive second proximal tubule segment PII (> 90% of proximal tubules), consisting of cells with numerous apical clear vesicles and extensive amplification of basolateral cell membranes. PII merged with the collecting tubule/ collecting duct (CT/CD) system without a distal segment. By RT-PCR, PII cells revealed high levels of NaPi-II related RNA; low levels were also observed in CTs. Previously characterized antisera against different epitopes of flounder NaPi-II specifically labeled the basolateral regions of PII and the apical cell portion of CT/CD cells and of some PII cells. These results suggest that tubular secretion of P(i) occurs in PII of teleost fish with modulation of urinary P(i) content in the subsequent CT/CD system.

Ischemia results in alterations in the integrity of the plasma membrane of renal epithelial cells, changes in cell polarity, and initiation of cell division. Because microtubules are implicated in these processes, we examined the effects of ischemia and reperfusion on the microtubular cytoskeleton in rat kidney. Major alterations in the microtubule network of S3 proximal tubules were detected after 40 min of ischemia followed by 1 h of reperfusion. There was fragmentation of microtubules and considerably less intense staining with antitubulin antibodies than with normal kidneys. Some thick ascending limbs of Henle close to medullary vascular bundles showed a variable loss of tubulin staining. After 24 and 48 h of reperfusion, tubulin labeling was again present in most proximal tubule cells in contact with the basement membrane but was not detectable in exfoliated cells. Numerous mitotic figures were present in kidneys 48 h after reperfusion. Kidneys subjected to 40 min of ischemia without reperfusion and contralateral kidneys studied after 1 h of reperfusion showed only mild microtubular disruption. Because of the established role of microtubules in the generation and maintenance of epithelial cell polarity, their loss may contribute to structural changes that occur after ischemia and reperfusion.

Bafilomycin A1 is a specific inhibitor of the brush-border membrane-bound H(+)-adenosinetriphosphatase (H(+)-ATPase) of the kidney cortex with no effect on the mitochondrial ATP synthetase or on the basolateral Na(+)-K(+)-ATPase activities. Bafilomycin A1 is thus a useful tool to estimate the contribution of the activity of the H(+)-ATPase to the cellular ATP turnover in a suspension of proximal tubules containing largely S1 and S2 segments. In dog proximal tubules incubated under control conditions, we found that 81% of the respiration is directly related to ATP synthesis, i.e., is sensitive to oligomycin (phosphorylative respiration). Of this amount, 29% is inhibited by 5 x 10(-7) M bafilomycin A1 alone and 90-95% by the combination of bafilomycin plus ouabain. These results indicate that the H(+)-ATPase activity is a significant energy-requiring process in dog proximal tubules. If bafilomycin is added after a 5- to 7-min preincubation with 1 mM ouabain, then the bafilomycin-sensitive ATP turnover is larger, reaching 44% of total phosphorylation. This may suggest that the H+ pump is stimulated by the indirect inhibition of the Na+/H+ exchanger produced by the exposure of tubules to ouabain. The contribution of the bafilomycin-sensitive H+ pump to the cell ATP turnover is also increased by acidification of the extracellular medium. In rabbit and hamster proximal tubules, the bafilomycin-sensitive ATP requirement involves only 5 and 10%, respectively, of the total ATP turnover. These results demonstrate that the metabolic cost of proton secretion by the membrane-bound H(+)-ATPase in suspensions of proximal tubules may be considerable but varies significantly from species to species.

