p53 Protects renal inner medullary cells from hypertonic stress by restricting DNA replication

Dmitrieva, Natalia, Luis Michea, and Maurice Burg. p53 Protects renal inner medullary cells from hypertonic stress by restricting DNA replication. Am J Physiol Renal Physiol 281: F522-F530, 2001.-We previously found that p53 upregulation by hypertonicity protected renal inner medullary collecting duct (mIMCD3) cells from apoptosis. The purpose of the present study was to investigate the mechanism by which p53 protects the cells. We now find that hypertonicity (NaCl added to a total of 500 mosmol) inhibits DNA replication and delays G 1-S transition as concluded from analysis of cell cycle distributions and bromodeoxyuridine (BrDU) incorporation rates. Lowering of p53 with p53 antisense oligonucleotide attenuated such effects of hypertonicity, resulting in an increased number of apoptotic cells in S phase and cells with Ͼ4 N DNA. Results with synchronized cells are similar, showing that cells in the early S phase are more sensitive to hypertonicity. Immunocytochemistry revealed that p53 becomes phosphorylated on Ser 15 and translocates to the nucleus in S both in isotonic and hypertonic conditions. Caffeine (2 mM) greatly reduces the p53 level and Ser 15 phosphorylation, followed by a remarkable increase of DNA replication rate, by failure of hypertonicity to inhibit it, and by reduction of cell number during hypertonicity. Finally, inhibition of DNA replication by the DNA polymerase inhibitor aphidicolin significantly improves cell survival, confirming that keeping cells in G1 and decreasing the rate of DNA replication is protective and that these actions of p53 most likely are what normally help protect cells against hypertonicity. cell cycle arrest; apoptosis; sodium chloride CELLS OF THE renal inner medulla are normally exposed to variable and often extreme hypertonicity as the result of the renal mechanism for concentrating the urine. This raises questions about the mechanisms that they employ to survive and function under such adverse conditions. p53 Is a tumor suppressor whose loss of function, observed in many types of cancer, contributes to genomic instability and malignancy In previous studies, we found that hypertonicity increased the amount of total and phosphorylated Ser 15 p53-and p53-dependent transcription in renal inner medullary collecting duct cells (mIMCD3; see Ref. 11). Under these conditions, reducing p53 with p53 antisense oligonucleotide (p53-AS) increased apoptosis, suggesting that activation of p53 is protective (11). Hypertonicity also arrests growth of mIMCD3 cells The purpose of present study was to analyze the mechanism by which p53 protects against hypertonicity. We found that phosphorylation of p53 on Ser 15 , previously noted to be protective during hypertonicity, occurs in the S phase of the cell cycle. Furthermore, reducing p53 expression with p53-AS or caffeine reversed both the G 1 -S arrest and reduction of DNA replication that are caused by hypertonicity. Under those conditions, apoptosis increased mainly in the cells in which DNA content had increased. We conclude that p53 protects cells against hypertonic stress by restricting DNA replication. EXPERIMENTAL PROCEDURES Cell culture. Subconfluent cultures of mIMCD3 cells (generously provided by S. Gullans; see Ref. 31) were used in passages 13-17. The medium contained 45% DME low glucose, 45% Coon's Improved Medium mF-12 (Irvine Scientific), and 10% FBS (Life Technologies). Osmolality of control ("isotonic") medium was 320 mosmol/kg. Hypertonic media, prepared by adding NaCl, were substituted for the control medium, as indicated. Cells were incubated at 37°C and gassed with 5% CO 2-95% air during growth and during all experiments. Antisense oligonucleotide experiments. For all experiments with p53-AS (Biognostik), cells were grown on eight-chamber plastic slides (Nalge Nunc International) and preincubated for 16 h with 2 M of p53-AS (sequence: CGT CAT GTG CTG TGA C) or control (CG-matched randomized-sequence phosphorothioate oligonucleotide: GAC TAC GAC CTA CGT G)

NFAT5 Is Activated by Hypoxia: Role in Ischemia and Reperfusion in the Rat Kidney

The current hypothesis postulates that NFAT5 activation in the kidney's inner medulla is due to hypertonicity, resulting in cell protection. Additionally, the renal medulla is hypoxic (10–18 mmHg); however there is no information about the effect of hypoxia on NFAT5. Using in vivo and in vitro models, we evaluated the effect of reducing the partial pressure of oxygen (PO2) on NFAT5 activity. We found that 1) Anoxia increased NFAT5 expression and nuclear translocation in primary cultures of IMCD cells from rat kidney. 2) Anoxia increased transcriptional activity and nuclear translocation of NFAT5 in HEK293 cells. 3) The dose-response curve demonstrated that HIF-1α peaked at 2.5% and NFAT5 at 1% of O2. 4) At 2.5% of O2, the time-course curve of hypoxia demonstrated earlier induction of HIF-1α gene expression than NFAT5. 5) siRNA knockdown of NFAT5 increased the hypoxia-induced cell death. 6) siRNA knockdown of HIF-1α did not affect the NFAT5 induction by hypoxia. Additionally, HIF-1α was still induced by hypoxia even when NFAT5 was knocked down. 7) NFAT5 and HIF-1α expression were increased in kidney (cortex and medulla) from rats subjected to an experimental model of ischemia and reperfusion (I/R). 7) Experimental I/R increased the NFAT5-target gene aldose reductase (AR). 8) NFAT5 activators (ATM and PI3K) were induced in vitro (HEK293 cells) and in vivo (I/R kidneys) with the same timing of NFAT5. 8) Wortmannin, which inhibits ATM and PI3K, reduces hypoxia-induced NFAT5 transcriptional activation in HEK293 cells. These results demonstrate for the first time that NFAT5 is induced by hypoxia and could be a protective factor against ischemic damage

