Cell proliferation and apoptosis in kidneys with chronic obstructive uropathy (COU) have not been adequately studied. Whether these fundamental cellular processes play any role in the pathogenesis and evolution of COU remains undetermined. Sprague-Dawley rats with COU induced by unilateral ureteral ligation were sacrificed at postoperative days 1, 6, 9, 15, 34, 43, 60, 75, and 90, and were compared with control, sham-operated rats sacrificed at days 0, 15, 43, and 90. The kidneys with ureteral ligation, the contralateral kidneys, and the control kidneys were submitted to in situ end-labeling of fragmented DNAs for the detection of apoptotic cells, and to immunostaining with many monoclonal antibodies directed against the nuclear antigens associated with cell proliferation for the detection of proliferating cells. Additional rats with COU were also submitted to BrdU labeling to detect proliferating cells. The tubular, interstitial, and glomerular cells showing either apoptosis or proliferation were separately quantitated and the obtained data were correlated with dry kidney weight, tubular diameter, glomerular surface area and interstitial volume. Apoptotic tubular cells in kidney with COU increased rapidly, reaching 30-fold that of control at day 25, which was followed by an equally rapid decrease to the control level. During the same period, both the dry kidney weight and the mean tubular diameter decreased markedly. These data suggest that apoptosis may play a significant role in tubular atrophy and renal weight loss. The rapid increase in tubular cell apoptosis was immediately preceded by a 37% gain in the dry kidney weight over the control; just before that increase, there was also an approximate 60-fold increase in the proliferation rate of tubular cells detected by immunostaining for proliferating nuclear antigen or by BrdU labeling. The significance of this intriguing temporal relationship of tubular cell apoptosis and proliferation remains to be elucidated, but it may have pathogenetic implications. In contrast to the rise and fall of the frequency of tubular cell apoptosis and proliferation, the frequency of interstitial cell apoptosis and proliferation displayed continuous increase toward the end of the experiment, with a roughly parallel increase in the interstitial damage. Apoptosis and proliferation of glomerular cells in kidneys with COU did not show any significant changes throughout the experiment. In conclusion, the obtained data suggest that tubular cell apoptosis may be pathogenetically related to the tubular atrophy and renal tissue loss in COU, and that proliferation and apoptosis of interstitial cells may play a role in the observed interstitial changes in this model. This study should provide the impetus for further exploration of the mechanisms of cell death and cell proliferation as a novel venue for understanding the pathogenesis of COU.

In renal cells, hypertonicity induces genes for heat shock proteins (HSP70, ␣B-crystallin), as well as enzymes and transporters directly involved in the metabolism and transport of protective organic osmolytes. While heat shock proteins are induced by many stresses including osmotic stress, the induction of the osmolytes genes appears to be specific to osmotic stress. These two adaptive mechanisms allow kidney cells to survive and function in the hypertonic environment that exists on routine basis in kidney medulla. In mammalian cells, hypertonicity induces three mitogen-activated protein kinase pathways: ERK (extracellular regulated kinase), JNK (Jun N-terminal kinase), and p38. ERK activation by osmotic stress is a consistent finding in many cells, but it is not essential for transcriptional regulation of mRNA for transporter of organic osmolyte betaine. While the growth of yeast cells on NaCl-supplemented medium is dependent on HOG1 pathway, it is still unclear which pathway mediates the adaptation to osmotic stress in mammalian cells. Here, we show that inhibition of p38 kinase activity, using the specific inhibitor SB203580 (4-(fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridyl) imidazole), abolishes the hypertonicity-mediated induction of mRNAs for HSP70 and betaine transporter in Madin-Darby canine kidney cells. The inhibition is dose-dependent and correlates with the in situ activity of native p38 kinase, determined as MAPKAPK-2 activity in cell extracts. As reported previously, the activities of ERK-1 and -2 were not affected by SB203580, but surprisingly, inhibition of native p38 kinase activity correlates with up-regulation of native JNK-1 activity in osmotically stressed cells. p38 mRNA is induced by hypertonic stress and is attenuated with p38 kinase inhibition. We also find that thermal induction of HSP70 mRNA is not affected by p38 kinase inhibition. Such findings suggest that p38 kinase activity is essential for the induction of genes involved in the adaptation of mammalian cells to osmotic stress and that the increased activity of JNK-1 during p38 kinase inhibition is consistent with regulation of JNK-1 by p38 kinase in osmotically stressed cells. In addition, the transduction pathways mediating HSP70 mRNA induction by different stresses appear to be divergent; osmotic induction of HSP70 is p38 kinase-dependent, while thermal induction is not.During water deprivation, the extracellular osmolality in the mammalian renal medulla can exceed 3000 mosmol/kg of H 2 O. While such an osmolality is incompatible with the survival of cells from other organs, kidney cells tolerate it well. The survival of kidney medullary cells in hypertonic environment is essential for the generation of the concentrating gradient, which is key for maintenance of body solute and water homeostasis.Many organisms, including bacteria, yeast, plants, and animals, adapt to sustained hyperosmotic stress by accumulating osmotically active organic solutes (compatible organic osmolytes) (1). These compounds do not perturb cellula...

