Over-expression of adenosine deaminase in mouse podocytes does not reverse puromycin aminonucleoside resistance

<p>Abstract</p> <p>Background</p> <p>Edema in nephrotic syndrome results from renal retention of sodium and alteration of the permeability properties of capillaries. Nephrotic syndrome induced by puromycin aminonucleoside (PAN) in rats reproduces the biological and clinical signs of the human disease, and has been widely used to identify the cellular mechanisms of sodium retention. Unfortunately, mice do not develop nephrotic syndrome in response to PAN, and we still lack a good mouse model of the disease in which the genetic tools necessary for further characterizing the pathophysiological pathway could be used. Mouse resistance to PAN has been attributed to a defect in glomerular adenosine deaminase (ADA), which metabolizes PAN. We therefore attempted to develop a mouse line sensitive to PAN through induction of normal adenosine metabolism in their podocytes.</p> <p>Methods</p> <p>A mouse line expressing functional ADA under the control of the podocyte-specific podocin promoter was generated by transgenesis. The effect of PAN on urinary excretion of sodium and proteins was compared in rats and in mice over-expressing ADA and in littermates.</p> <p>Results</p> <p>We confirmed that expression of ADA mRNAs was much lower in wild type mouse than in rat glomerulus. Transgenic mice expressed ADA specifically in the glomerulus, and their ADA activity was of the same order of magnitude as in rats. Nonetheless, ADA transgenic mice remained insensitive to PAN treatment in terms of both proteinuria and sodium retention.</p> <p>Conclusions</p> <p>Along with previous results, this study shows that adenosine deaminase is necessary but not sufficient to confer PAN sensitivity to podocytes. ADA transgenic mice could be used as a background strain for further transgenesis.</p

Platelet activating factor inhibits Cl and K transport in the medullary thick ascending limb

Platelet activating factor inhibits Cl and K transport in the medullary thick ascending limb. Since the kidney medulla was reported to generate platelet activating factor (PAF), we investigated a possible effect of this agent on the reabsorptive function of in vitro microperfused medullary thick ascending limbs from mouse kidney (mTAL). PAF, 10-7 M in the bath, significantly decreased the net chloride flux (JCl) from 48.8 ± 7.1 to 27.4 ± 5.7 pmol/min. This effect was reversible, blocked by the antagonist BN 50730, and not reproduced by the inactive metabolite lyso-PAF. PAF inhibited the transepithelial potential difference with a threshold at 10-9 M. In the presence of isoproterenol, the PAF-induced decrease of JCl was not significantly different from that observed in basal conditions; moreover, PAF did not modify the adenylate cyclase activity in isolated mTALs, either in basal condition or under stimulation by isoproterenol. The effect of PAF on JCl was not prevented by mepacrine, NDGA associated with proadifen, or adenosine desami-nase. When the apical Na-K-2Cl cotransport was blocked by furosemide or bumetanide, a net K secretion occurred (-1.1 ± 0.2 pmol/min), which was significantly decreased by PAF (-0.06 ± 0.3 pmol/min). Moreover, it was verified on isolated mTALs that PAF did not modify the Na,K-ATPase activity. It is concluded that PAF inhibits the reabsorptive function of the mTAL, as indicated by the decrease of Cl reabsorption and K secretion. This effect could not be accounted for by adenosine or arachidonic acid metabolite action, and was not mediated by an inhibition of the adenylate cyclase activity

Effect of metabolic acidosis and alkalosis on NEM-sensitive ATPase in rat nephron segments

International audienceAn N-ethylmaleimide (NEM)-sensitive adenosinetriphosphatase (ATPase) displaying the kinetic and pharmacological properties of an electrogenic proton pump has been described in the different segments of rat nephron, where it mediates part of the active tubular proton secretion. This study was therefore designed to evaluate whether changes in urinary acidification observed during metabolic acidosis or alkalosis were associated with alterations of the activity of tubular NEM-sensitive ATPase, and if so, to localize the nephron segments responsible for these changes. Within 1 wk after the onset of ammonium chloride treatment, rats developed a metabolic acidosis, and NEM-sensitive ATPase activity was markedly increased in the medullary thick ascending limb of Henle's loop and outer medullary collecting tubule, and slightly increased in the cortical collecting tubule. Conversely, treatment with sodium bicarbonate induced a metabolic alkalosis that was accompanied by decreased NEM-sensitive ATPase activity in medullary thick ascending limb and outer medullary collecting tubule. NEM-sensitive ATPase activity was not altered in any other nephron segment tested in alkalotic and acidotic rats, i.e., the proximal tubule and the cortical thick ascending limb of Henle's loop. Changes qualitatively similar were observed as soon as 3 h after the onset of NaHCO3 or NH4Cl-loading. In the medullary collecting tubule, alterations of NEM-sensitive ATPase activity are in part due to hyperaldosteronism observed in both acidotic and alkalotic rats.(ABSTRACT TRUNCATED AT 250 WORDS

Characterization and control of proton-ATPase along the nephron.

International audienceSince it is now well established that the Na-H exchanger accounts for only part of tubular proton secretion, we attempted to characterize the molecular processes responsible for the remaining moiety. In particular, we evaluated the possible roles of proton pumps in urinary acidification. For this purpose, we characterized ATPase activities associated with the electrogenic H pump, on the one hand, and with H-K-ATPase, on the other. In order to circumvent the axial heterogeneity of nephron, this study was carried out on microdissected segments of nephron. The present report summarized experiments which aimed: (1) at characterizing H-ATPase and H-K-ATPase on kinetic and pharmacologic bases in the successive segments of mammalian nephrons; (2) at evaluating the ionic transport mediated by these two ATPases; and (3) at determining the factors which control the activity of these pumps

Difference in the Na affinity of Na(+)-K(+)-ATPase along the rabbit nephron: modulation by K

International audienceThe sensitivity of Na(+)-K(+)-ATPase to Na was determined in single segments of rabbit nephron isolated by microdissection. In the cortical collecting tubule (CCT), Na(+)-K(+)-ATPase was threefold more sensitive to Na (apparent K0.5 approximately 3 mM) than in proximal convoluted tubule and cortical thick ascending limb (apparent K0.5 approximately 10 mM). Furthermore, increasing K concentration from 5 to greater than 100 mM markedly reduced the affinity of the pump for Na in all three nephron segments. In fact, the main shift in Na affinity occurred when K changed from 100 to 120 mM; in the CCT, increasing K concentration from 100 to 120 mM while maintaining Na concentration at 10 mM reduced Na(+)-K(+)-ATPase activity by greater than 35%. These findings confirm that, in kidney cells as in other cells, intracellular Na limits the rate of Na(+)-K(+)-ATPase. Thus any alteration of intracellular Na concentration modifies the pump activity in a way that contributes to the restoration of intracellular Na homeostasis. This adaptive property is particularly efficient in the collecting tubule in which the apparent K0.5 of the pump for Na is close to normal intracellular Na concentration. Furthermore, changes in intracellular K concentration, which usually accompany those of Na so as to maintain the total cation concentration constant, potentiate the regulatory role of Na through modifications of its affinity for the pump