NADPH oxidase 4-derived superoxide mediates flow-stimulated NKCC2 activity in thick ascending limbs

Luminal flow augments Na+ reabsorption in the thick ascending limb more than can be explained by increased ion delivery. This segment reabsorbs 30% of the filtered load of Na+, playing a key role in its homeostasis. Whether flow elevations enhance Na+-K+-2Cl− cotransporter (NKCC2) activity and the second messenger involved are unknown. We hypothesized that raising luminal flow augments NKCC2 activity by enhancing superoxide ([Formula: see text]) production by NADPH oxidase 4 (NOX4). NKCC2 activity was measured in thick ascending limbs perfused at either 5 or 20 nl/min with and without inhibitors of [Formula: see text] production. Raising luminal flow from 5 to 20 nl/min enhanced NKCC2 activity from 4.8 ± 0.9 to 6.3 ± 1.2 arbitrary fluorescent units (AFU)/s. Maintaining flow at 5 nl/min did not alter NKCC2 activity. The superoxide dismutase mimetic manganese (III) tetrakis (4-benzoic acid) porphyrin chloride blunted NKCC2 activity from 3.5 ± 0.4 to 2.5 ± 0.2 AFU/s when flow was 20 nl/min but not 5 nl/min. When flow was 20 nl/min, NKCC2 activity showed no change with time. The selective NOX1/4 inhibitor GKT-137831 blunted NKCC2 activity when thick ascending limbs were perfused at 20 nl/min from 7.2 ± 1.1 to 4.5 ± 0.8 AFU/s but not at 5 nl/min. The inhibitor also prevented luminal flow from elevating [Formula: see text] production. Allopurinol, a xanthine oxidase inhibitor, had no effect on NKCC2 activity when flow was 20 nl/min. Tetanus toxin prevents flow-induced stimulation of NKCC2 activity. We conclude that elevations in luminal flow enhance NaCl reabsorption in thick ascending limbs by stimulating NKCC2 via NOX4 activation and increased [Formula: see text]. NKCC2 activation is primarily the result of insertion of new transporters in the membrane.

Adaptive changes in GFR, tubular morphology, and transport in subtotal nephrectomized kidneys: modeling and analysis

Removal of renal mass stimulates anatomical and functional adaptations in the surviving nephrons, including elevations in single-nephron glomerular filtration rate (SNGFR) and tubular hypertrophy. A goal of this study is to assess the extent to which the concomitant increases in filtered load and tubular transport capacity preserve homeostasis of water and salt. To accomplish that goal, we developed computational models to simulate solute transport and metabolism along nephron populations in a uninephrectomized (UNX) rat and a 5/6-nephrectomized (5/6-NX) rat. Model simulations indicate that nephrectomy-induced SNGFR increase and tubular hypertrophy go a long way to normalize excretion, but alone are insufficient to fully maintain salt balance. We then identified increases in the protein density of Na+-K+-ATPase, Na+-K+-2Cl− cotransporter, Na+-Cl− cotransporter, and epithelial Na+ channel, such that the UNX and 5/6-NX models predict urine flow and urinary Na+ and K+ excretions that are similar to sham levels. The models predict that, in the UNX and 5/6-NX kidneys, fractional water and salt reabsorption is similar to sham along the initial nephron segments (i.e., from the proximal tubule to the distal convoluted tubule), with a need to further reduce Na+ reabsorption and increase K+ secretion primarily along the connecting tubules and collecting ducts to achieve balance. Additionally, the models predict that, given the substantially elevated filtered and thus transport load among each of the surviving nephrons, oxygen consumption per nephron segment in a UNX or 5/6-NX kidney increases substantially. But due to the reduced nephron population, whole animal renal oxygen consumption is lower. The efficiency of tubular Na+ transport in the UNX and 5/6-NX kidneys is predicted to be similar to sham.

Fluid reabsorption in proximal convoluted tubules of mice with gene deletions of claudin-2 and/or aquaporin1

Deletions of claudin-2 (Cldn2) and aquaporin1 (AQP1) reduce proximal fluid reabsorption (PFR) by about 30% and 50%, respectively. Experiments were done to replicate these observations and to determine in AQP1/claudin-2 double knockout mice (DKO) if the effects of deletions of these established water pores are additive. PFR was determined in inactin/ketamine-anesthetized mice by free-flow micropuncture using single-nephron I125-iothalamate (io) clearance. Animal means of PFR [% of glomerular filtration rate (GFR)] derived from TF/Piothalamate ratios in 12 mice in each of four groups [wild type (WT), Cldn2−/−, AQP1−/−, and DKO) were 45.8 ± 0.85 (51 tubules), 35.4 ± 1 (54 tubules; P < 0.01 vs. WT), 36.8 ± 1 (63 tubules; P < 0.05 vs. WT), and 33.9 ± 1.4 (69 tubules; P < 0.01 vs. WT). Kidney and single-nephron GFRs (SNGFR) were significantly reduced in all mutant strains. The direct relationship between PFR and SNGFR was maintained in mutant mice, but the slope of this relationship was reduced in the absence of Cldn2 and/or AQP1. Transtubular osmotic pressure differences were not different between WT and Cldn2−/− mice, but markedly increased in DKO. In conclusion, the deletion of Cldn2, AQP1, or of both Cldn2 and AQP1 reduces PFR by 22.7%, 19.6%, and 26%, respectively. Our data are consistent with an up to 25% paracellular contribution to PFR. The reduced osmotic water permeability caused by absence of AQP1 augments luminal hypotonicity. Aided by a fall in filtered load, the capacity of non-AQP1-dependent transcellular reabsorption is sufficient to maintain PFR without AQP1 and claudin-2 at 75% of control.

