Molecular mechanisms in proximal tubular and small intestinal phosphate reabsorption (Plenary Lecture)

Renal and small intestinal (re-)absorption contribute to overall phosphate(P_i)-homeostasis. In both epithelia, apical sodium (Na⁺)/P_i-cotransport across the luminal (brush border) memi brane is rate limiting and the target for physiological/pathophysiological alterations. Three different Na/P_i-cotransporters have been identified: (i) type I cotransporter(s) - present in the proximal tubule - also show anion channel function and may play a role in secretion of organic anions; in the brain, it may serve vesicular glutamate uptake functions; (ii) type II cotransporter(s) seem to serve rather specific epithelial functions; in the renal proximal tubule (type IIa)and in the small intestine (type IIb), isoform determines Na⁺-dependent transcellular P_i-movements; (iii) type III cotransporters are expressed in many different cells/tissues where they could serve housekeeping functions. In the small intestine, alterations in P_i-absorption and, thus, apical expression of IIb protein are mostly in response to longer term (days) situations (altered P_i-intake, levels of 1.25 (OH₂) vitamin D₃, growth, etc), whereas in renal proximal tubule, in addition, hormonal effects (e.g. Parathyroid Hormone, PTH) acutely control (minutes/hours) the expression of the IIa cotransporter. The type II Na/P_i-cotransporters operate (as functional monomers) in a 3 Na⁺:1 P_i stoichiometry, including transfer of negatively charged (-1) empty carriers and electroneutral transfers of partially loaded carriers (1 Na⁺, slippage)and of the fully loaded carriers (3 Na⁺, 1 P_i). By a chimera (IIa/IIb) approach, and by site-directed mutagenesis (including cysteine-scanning), specific sequences have been identified contributing to either apical expression, PTH-induced membrane retrieval, Na⁺-interaction or specific pH-dependence of the IIa and IIb cotransporters. For the COOH-terminal tail of the IIa Na/P_i -cotransporter, several interacting PDZ-domain proteins have been identified which may contribute to either its apical expression (NaPi-Cap1) or to its subapical/lysosomal traffic (NaPi-Cap2).     

Molecular mechanisms in proximal tubular and small intestinal phosphate reabsorption (plenary lecture)

Renal and small intestinal (re-)absorption contribute to overall phosphate(Pi)-homeostasis. In both epithelia, apical sodium (Na+)/Pi-cotransport across the luminal (brush border) membrane is rate limiting and the target for physiological/pathophysiological alterations. Three different Na/Pi-cotransporters have been identified: (i) type I cotransporter(s)--present in the proximal tubule--also show anion channel function and may play a role in secretion of organic anions; in the brain, it may serve vesicular glutamate uptake functions; (ii) type II cotransporter(s) seem to serve rather specific epithelial functions; in the renal proximal tubule (type Ila) and in the small intestine (type IIb), isoform determines Na+-dependent transcellular Pi-movements; (iii) type III cotransporters are expressed in many different cells/tissues where they could serve housekeeping functions. In the small intestine, alterations in Pi-absorption and, thus, apical expression of IIb protein are mostly in response to longer term (days) situations (altered Pi-intake, levels of 1.25 (OH2) vitamin D3, growth, etc), whereas in renal proximal tubule, in addition, hormonal effects (e.g. Parathyroid Hormone, PTH) acutely control (minutes/hours) the expression of the IIa cotransporter. The type II Na/Pi-cotransporters operate (as functional monomers) in a 3 Na+:1 Pi stoichiometry, including transfer of negatively charged (-1) empty carriers and electroneutral transfers of partially loaded carriers (1 Na+, slippage) and of the fully loaded carriers (3 Na+, 1 Pi). By a chimera (IIa/IIb) approach, and by site-directed mutagenesis (including cysteine-scanning), specific sequences have been identified contributing to either apical expression, PTH-induced membrane retrieval, Na+-interaction or specific pH-dependence of the IIa and IIIb cotransporters. For the COOH-terminal tail of the IIa Na/Pi-cotransporter, several interacting PDZ-domain proteins have been identified which may contribute to either its apical expression (NaPi-Cap1) or to its subapical/lysosomal traffic (NaPi-Cap2).     

