Mechanism of Fluid Reabsorption in Kidney Proximal Tubule: Interplay Between Lateral Na+/K+-ATPase and AQP1 and SGLT1 Mediated Water Fluxes.

Nitric oxide prevents hypertension yet enhances proximal tubule Na+ reabsorption. Nitric oxide synthase is inhibited by asymmetric dimethylarginine (ADMA) that is metabolized by dimethylarginine dimethylaminohydrolase (DDAH) whose type 1 isoform is expressed abundantly in the proximal tubule (PT). We hypothesize that ADMA metabolized by DDAH-1 inhibits fluid reabsorbtion (Jv) by the proximal tubule. S2 segments of the PT were microperfused between blocks in vivo to assess Jv in anesthetized rats. Compared with vehicle, microperfusion of ADMA or Nω-nitro-l-arginine methyl ester (l-NAME) in the proximal tubule reduced Jv dose dependently. At 10-4 mol/l both reduced Jv by ~40% (vehicle: 3.2 ± 0.7 vs. ADMA: 2.1 ± 0.5, P < 0.01 vs. l-NAME: 1.9 ± 0.4 nl·min-1·mm-1, P < 0.01; n = 10). Selective inhibition of DDAH-1 in rats with intravenous L-257 (60 mg/kg) given 2 h before and L-257 (10-5 mol/l) perfused in the proximal tubule for 5 min reduced Jv by 32 ± 4% (vehicle: 3.2 ± 0.5 vs. L-257: 2.2 ± 0.5 nl·min-1·mm-1; P < 0.01) and increased plasma ADMA by ≈50% (vehicle: 0.46 ± 0.03 vs. L-257: 0.67 ± 0.03 µmol/l, P < 0.0001) without changing plasma symmetric dimethylarginine. Compared with nontargeted control small-interference RNA, knock down of DDAH-1 in mice by 60% with targeted small-interference RNAs (siRNA) reduced Jv by 29 ± 5% (nontargeted siRNA: 2.8 ± 0.20 vs. DDAH-1 knockdown: 1.9 ± 0.31 nl·min-1·mm-1, P < 0.05). In conclusion, fluid reabsorption in the proximal tubule is reduced by tubular ADMA or by blocking its metabolism by DDAH-1. L-257 is a novel regulator of proximal tubule fluid reabsorption.

Electrophysiology of proximal and distal tubules in the autoperfused dog kidney.

The acute effects on kidney function of acetazolamide (250 mg) given intravenously were evaluated in seven healthy subjects. Glomerular filtration rate was measured as the renal clearance of 51Cr-EDTA, and fluid flow rate out of the proximal tubules was assessed by measurement of the renal lithium clearance. An 18% decline in glomerular filtration rate (ml/min) was observed after acetazolamide administration (109 +/- 16 vs 89 +/- 14, p less than 0.02), while lithium clearance (ml/min) increased by 35% (30 +/- 5 vs 38 +/- 8, p less than 0.02). Absolute proximal tubular reabsorption of water (ml/min) was reduced by about one third (79 +/- 12 vs 51 +/- 9, p less than 0.02), and fractional proximal reabsorption of water and sodium (%) declined (73 +/- 2 vs 58 +/- 6, p less than 0.02). Renal sodium clearance and absolute distal reabsorption of sodium increased, while fractional distal reabsorption of sodium declined. Acetazolamide reduces absolute and fractional proximal tubular reabsorption of sodium and water, and glomerular filtration rate. Primarily, this induces an increase in the output of fluid from the proximal tubules accounting for the diuretic effect of the drug. The acute fall in glomerular filtration rate is probably mediated by a temporary increase in proximal intratubular pressure and activation of the tubuloglomerular feedback mechanism.