Characterization of the Mechanisms Involved in the Increased Renal Elimination of Bromosulfophthalein During Cholestasis: Involvement of Oatp1

The kidneys and liver are the major routes for organic anion elimination. We have recently shown that acute obstructive jaundice is associated with increased systemic and renal elimination of two organic anions, p-aminohippurate and furosemide, principally excreted through urine. This study examined probable adaptive mechanisms involved in renal elimination of bromosulfophthalein (BSP), a prototypical organic anion principally excreted in bile, in rats with acute obstructive jaundice. Male Wistar rats underwent bile duct ligation (BDL rats). Pair-fed sham-operated rats served as controls. BSP renal clearance was performed by conventional techniques. Renal organic anion-transporting polypeptide 1 (Oatp1) expression was evaluated by immunoblotting and IHC. Excreted, filtered, and secreted loads of BSP were all higher in BDL rats compared with Sham rats. The higher BSP filtered load resulted from the increase in plasma BSP concentration in BDL rats, because glomerular filtration rate showed no difference with the Sham group. The increase in the secreted load might be explained by the higher expression of Oatp1 observed in apical membranes from kidneys of BDL animals. This likely adaptation to hepatic injury, specifically in biliary components elimination, might explain, at least in part, the huge increase in BSP renal excretion observed in this experimental model. (J Histochem Cytochem 57:449–456, 2009)

Evidence that postprandial reduction of renal calcium reabsorption mediates hypercalciuria of patients with calcium nephrolithiasis

Idiopathic hypercalciuria (IH) is common among calcium stone formers (IHSF). The increased urinary calcium arises from increased intestinal absorption of calcium, but it is unclear whether increased filtered load or decreased renal tubular reabsorption of calcium is the main mechanism for the increased renal excretion. To explore this question, 10 IHSF and 7 normal subjects (N) were studied for 1 day. Urine and blood samples were collected at 30- to 60-min intervals while subjects were fasting and after they ate three meals providing known amounts of calcium, phosphorus, sodium, protein, and calories. Fasting and fed, ultrafiltrable calcium levels, and filtered load of calcium did not differ between N and IHSF. Urine calcium rose with meals, and fractional reabsorption fell in all subjects, but the change was significantly higher in IHSF. The changes in calcium excretion were independent of sodium excretion. Serum parathyroid hormone levels did not differ between N and IHSF, and they could not account for the greater fall in calcium reabsorption in IHSF. Serum magnesium and phosphorus levels in IHSF were below N throughout the day, and tubule phosphate reabsorption was lower in IHSF than N after meals. The primary mechanism by which kidneys ferry absorbed calcium into the urine after meals is via reduced tubule calcium reabsorption, and IHSF differ from N in the magnitude of the response. Parathyroid hormone is not likely to be a sufficient explanation for this difference.

Model of albumin reabsorption in the proximal tubule

Normally, the small amount of albumin which passes through the glomerular capillary wall is almost completely reabsorbed in the proximal tubule, via an endocytic mechanism, but the reabsorptive process can be overwhelmed if the filtered load of albumin is too large. To examine the factors that control the fractional reabsorption of albumin ( f), we developed a mathematical model which assumes saturable endocytosis kinetics with a maximum reabsorptive capacity, V max, and which includes the effects of flow and diffusion in the lumen. Limitations in albumin transport from the bulk tubule fluid to the endocytic sites at the bases of the microvilli had only a modest (8%) effect on the value of V max needed to fit micropuncture data on tubule albumin concentrations in rats. For moderate changes in filtered load, there was much greater sensitivity of f to SNGFR than to the albumin concentration of the filtrate ( C0). A 50% increase in SNGFR was predicted to cause four- to fivefold increases in albumin excretion in rats or humans. For large increases in C0, as might result from defects in glomerular sieving, there was a threshold at which the reabsorptive process became saturated and f fell sharply. That threshold corresponded to sieving coefficients of 10−3 to 10−2, the higher values occurring at reduced SNGFR. The predictions of the present model contrast with those of one proposed recently by Smithies ( 32 ), which does not include the effects of tubule flow rate.