Effects of angiotensin on proximal tubular reabsorption

Effects of angiotensin II on rat, rabbit, and bovine proximal tubular reabsorption have been demonstrated with a variety of techniques, including in vivo microperfusion, free-flow micropuncture of surface and juxtamedullary nephrons, perfusion of isolated tubules in vitro, and cell culture. Blockade of endogenous angiotensin production in vivo with converting-enzyme inhibition, or of receptors with saralasin, consistently inhibits proximal reabsorption of fluid in both superficial and juxtamedullary proximal tubules. Angiotensin effects on the proximal tubule are not neurally mediated, for they persist in denervated kidneys and are seen in nerve-free isolated tubules. Physiological concentrations of angiotensin (10(-11)-10(-9) M) stimulate electroneutral sodium transport from the basolateral membrane, whereas pharmacological doses (10(-7) M and above) inhibit reabsorption. The stimulatory effects appear to be receptor mediated. In addition to these direct effects of angiotensin on the proximal tubule epithelium, endogenous angiotensin may also alter peritubular physical forces to further enhance proximal reabsorption. These effects of angiotensin may represent an important homeostatic mechanism during states of extracellular fluid volume depletion.     

Flash photolysis of caged nitric oxide inhibits proximal tubular fluid reabsorption in free-flow nephron

A nonobstructing optical method was developed to measure proximal tubular fluid reabsorption in rat nephron at 0.25 Hz. The effects of uncaging luminal nitric oxide (NO) on proximal tubular reabsorption were investigated with this method. Proximal fluid reabsorption rate was calculated as the difference of tubular flow measured simultaneously at two locations (0.8-1.8 mm apart) along a convoluted proximal tubule. Tubular flow was estimated on the basis of the propagating velocity of fluorescent dextran pulses in the lumen. Changes in local tubular flow induced by intratubular perfusion were detected simultaneously along the proximal tubule, indicating that local tubular flow can be monitored in multiple sites along a tubule. The estimated tubular reabsorption rate was 5.52 +/- 0.38 nl.min(-1).mm(-1) (n = 20). Flash photolysis of luminal caged NO (potassium nitrosylpentachlororuthenate) was induced with a 30-Hz UV nitrogen-pulsed laser. Release of NO from caged NO into the proximal tubule was confirmed by monitoring intracellular NO concentration using a cell-permeant NO-sensitive fluorescent dye (DAF-FM). Emission of DAF-FM was proportional to the number of laser pulses used for uncaging. Photolysis of luminal caged NO induced a dose-dependent inhibition of proximal tubular reabsorption without activating tubuloglomerular feedback, whereas uncaging of intracellular cGMP in the proximal tubule decreased tubular flow. Coupling of this novel method to measure reabsorption with photolysis of caged signaling molecules provides a new paradigm to study tubular reabsorption with ambient tubular flow.     

Acute arterial hypertension inhibits proximal tubular fluid reabsorption in normotensive rat but not in SHR

The effect of acute arterial hypertension on proximal tubular fluid reabsorption was investigated in Sprague-Dawley rats and spontaneously hypertensive rats (SHR) by measuring proximal tubular flow with a nonobstructive optical method. Under control conditions, spontaneous tubular flow was oscillating at 0.02-0.03 Hz in Sprague-Dawley rats. Acute hypertension induced an immediate increase of mean tubular flow (50% increase after 20 min of hypertension) and augmentation of oscillatory amplitude. Acute hypertension did not alter single-nephron blood flow as measured by laser-Doppler velocimetry (n = 12), suggesting that the increase of tubular flow was due to inhibition of reabsorption but not increase of filtration. By contrast, spontaneous tubular flow was fluctuating aperiodically in SHR. Acute hype tension did not induce a continuous increase of tubular flow or an increase in amplitude of fluctuations (n = 15). When apical Na(+)/H(+) exchange activity of proximal tubule was monitored, acute hypertension did not alter the activity in SHR (n = 8), while similar procedures had been shown to inhibit apical Na(+)/H(+) exchange activity of proximal tubules by more than 40% in Sprague-Dawley rats. These observations suggest that acute hypertension inhibits proximal tubular fluid reabsorption by inhibiting apical Na(+)/H(+) exchange activity in Sprague-Dawley rats and that this mechanism is impaired in SHR.     