Biochemical evidence for adhesion-promoting role of major intrinsic protein isolated from both normal and cataractous human lenses

In this study, we tested the adhesion-promoting role of major intrinsic protein from both normal human (cadaver) and senile cataractous lenses. Junctional membrane solubilized proteins and pure major intrinsic protein obtained from both type of lenses were reconstituted in neutral phosphatidylcholine liposomes. The interaction of these liposomes with phosphatidylserine vesicles was studied by resonance energy transfer. Our results show that normal human lens junction solubilized proteins and pure major intrinsic protein isolated from them promote adhesion. No quenching effect was observed when major intrinsic protein was omitted in the vesicle reconstitution, no other intrinsic protein of normal human junctional membrane provoked the adhesive effect. In contrast, major intrinsic protein isolated from human senile cataractous lens fails to induce adhesion. The proteolytic cleavages that in vitro originate major intrinsic protein 22 000 Da did not blunt its adhesive capability, suggesti

Regulation of the sodium-phosphate cotransporter Pit-1 and its role in vascular calcification

Vascular calcification is caused by the deposition of basic calcium phosphate crystals in blood vessels, myocardium, and/or cardiac valves. Calcification decreases artery wall compliance, and arterial calcification is associated to mortality in hyperphosphatemic renal failure and diabetes mellitus. The calcification of the tunica media characterizes the arteriosclerosis observed with age, diabetes and end stage-renal disease, and it can develop independently from intima calcification. As part of the vascular calcification mechanism, vascular smooth muscle cells (VSMC) experience a transition from a contractile to an osteochondrogenic phenotype and a sequence of molecular events that are typical of endochondral ossification. The current evidence indicates a key role of increased phosphate uptake by VSMC for calcification, which supplies the substrate for hydroxyapatite formation and could trigger or potentiate VSMC transdiferentiation. The present review analyzes the sodium-phosphate c

Cambios fisiológicos asociados al envejecimiento

La población envejece en forma acelerada, y la comprensión de los cambios fisiológicos asociados al envejecimiento es una herramienta importante para enfrentar las demandas biomédicas y sociales de ese grupo etario. El objetivo de la presente revisión es definir los principales cambios morfológicos y funcionales en los sistemas cardiovascular, renal, nervioso central, muscular y metabolismo de la glucosa asociados a la edad. La evidencia de estudios clínicos y experimentales muestra que el envejecimiento de los vasos sanguíneos y el corazón se asocia a la pérdida de células musculares y menor distensibilidad. La fracción de eyección se mantiene constante. El riñón muestra disminución moderada de la velocidad de filtración glomerular, esclerosis vascular y glomerular, menor capacidad de concentración/dilución y de hidroxilación de la vitamina D. El cerebro disminuye su volumen, pero no por una pérdida generalizada de neuronas ni de arborización dendrítica. Hay menor capacidad de atención, memoria de trabajo y trastornos motores. La masa muscular disminuye y aumenta su infiltración grasa, asociado a disminución progresiva de la fuerza. El aumento de grasa corporal, especialmente visceral, participaría en una mayor resistencia insulínica que asociada a la disminución de la masa de células beta facilitaría el desarrollo de diabetes. La evidencia disponible muestra importantes cambios morfológicos y funcionales asociados a la edad. El conocimiento de la población en edad media de la vida no debiera generalizarse a los adultos mayores. El reconocimiento de cambios debidos al envejecimiento normal es difícil por la gran variabilidad entre sujetos y la alta prevalencia de comorbilidad

Effect of Casein-Derived Peptides on D-Xylose Absorption Assessed by H2 Breath Test in Normal Volunteers

Studies have shown a promoting effect of food on small intestinal absorption. Casein hydrolysate seems be more effective in increasing of D-xylose absorption in dogs than the whole protein and lactulose. The purpose this study was to analyze the effect of groups of peptides derived from casein hydrolysate on the absorption of D-Xylose and intestinal transit time in normal subjects. Seven normal volunteers participated in the study. Three peptide fractions were isolated from casein enzymatic hydrolysate by means of a preparative HPLC silica column. On separate days subjets drank test solutions containing lactulose, D-xylose, and D-xylose with one of three peptide groups. The hydrogen breath test was used to indirectly estimate D-xylose absorption and orocecal transit time. Two peptide fractions when added to D-xylose were followed by an increased absorption characterized by decreased H2 production. A nonstatistically significant increase of orocecal transit time was observed with these peptides