Formate stimulates sodium chloride and fluid reabsorption in kidney proximal tubule; however, the exact cellular mechanism of this effect remains unknown. We hypothesized that the primary target of formate is the apical Na(+)/H(+) exchanger. Here, we demonstrate that formate directly enhances the apical Na(+)/H(+) exchanger (NHE3) activity in mouse kidney proximal tubule. In the absence of CO(2)/HCO(3)(-), addition of formate (500 microM) to the bath and lumen of microperfused mouse kidney proximal tubule caused significant intracellular alkalinization, with intracellular pH (pH(i)) increasing from baseline levels 7.17 +/- 0.01 to 7.55 +/- 0.01 (P < 0.001, n = 14), with a Delta pH of 0.38 +/- 0.02. Removal of luminal chloride did not block cell pH alkalinization by formate (baseline pH of 7.26 +/- 0.01 to 7.53 +/- 0.01 with formate, P < 0.001, n = 10), indicating that the apical Cl(-)/OH(-) exchanger was not the primary mediator of the effect of formate on cell pH. However, removal of sodium from the lumen or addition of EIPA completely prevented cell pH alkalinization. Addition of formate to the lumen and bath in the outer medullary collecting duct, which does not express any apical Na(+)/H(+) exchanger, did not cause any cell pH alkalinization. At lower concentrations (50 microM), formate caused significant pH(i) alkalinization in proximal tubule cells, with pH(i) increasing from baseline levels 7.15 +/- 0.02 to 7.36 +/- 0.02 (P < 0.02, n = 11). Acetate, at 50 microM, had no effect on pH(i). Formate's effect was observed both in the absence and presence of CO(2)/HCO(3)(-) in the media. We conclude that formate stimulates the apical Na(+)/H(+) exchanger NHE3 in the kidney proximal tubule. We propose that formate stimulation of chloride reabsorption in the proximal tubule is indirect and is secondary to the activation of apical Na(+)/H(+) exchanger NHE3, which then leads to the stimulation of the apical chloride/base exchanger.

The calcium sensing receptor modulates fluid reabsorption and acid secretion in the proximal tubule

The mechanism of HCO3- reabsorption in proximal and distal tubules was examined in rats undergoing NaHCO3 diuresis. The steady-state intratubular pH was measured with pH-sensitive glass microelectrodes and compared with the equilibrium pH calculated from the HCO3- concentration of the tubular fluid (measured with quinhydrone electrodes) and plasma Pco2. In the proximal tubule the intratubular pH and the equilibrium pH were identical, indicating no accumulation of excess H2CO3. After inhibition of carbonic anhydrase, however, intratubular pH was significantly lower (0.85 pH U) than the equilibrium pH. It was concluded that HCO3- reabsorption in the proximal tubule was mediated by H+ secretion, but that carbonic anhydrase located in the luminal membrane of the cell prevented H2CO3 from accumulating in the tubular fluid. In the distal tubule the intratubular pH was 0.85 U lower than the equilibrium pH. This difference could be obliterated by an intravenous injection of carbonic anhydrase. It was concluded that HCO3- reabsorption in this segment was also accomplished by H+ secretion. The accumulation of excess H2CO3 in the tubular fluid indicated that, in contrast to the proximal tubule, carbonic anhydrase was not located in the luminal membrane of distal tubular cells.

Background: Proximal tubule (PT) fluid reabsorption (Jv) is diminished by knockout of NOS-I or III. ADMA is an endogenous inhibitor of NOS, but its effects on tubular function are unknown. ADMA is degradated by DDAH whose type 1 isoform (DDAH-1) is richly expressed in the PT. SNIPs of DDAH-1 predict the decline of GFR in chronic kidney disease (CKD). Hypothesis: that DDAH-1 metabolizes ADMA in the PT thereby reducing its plasma concentration and enhancing Jv. Methods: Jv was measured in anesthetized rats and mice in S2 segments of the PT by direct in vivo micropuncture and microperfusion of artificial tubular fluid (ATF). Results: Addition of L-257 (10 -4 M; DDAH-1 inhibitor) vs vehicle to ATF perfusing the PT of rats for 10 mins did not affect Jv (3.4 ± 0.20 vs 3.1±0.39 nl/min/mm). However, a bolus i.v. injection (60mg/kg) of L-257 2hrs before and 10 mins of PT perfusion significantly (P&lt; 0.005) reduced Jv by 33% to 2.3±0.17 nl/min/mm and increased plasma ADMA (systemic Veh: 0.46 ± 0.030 vs. systemic L-257: 0.67 ± 0.029 μmol/l; P &lt; 0.0001) without changing SDMA. Microperfusion of ADMA (10 -4 M) or L-NAME (10 -4 M) in rats both reduced Jv significantly by &gt;40% to 2.0±0.24 and 1.8±0.22 nl/min/mm, respectively (P&lt;0.05). An IV 1 ml bolus of saline containing Transit and siRNA targeted to DDAH-1 (hydrodynamic in vivo gene silencing in mice) three days later reduced Jv significantly by 36% compared to scrambled siRNA (2.2 ± 0.16 vs 1.4 ± 0.26 nl/min/mm; P&lt;0.05). Surprisingly, PT cell-specific DDAH-1 knockdown in mice did not affect Jv significantly compared to wild type (PTC-D1WT: 1.7 ± 0.21 vs PTC-D1KO: 2.0 ± 0.15). Conclusions: PT fluid reabsorption is regulated by ADMA and its tubular metabolism by DDAH-1. The two-hour delay for L-257 to diminish Jv likely was required for accumulation of sufficient tubular ADMA to impair PT reabsorption. Whereas systemic DDAH-1 gene knockdown in mice was as effective as systemic DDAH-1 blockade with L-257 in rats in reducing Jv in the PT, lifelong deletion of PT DDAH-1 was ineffective, suggesting robust adaptive mechanisms that restore PT reabsorption. Thus, L-257 is a novel regulator of PT function. SNIPs that alter DDAH-1 in the PT may change tubular function and thereby contribute to CKD progression.