Cellular mechanisms of calcium phosphate ceramic degradation.

Calcium phosphate (CaP) ceramics are widely used for bone substitution in orthopedic, maxillofacial and dental surgery. Many environmental factors are involved in the gradual degradation of calcium phosphate ceramic after implantation, including physiocochemical processes (dissolution-precipitation) and the effects of various cell types. Several of these cell types degrade ceramics by phagocytotic mechanisms (fibroblasts, osteoblasts, monocytes/macrophages) or by an acidic mechanism with a proton pump to reduce the pH of the microenvironment and resorb these synthetic substrates (osteoclasts). Various mesenchymal cells located at the implantation sites can induce the solubilization of CaP ceramics. Crystal-cell contacts were required to induce such crystal dissolution. Mesenchymal cells such as fibroblastic cells are also actively involved in the ceramic degradation process. In this context, CaP crystals underwent dissolution into the phagosome. If osteoclasts resorb CaP ceramics similarly to the natural bone, they possess a phagocytic capability. This phagocytosis mechanism consisted of three steps: crystal phagocytosis, disappearance of the endophagosome envelope membrane and fragmentation of phagocytosed crystals within the cytoplasm. Similar phenomenons have been observed during the phagocytic mechanism induced by monocytes/macrophages. The cellular mechanisms of CaP ceramic degradation are modulated by various parameters, such as the properties of the ceramic itself, the implantation sites and the presence of various proteins (cytokines, hormones, vitamins, ions, etc.). The cells involved in these mechanisms could intervene directly or indirectly through their cytokine/growth factor secretions and their sensitivity to the same molecules. This article reviews recent knowledge on the cellular mechanisms of calcium phosphate ceramic degradation.     

Ultrastructure of proximal tubular cells of the rat nephron in the period of intense glucose reabsorption

It has been demonstrated in rat experiment that maximal glucose reabsorption is accompanied by the increased electron density of mitochondrial matrix and the enlargement of the size of microbodies. No vacuoles have been recorded, which is at variance with the concept of the pinocytosis involvement into glucose reabsorption.     

Isosmotic volume reabsorption in rat proximal tubule

A theoretical model incorporation both active and passive forces has been developed for fluid reabsorption from split oil droplets in rat intermediate and late proximal tubule. Of necessity, simplifying assumptions have been introduced; we have assumed that the epithelium can be treated as a single membrane and that the membrane "effective" HCO3 permeability is near zero. Based on this model with its underlying assumptions, the following conclusions are drawn. Regardless of the presence or absence of active NaCl transport, fluid reabsorption from the split oil droplet is isosmotic. The reabsorbate osmolarity can be affected by changes in tubular permeability parameters and applied forces but is not readily altered from an osmolarity essentially equal to that of plasma. In a split droplet, isosmotic flow need not be a special consequence of active Na transport, is not the result of a particular set of permeability properties, and is not merely a trivial consequence of a very high hydraulic conductivity; isosmotic flow can be obtained with hydraulic conductivity nearly an order of magnitude lower than that previously measured in the rat proximal convoluted tubule. Isosmotic reabsorption is, in part, the result of the interdependence of salt and water flows, their changing in parallel, and thus their ratio, the reabsorbate concentration being relatively invariant. Active NaCl transport can cause osmotic water flow by reducing the luminal fluid osmolarity. In the presence of passive forces the luminal fluid can be hypertonic to plasma, and active NaCl transport can still exert its osmotic effect on volume flow. There are two passive forces for volume flow: the Cl gradient and the difference in effective osmotic pressure; they have an approximately equivalent effect on volume flow. Experimentally, we have measured volume changes in a droplet made hyperosmotic by the addition of 50 mM NaCl; the experimental results are predicted reasonably well by our theoretical model.