The rabbit proximal tubule (PT) has been widely utilized to study the direct effects of angiotensin II (ANG II) on PT function. The purpose of the present study was to characterize the binding properties of PT ANG II receptors, using nonpeptide antagonists, and to clone a rabbit PT ANG II receptor. In rat and rabbit kidney cortical brush-border and basolateral membranes, specific binding of 125I-ANG II was inhibited by the AT1 ANG II-receptor antagonist DuP 753, but not by the AT2 antagonist PD 123319. Using a rabbit kidney cortex cDNA library, we isolated cDNA encoding an ANG II receptor, with an open-reading frame sharing a high degree of sequence homology to previously cloned AT1 ANG II receptors. In transfected COS-1 cells, this rabbit ANG II receptor had properties of the AT1 class. Northern analysis revealed high levels of mRNA expression for this receptor in rabbit kidney cortex and adrenal gland. Within the kidney, message was detected in primary cultures of rabbit PT cells, as well as in freshly isolated rabbit PT segments. Message was also present in cells of the mouse PT line, MCT, and in rat glomerular mesangial cells. Utilizing polymerase chain reaction (PCR) with primers derived from the 1st and 4th transmembrane domains of the rat AT1A ANG II receptor, a 279-bp DNA fragment was amplified from reverse-transcribed RNA from rabbit PT cells. This DNA encoded an amino acid sequence identical to that encoded by the rabbit kidney cDNA clone in the corresponding region and differed by a single base substitution. Southern analysis of rabbit genomic DNA restriction digests with the rabbit ANG II receptor probe revealed hybridization to a single band in each lane. These results indicate that an AT1 ANG II receptor is present in the PT and that a single gene codes for the AT1 receptor in rabbit. The clone isolated in the present study should provide a useful tool with which to study the regulation of the PT renin-angiotensin system.

The coupling between the dopamine1 (DA1) receptor and the G protein/adenylyl cyclase (AC) enzyme complex is defective in the proximal convoluted tubule (PCT) of 20-wk-old spontaneously hypertensive rats (SHRs). Because this coupling defect could have been due to desensitization secondary to elevated renal dopamine levels in the adult animal, we studied the interaction between DA1 receptors and AC in PCT of rats as early as 3 wk of age, a time when renal dopamine levels are similar in SHRs and their normotensive controls (Wistar-Kyoto rats, WKYs). Maximum receptor density did not change with age and was similar in WKYs and SHRs in all the age groups studied (3, 8, and 20 wk). Basal-, forskolin-, and guanyl nucleotide-stimulated AC activities were also similar in WKYs and SHRs and did not change with age. However, the DA1 agonist-stimulated AC activity was greater in WKYs than in SHRs and increased with age in WKYs but not in SHRs. Moreover, the ability of a nonhydrolyzable analogue of GTP, Gpp(NH)p, to enhance DA1 agonist (SND-919-C12, 1 microM)-stimulated AC activity increased with age in WKY but not in SHRs. To determine if the defect noted in the PCT of SHRs is due to a defective D1A receptor gene, parallel studies were performed in the striatum, since this receptor is expressed predominantly in the latter tissue. In contrast to the results in PCT, radioligand binding and AC studies in striatum revealed no differences between WKYs and SHRs.(ABSTRACT TRUNCATED AT 250 WORDS)

In dual micropuncture experiments the shrinking drop technique was used to measure volume flux with artificial tubular fluid (AF) alternating with harvested tubular fluid (HTF) during saline volume-expanded (VE) and nonexpanded (NE) periods. In VE rats, volume flux (Jv) (nl.mm-1.min-1) with AF was 1.78 +/- 0.08 (means +/- SE) during NE and was reduced to 1.39 +/- 0.09 (P = 0.01) during subsequent VE, whereas with randomly alternating HTF during VE it was 1.07 +/- 0.08 (P less than 0.0001 and less than 0.03, respectively). Jv with HTF from NE rats tested in the VE rats was 1.20 +/- 0.06, which is significantly higher than that measured with their own HTF (Wilcoxon rank, P = 0.05). In NE rats Jv was 1.65 +/- 0.10 and 1.69 +/- 0.10 with AF and HTF and was 1.28 +/- 0.07 (P less than 0.001 from both) with HTF obtained from VE rats. Elemental analysis of reaspirated tubular fluids showed no significant differences in Na or Cl concentrations among any of the fluids. It is concluded that during VE, a transferable Na transport inhibitor appears in proximal tubular fluid, which, together with a changed proximal tubular epithelium, possibly due to physical forces, accounts for proximal tubular Na transport inhibition during VE.