To examine to what extent the reabsorbate concentrations, calculated as the flux ratios between solutes and water, represent the fluid composition in the lateral intercellular space (LIS) in the proximal tubules, reabsorption was stimulated by elevating PCO2 from 5 to 13 kPa before and during infusion of mannitol to a plasma concentration of 70 mM in volume-expanded dogs receiving ethacrynic acid. The reabsorbate concentration of NaHCO3 increased by 50 mM during mannitol infusion. The real concentration of NaHCO3 in LIS could not, however, be elevated by this amount, since the driving forces for fluid reabsorption then would have increased during osmotic diuresis due to diffusion of mannitol into LIS from plasma. A model analysis of diffusion in LIS showed that transcellular transport can only lead to trivial increases of LIS concentrations compared to plasma, whereas diffusion across tight junctions can increase LIS concentrations by several mM. NaCl diffusion and coupled transcellular water transport may therefore represent a significant contribution to total bicarbonate-dependent NaCl and water reabsorption in the proximal tubules.

Proteinuria can result from increased glomerular protein filtration, but insufficient or reduced protein reabsorption in the proximal tubule (PT) may also play a role. Protein is reabsorbed via receptor-mediated endocytosis, a process that is pH-dependent and requires energy in the form of ATP. Specifically, ATP is hydrolyzed by V-type ATPase, and the resulting energy is used to acidify the endocytic vesicle by pumping H+ ions into the endosome. This reduces the internal endosome pH and causes protein-receptor complex dissociation, which allows the protein to be further processed by the cell. Alterations in intracellular pH or ATP availability may impact the PT epithelial cells’ ability to form endosomes and reabsorb protein. To quantify the relationship between cellular pH, protein reabsorption, and ATP utilization, we developed a mathematical model that quantifies the energy demands of protein reabsorption in the kidney proximal tubule. The model assumes normal kidney function and focuses on albumin reabsorption for simplification. To determine the energy required to acidify a single endosome (ATP/endosome), we expanded upon a published mathematical model of general lysosome acidification that quantifies the H+ ions required to acidify a vesicle and the final pH reached, accounting for ClC-7 antiporters and proton leak, as a function of intracellular pH (Ishida et al. J Gen Phys 2013). The Nernst equation was used to determine the cumulative energy (ΔG) required to pump H+ ions into one endosome until a final stable internal pH is reached. To determine the rate of endosome formation required to reabsorb a normal filtered albumin load, we first calculated the albumin reabsorption rate per unit length of the proximal tubule as a function of albumin filtration rate, nephron number, and tubular dimensions. Next, using previous experimentally measured values for membrane internalization velocity and early endocytic radius, the rate of endosome formation required to reabsorb the filtered albumin load was calculated (total endosomes formed /sec). Total rate of ATP required to reabsorb a filtered load of albumin was then determined as the product of ATP/endosomes and total endosomes formed / second. At normal intracellular pH and normal filtered albumin load, it is estimated that 83,600 endosomes, with a radius of 0.34 µm, and 25,000 molecules of ATP are needed to reabsorb 740 µg of albumin per second for one proximal tubule. This is a very small fraction of total ATP utilized by the cell. To our knowledge, this model is the first to quantify the energetics of PT endosome formation and protein reabsorption. It provides a tool to quantitatively evaluate the impact of variations in factors such as intracellular pH, filtered protein load, and energy availability on the PT's ability to acidify endosomes and reabsorb protein. This may aid in future elucidation of pathological mechanisms of proteinuria.

