RESUMO
BACKGROUND: Phospholipase C (PLC) is an important factor in signal transduction because this enzyme is activated by several hormones and growth factors. Eight PLC isoforms have been described raising the possibility that different cells express a single isoform or activate specific isoforms in different cells. Therefore, the goal of this study was to determine which PLC isoforms are expressed in specific regions of rat kidney. METHODS: Western blot analysis was performed in microdissected nephron segments of rat kidney, while immunohistochemical analysis was performed on whole rat kidney slices using PLC isoform-specific antibodies. RESULTS: All three families of PLC isoforms (beta, gamma, and delta) were present throughout the cortical and medullary regions of the kidney. Only the PLC-beta1 isoform was observed in the brush border of the proximal tubule, but all isoforms were present in glomeruli and in the cytoplasm of tubular epithelial cells. In addition, only the PLC-gamma1 isoform was expressed in the internal elastic lamina of the renal artery, while vasa recta expressed PLC-beta1 most intensely. Medullary thick ascending limbs showed an intense level of expression of all three isoforms. CONCLUSION: Multiple PLC isoforms are present in glomeruli, renal tubules, and renal vasculature in vivo, but with some segment-specific differences. These findings suggest that the response of a specific cell is not determined by expression of only one PLC isoform, with the exception of the brush border of the proximal tubule and the renal arteries. Instead, the presence of multiple PLC isoforms in specific regions of the kidney suggests that hormonal regulation in vivo involves mechanisms beyond cell-specific isoforms of PLC.
Assuntos
Isoenzimas/análise , Rim/enzimologia , Fosfolipases Tipo C/análise , Animais , Western Blotting , Imuno-Histoquímica , Isoenzimas/imunologia , Masculino , Ratos , Ratos Sprague-Dawley , Fosfolipases Tipo C/imunologiaRESUMO
Heat shock proteins (HSP) are components of the steroid receptor complex and are released into the cell cytosol after hormone binding. We tested whether HSPs released from steroid receptors mediate an increase in calcineurin phosphatase activity by steroid hormones. Aldosterone increased calcineurin activity in microdissected rat cortical collecting ducts (CCD) and connecting tubules, but not in proximal tubules, medullary thick ascending limb, or outer medullary collecting ducts. In contrast, 5 microM dexamethasone increased calcineurin activity in both CCD and proximal tubules. Aldosterone increased CCD calcineurin activity after 30 min and this response was blocked by spironolactone, but not by actinomycin D. An antibody recognizing HSP-56 did not change basal calcineurin activity, but completely blocked the stimulation of calcineurin by aldosterone. Rapamycin, an immunosuppressive drug that stabilizes the HSP-steroid receptor complex, also blocked the aldosterone response, whereas HSP-90 or HSP-70 increased calcineurin activity in permeabilized CCD. In summary, (a) aldosterone increases calcineurin activity in CCD through a transcription-independent process; (b) maneuvers inactivating HSP-56 or slowing HSP disassociation from the receptor complex blocks stimulation of calcineurin by steroid hormones; (c) HSP-90 and HSP-70 increase CCD calcineurin activity in the absence of steroid hormone. We conclude that HSPs released from transformed steroid receptors can stimulate calcineurin activity through a transcription-independent pathway.
Assuntos
Aldosterona/farmacologia , Proteínas de Ligação a Calmodulina/metabolismo , Dexametasona/farmacologia , Proteínas de Choque Térmico/fisiologia , Fosfoproteínas Fosfatases/metabolismo , Receptores de Esteroides/fisiologia , Transcrição Gênica , Adrenalectomia , Animais , Calcineurina , Proteínas de Ligação a Calmodulina/efeitos dos fármacos , Proteínas de Ligação a Calmodulina/genética , Ativação Enzimática/efeitos dos fármacos , Isomerismo , Túbulos Renais Coletores/efeitos dos fármacos , Túbulos Renais Coletores/enzimologia , Masculino , Néfrons/efeitos dos fármacos , Néfrons/enzimologia , Especificidade de Órgãos , Fosfoproteínas Fosfatases/efeitos dos fármacos , Fosfoproteínas Fosfatases/genética , Ratos , Ratos Sprague-DawleyRESUMO