In rabbit renal cortical collecting tubule (CCT), perfused in vitro at 38 degrees C, ATP in concentrations of 10(-7) M and greater inhibits arginine vasopressin (AVP)-stimulated osmotic water permeability (Pf). The P1-purinergic receptor antagonist 8-phenyltheophylline did not attenuate the inhibitory action of ATP, and the poorly hydrolyzable ATP analogue, 5'-adenylylimidodiphosphate (AMP-PNP), mimicked the effect of ATP, arguing against an effect of ATP on a P1 receptor or the "P site." Purinergic receptor agonists inhibited AVP-stimulated Pf with the following rank order efficacy: ATP = ADP = UTP = AMP-PNP = alpha, beta-methylene-ATP > 2-methylthio-ATP >> AMP > adenosine, consistent with the pharmacology of a "nucleotide" receptor subtype. Pertussis toxin pretreatment attenuated the action of 10(-5) and 10(-6) MATP; however, 10(-4) MATP failed to inhibit the hydrosmotic action of forskolin or 8-bromoadenosine 3',5'-cyclic monophosphate. Pretreatment with the phosphodiesterase inhibitor RO20-1724 or indomethacin did not inhibit the action of ATP. Staurosporin and 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester significantly attenuated the inhibition of Pf by lower concentrations of ATP. These data suggest that ATP activates nucleotide receptors on the CCT, mobilizing intracellular Ca2+, which inhibits the hydrosmotic action of AVP.

A B S T R A C T Calcium transport was studied in medullary and cortical segments ofthe thick ascending limb of Henle perfused in vitro. 45Ca was added to the perfusate for measuring lumen-to-bath flux (JCa), to the bath for measuring bath-to-lumen flux (Jca), or to both perfusate and bath for measuring net flux (Jnt). In the medullary segment J exceeded Jca and the efflux:influx coefficient ratio was not different from the value predicted from the observed potential difference (PD). In the cortical segments, however, efflux:influx coefficient ratio was greater than the value predicted from the PD, suggesting that calcium transport in this segment may be active, while it is passive in the medullary segment. Furosemide, which reversibly decreases PD in both cortical and medullary segments, inhibited Ja,lb only in the medullary segment. Parathyroid hormone (PTH), on the other hand, had no effect on Jca in the medullary segment, but it significantly augmented Jc" in the cortical segment. These results indicate that calcium transport in the thick ascending limb is heterogeneous. In the medullary segment it is passive, inhibited by furosemide and not influenced by PTH. In the cortical segment, however, calcium transport appears to be active, not inhibited by furosemide and stimulated by PTH.

Abstract. Calcium transport was studied in isolated S2 segments of rabbit superficial proximal convoluted tubules. '"Ca was added to the perfusate for measurement of lumen-to-bath flux (JibCa), to the bath for bath-to-lumen flux (JbIc), and to both perfusate and bath for net flux (Jnetc). In these studies, the perfusate consisted of an equilibrium solution that was designed to minimize water flux or electrochemical potential differences (PD). Under these conditions, JibCa (9.1 ± 1.0 peq/mm min) was not different from JblCa (7.3±1.3 peq/mm min), and Jntc1 was not different from zero, which suggests that calcium transport in the superficial proximal convoluted tubule is due primarily to passive transport. The efflux coefficient was 9.5±1.2 X 10-5 cm/ s, which was not significantly different from the influx coefficient, 7.0±1.3 X 10-5 cm/s. When the PD was made positive or negative with use of different perfusates, net calcium absorption or secretion was demonstrated, respectively, which supports a major role for passive transport. These results indicate that in the superficial proximal convoluted tubule ofthe rabbit, passive driving forces are the major determinants of calcium transport.