Water reabsorption in the proximal convoluted tubule of the rat kidney was examined by in vivo microperfusion techniques in order to examine the effect of D-glucose within the tubular lumen. When tubules were perfused with a balanced artificial solution containing Na, K, Cl, HCO3, urea, and D-glucose, absolute reabsorption averaged 4.01 +/- 0.24 nl/min per mm. Addition of D-glucose to the NaCl perfusate enhanced water reabsorption to values similar to those obtained with the balanced artificial perfusate. The enhanced water reabsorption consequent to the addition of D-glucose to the NaCl perfusion solution was completely inhibited by addition of phloridzin to the perfusate. The addition of an unabsorbed hexose, 2-deoxy-D-glucose, to the NaCl perfusate failed to enhance water reabsorption, whereas the addition of an incompletely reabsorbed sugar that is not metabolized, 3-O-methyl-D-glucose, resulted in partial enhancement of theabsolute rate of water reabsorption. These studies demonstrate that D-glucose has the specific effect of augmenting water reabsorption in the proximal tubule of the rat kidney.

Ochratoxin A (OTA) is a mycotoxin produced by Aspergillus and Penicillium that represent toxic real threat for human beings and animal health. In this study we evaluated the effect of a new recombinant mitochondrial manganese containing superoxide dismutase (rMnSOD) on oxidative stress and on the alterations of fluid reabsorption in renal proximal tubule (PT) as possible causes of OTA nephrotoxicity. Finally, we have measured the concentration of O2- in the kidney through dihydroethidium assay (DHE) and nitric oxide (NO) concentration through nitrites and nitrates assay. Male Sprague Dawley rats weighing 120-150 g were treated for 14 days by gavage, as follows: Control group, 12 rats received a corresponding amount of saline solution (including 10% DMSO); rMnSOD group, 12 rats treated with rMnSOD (10 µg/kg bw); OTA group, 12 rats treated with OTA (0.5 mg/kg bw) dissolved in 10% DMSO and then scaled to required volume with corn oil; rMnSOD + OTA, 12 rats treated with rMnSOD (10 µg/kg bw) plus OTA (0.5 mg/kg bw). Our results have shown that rMnSOD restores the alteration of reabsorption in PT in rats treated with OTA plus rMnSOD, probably through the response to pressure natriuresis, where nitric oxide plays a key role. Moreover, rMnSOD prevents the nephrotoxicity induced by OTA probably restoring the balance between superoxide and NO that is most probably the cause of hypertension and renal functional alterations through the inhibition of NO synthase. In conclusion these data provide important information for understanding of mechanism of toxic action of OTA. J. Cell. Biochem. 119: 424-430, 2018. © 2017 Wiley Periodicals, Inc.

The effects of lysine on bicarbonate and fluid reabsorption in the rat proximal tubule were studied by luminal and capillary perfusion in situ. The proximal tubule and peritubular capillaries were perfused with bicarbonate Ringer solution containing [14C]inulin. The rate of bicarbonate reabsorption (JHCO3) was estimated to be 124 +/- 9.5 peq.min-1.mm-1 using a pH membrane glass electrode. The rate of net fluid reabsorption (Jv) was 2.6 +/- 0.21 nl.min-1.mm-1. When 10 mM L-lysine was added to the luminal perfusate, a 35% reduction in JHCO3 and no change in Jv were observed. Increase of L-lysine concentration in the luminal perfusate to 20 mM did not reduce JHCO3 further nor did it influence Jv.l When 10 mM L-lysine was added to the capillary perfusate, a 13% reduction in JHCO3 was observed (NS). Increase of lysine concentration in the capillary perfusate to 20 mM significantly reduced JHCO3 by 26% (P less than 0.01). There was no significant change in Jv under both conditions. The effect of L-lysine in the lumen was related to its reabsorption kinetics, D-Lysine, which was not reabsorbed significantly, did not affect bicarbonate reabsorption in the proximal tubule. These results indicate that the inhibitory effect of L-lysine is related to the entry of lysine into the cell from the lumen.