Calcineurin activity and alpha-subunit expression were studied in microdissected proximal tubules (S2), medullary thick ascending limbs (MTAL), cortical collecting ducts (CCD), connecting tubules (CNT), and outer medullary collecting ducts (OMCD). We have shown that cyclosporin A (CsA) and FK-506 inhibit sodium-potassium-adenosinetriphosphatase (Na-K-ATPase) activity in CCD, OMCD, and MTAL but did not uncover the mechanism for resistance of proximal tubule segments to these drugs. Because cells expressing high calcineurin activity are relatively resistant to the biological effects of CsA and FK-506, we hypothesized that the resistance of proximal tubules may be linked to increased calcineurin expression. Consequently, we measured calcineurin activity in microdissected tubules using a calcineurin-specific substrate. Calcineurin activity in S2 proximal tubule segments was 10-fold higher than in CCD, CNT, OMCD, or MTAL. FK-506 (6.0 ng/ml) inhibited calcineurin activity in CCD, CNT, and MTAL but not S2; 250 ng/ml FK-506 inhibited S2 calcineurin activity by 50%. Likewise, high concentrations of CsA (25 micrograms/ml) and FK-506 (250 ng/ml) inhibited S2 Na-K-ATPase activity by 77 and 73%, respectively. To investigate whether the resistance of S2 segments might be due to differential expression of calcineurin alpha-subunit isoforms, we determined the isoform expression by Western blot analysis using isoform-specific antibodies against the alpha 1-, alpha 2-, and alpha 3-isoforms. We found that alpha 1 expression in S2 was significantly greater than in the CCD and MTAL, whereas alpha 2 expression in the S2 was significantly less than in CCD and MTAL. No alpha 3 was detected in any nephron segment tested.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Proteínas de Ligação a Calmodulina/metabolismo , Néfrons/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Animais , Western Blotting , Calcineurina , Proteínas de Ligação a Calmodulina/antagonistas & inibidores , Ciclosporina/farmacologia , Isomerismo , Túbulos Renais/metabolismo , Masculino , Fosfoproteínas Fosfatases/antagonistas & inibidores , Ratos , Ratos Sprague-Dawley , Tacrolimo/farmacologia , Distribuição TecidualRESUMO
We reported that feeding rats 8% protein for 3 wk induces net urea transport and morphologic changes in initial inner medullary collecting ducts (IMCDs) which are not present in rats fed 18% protein. In this study, we measured net urea transport in microperfused initial IMCDs from rats fed 8% protein for > or = 3 wk and tested the effect of inhibiting Na+/K(+)-ATPase activity and found that adding 1 mM ouabain to the bath reversibly inhibited net urea transport from 14 +/- 3 to 6 +/- 2 pmol/mm per min (P < 0.01), and that replacing potassium (with sodium) in the bath reversibly inhibited net urea transport from 18 +/- 3 to 5 +/- 0 pmol/mm per min (P < 0.01). Replacing perfusate sodium with N-methyl-D-glucamine reversibly inhibited net urea transport from 12 +/- 2 to 0 +/- 1 pmol/mm per min (P < 0.01), whereas replacing bath sodium had no significant effect on net urea transport. Adding 10 nM vasopressin to the bath exerted no significant effect on net urea transport. Finally, we measured Na+/K(+)-ATPase activity in initial and terminal IMCDs from rats fed 18% or 8% protein and found no significant difference in either subsegment. Thus, net urea transport in initial IMCDs from rats fed 8% protein for > or = 3 wk requires sodium in the lumen, is reduced by inhibiting Na+/K(+)-ATPase, and is unchanged by vasopressin or phloretin. These results suggest that net urea transport may occur via a novel, secondary active, sodium-urea cotransporter.
Assuntos
Medula Renal/metabolismo , Túbulos Renais Coletores/metabolismo , Sódio/fisiologia , Ureia/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Dieta , Técnicas In Vitro , Masculino , Ouabaína/farmacologia , Perfusão , Potássio/fisiologia , Proteínas , Ratos , Ratos Sprague-Dawley , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Organismos Livres de Patógenos Específicos , Ureia/urina , Vasopressinas/farmacologiaRESUMO
We reported that cyclosporin A (CsA) inhibits Na+/K(+)-ATPase activity in specific segments of the rat nephron. In this study, we tested the hypothesis that cyclosporin A reduces Na+/K(+)-ATPase activity through inhibition of calcineurin. In T cells, cyclosporin A and FK506 bind to immunophilins and inhibit the phosphatase activity of calcineurin; Rapamycin and SDZ 220-384 also bind to immunophilins but do not change calcineurin activity. Na+/K(+)-ATPase activity was measured in microdissected rat proximal tubule (S2 subsegment), medullary thick ascending limb (mTAL), and cortical collecting duct (CCD). First we found that two inhibitors of calcineurin, pentafluorophenol (PFP, 100 mM) and peptide 412 (1 mM), significantly reduced Na+/K(+)-ATPase activity in the CCD by 78% and 70%, respectively. In CCDs, FK506 inhibited Na+/K(+)-ATPase activity by 61 to 85% at concentrations of 1.5 to 6 ng/ml, but not at 0.5 ng/ml. FK506 (6 ng/ml) inhibited Na+/K(+)-ATPase activity in mTALs by 56% but did not inhibit it in S2s or glomeruli. In contrast, Rapamycin (12.5 ng/ml) did not change Na+/K(+)-ATPase activity in CCDs or mTALs, but at a concentration of 12.5 micrograms/ml did block the inhibitory effect of FK506 (6 ng/ml) in both segments. SDZ 220-384 (600 ng/ml) did not change Na+/K(+)-ATPase activity in CCDs. Thus, in CCDs and mTALs: (1) FK506, like cyclosporin A, inhibits Na+/K(+)-ATPase activity; (2) Rapamycin and SDZ 220-384 do not inhibit Na+/K(+)-ATPase activity; and (3) Rapamycin prevents FK506-induced inhibition of Na+/K(+)-ATPase activity. These responses may be explained by a direct inhibition of calcineurin activity yielding lower Na+/K(+)-ATPase activity in CCDs and mTALs.