Inhibition of the formation of EETs and 20-HETE with 1-aminobenzotriazole attenuates pressure natriuresis

This study examined the effects of chronic blockade of the renal formation of epoxyeicosatrienoic acids and 20-hydroxyeicosatetraenoic acid with 1-aminobenzotriazole (ABT; 50 mg·kg−1· day−1 ip for 5 days) on pressure natriuresis and the inhibitory effects of elevations in renal perfusion pressure (RPP) on Na+-K+-ATPase activity and the distribution of the sodium/hydrogen exchanger (NHE)-3 in the proximal tubule of rats. In control rats ( n = 15), sodium excretion rose from 2.3 ± 0.4 to 19.4 ± 1.8 μeq·min−1·g kidney weight−1 when RPP was increased from 114 ± 1 to 156 ± 2 mmHg. Fractional excretion of lithium rose from 28 ± 3 to 43 ± 3% of the filtered load. Chronic treatment of the rats with ABT for 5 days ( n = 8) blunted the natriuretic response to elevations in RPP by 75% and attenuated the increase in fractional excretion of lithium by 45%. In vehicle-treated rats, renal Na+-K+-ATPase activity fell from 31 ± 5 to 19 ± 2 μmol Pi·mg protein−1·h−1 and NHE-3 protein was internalized from the brush border of the proximal tubule after an elevation in RPP. In contrast, Na+-K+-ATPase activity and the distribution of NHE-3 protein remained unaltered in rats treated with ABT. These results suggest that cytochrome P-450 metabolites of arachidonic acid contribute to pressure natriuresis by inhibiting Na+-K+-ATPase activity and promoting internalization of NHE-3 protein from the brush border of the proximal tubule.

Mechanisms used to dispose of progressively increasing alkali load in rats

Our objective was to describe the process of alkali disposal in rats. Balance studies were performed while incremental loads of alkali were given to rats fed a low-alkali diet or their usual alkaline ash diet. Control groups received equimolar NaCl or KCl. Virtually all of the alkali was eliminated within 24 h when the dose exceeded 750 μmol. The most sensitive response to alkali input was a decline in the excretion of NH[Formula: see text]. The next level of response was to increase the excretion of unmeasured anions; this rise was quantitatively the most important process in eliminating alkali. The maximum excretion of citrate was ∼70% of its filtered load. An even higher alkali load augmented the excretion of 2-oxoglutarate to >400% of its filtered load. Only with the largest alkali load did bicarbonaturia become quantitatively important. We conclude that renal mechanisms eliminate alkali while minimizing bicarbonaturia. This provides a way of limiting changes in urine pH without sacrificing acid-base balance, a process that might lessen the risk of kidney stone formation.

Regulatory responses to an orald-glutamate load: formation ofd-pyrrolidone carboxylic acid in humans

Previously published studies have shown d-glutamate to be the most potent natural inhibitor of glutathione synthesis known, yet how d-glutamate is handled in humans is unknown. Therefore, we administered an oral d-glutamate load to four healthy volunteers and monitored the plasma d-glutamate concentration and excretion over a 3-h postload period. Compared with time controls, the plasma d-glutamate concentration increased 10-fold in the 1st h and then reached a plateau over the remaining time course. In contrast, plasma d-pyrrolidone carboxylic acid increased progressively throughout the 3-h time course to a level 10-fold higher than the d-glutamate plasma concentration. Excretion of d-glutamate progressively increased despite a constant filtered d-glutamate load rising from only 5 to 95% of the filtered amount. Excretion ofd-pyrrolidone carboxylic acid increased with the rise in filtered load without significant reabsorption. The amount ofd-pyrrolidone carboxylic acid excreted over the 3-h time course was 10 times the amount excreted as d-glutamate and accounted for almost 20% of the administered d-glutamate. These findings indicate that plasma d-glutamate concentration is tightly regulated through two mechanisms: 1) the transport into cells and metabolic conversion tod-pyrrolidone carboxylic acid and excretion, and 2) the enhancement of d-glutamate clearance by the kidneys.

Interstitial water and solute recovery by inner medullary vasa recta

A recent model of volume and solute microvascular exchange in the renal medulla was extended by simulating the deposition of NaCl, urea, and water into the medullary interstitium from the loops of Henle and collecting ducts with generation rates that undergo spatial variation within the inner medullary interstitium. To build an exponential osmolality gradient in the inner medulla, as suggested by Koepsell et al. (H. Koepsell, W. E. A. P. Nicholson, W. Kriz, and H. J. Höhling. Pflügers Arch. 350: 167–184, 1974), the ratio of the interstitial area-weighted generation rate of small solutes to that of water must increase along the corticomedullary axis. We satisfied this condition either by holding the area-weighted generation rate of water constant while increasing that of NaCl and urea or by reducing the input rate of water with medullary depth. The latter case, in particular, yielded higher solute concentrations at the papillary tip. Assuming that the fraction of the filtered load recovered by inner medullary vasa recta for water, NaCl, and urea is 1%, 1%, and 40%, respectively, papillary tip osmolality is 1,470 mosmol/kgH2O when urea generation and NaCl generation per unit volume of interstitium increase exponentially and linearly, respectively. The inner medullary osmolar gradient also increases further when 1) medullary blood flow is reduced, 2) hydraulic conductivity of descending vasa recta (DVR) is lowered, and 3) vasa recta permeability to NaCl and urea is maximized. The coupling between water and small solute transport, resulting from aquaporin-1-mediated transcellular flux in DVR, also enhances tip osmolality.