Bicarbonate (HCO3−) is an important component of acid/base regulation. Therefore, it is crucial to understand the mechanism of bicarbonate reabsorption in the kidney proximal tubule (PT). In mammals HCO3−reabsorption is well understood, but evidence suggests that birds may use a different mechanism. We hypothesize that in the avian PT HCO3−crosses the luminal membrane in ionic form, rather than as CO2, and that HCO3− ions then leave the basolateral side via an NBC1‐like transporter, as in the mammalian PT. Experiments used primary cell cultures of chick PT and electrophysiological studies to measure currents (ISC) associated with ion transport. Monolayers were first stimulated with 1μM forskolin, which activated a chloride secretory current in the avian PT. This was followed by basolateral application of 100 μM DIDS, an inhibitor of NBC1. With bicarbonate in the bathing solution DIDS caused an increase in ISC of 6.25 ± 1.55 μAmps/cm2 (n = 6), but only 2.30 ± 0.58 μAmps/cm2 (n = 5) in the absence of bicarbonate. The increased ISC is consistent with inhibition of electrogenic transport of HCO3− ions via the NBC1 transporter, and the decreased effect of DIDS in the nominal absence of bicarbonate further supports this. Additional studies support both mRNA expression and NBC1 protein expression (western blotting) in chick PT cultures and native tissue. Experiments are underway to test for an alternative apical transporter.

Kinetic study of bicarbonate reabsorption in proximal tubule of the rat.

Nitric oxide (NO) stimulates proximal tubule (PT) Na + and fluid reabsorption. Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthase (NOS) but its effects on tubular function are unknown. ADMA is metabolized by dimethylarginine dimethylaminohydrolase (DDAH) whose type 1 isoform is predominant in the PT. SNIPs of DDAH-1 predicted the rate of decline of renal function in patients with chronic kidney disease (CKD). Therefore, we tested the hypothesis that DDAH-1 metabolizes ADMA in the proximal tubule and thereby enhances PT fluid reabsorption (J v ). J v was measured in anesthetized rats by direct in vivo microperfusion and recollection of artificial tubular fluid (ATF) in S2 segments of the PT isolated between oil blocks. Addition of a selective inhibitor of DDAH-1, L-257 (10 -4 M) to ATF did not effect J v when compared to addition of vehicle (3.40.6 vs 3.10.3 nl/min/mm). However, L-257 administered as a bolus intravenous injection (60mg/kg) 2hrs before microperfusion of the PT with L-257 significantly reduced J v to 1.80.2 nl/min/mm, (P&lt;0.05) and significantly enhanced urine flow rate (2.1±0.3 vs 9.0±1.8 μl/min; P&lt;0.05). Microperfusion of ADMA (10 -4 M) or L-NAME (10 -4 M) into the PT both reduced J v significantly to 2.00.2 and 1.80.2 nl/min/mm, respectively (P&lt;0.05). Rats pretreated with a rapid intravenous injection of siRNA targeted to DDAH-1 that reduced its renal mRNA expression significantly also had reduced J v (3.1±0.2 vs 2.2±6.2 nl/min/mm; P&lt;0.05) and had a further reduction in J v with 10 -4 M ADMA added to ATF to 1.4±0.1 nl/min/mm; (P&lt;0.005). In conclusion, PT fluid reabsorption is regulated by ADMA and its tubular metabolism by DDAH-1. The two hour delay in the effects of a bolus injection of L-257 to diminish J v indicated the time required after inhibition of DDAH-1 for accumulation of sufficient tubular ADMA to impair PT reabsorption. Thus, L-257 is a novel regulator of PT function and is a proximal tubule diuretic. SNIPs that alter DDAH-1 in the PT may change renal function and contribute to CKD progression.