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The human kidney is composed of many cell types that vary in their abundance and distribution from normal to diseased organ. As these cell types perform unique and essential functions, it is important to confidently label each within a single tissue to accurately assess tissue architecture and microenvironments. Towards this goal, we demonstrate the use of co-detection by indexing (CODEX) multiplexed immunofluorescence for visualizing 23 antigens within the human kidney. Using CODEX, many of the major cell types and substructures, such as collecting ducts, glomeruli, and thick ascending limb, were visualized within a single tissue section. Of these antibodies, 19 were conjugated in-house, demonstrating the flexibility and utility of this approach for studying the human kidney using custom and commercially available antibodies. We performed a pilot study that compared both fresh frozen and formalin-fixed paraffin-embedded healthy non-neoplastic and diabetic nephropathy kidney tissues. The largest cellular differences between the two groups was observed in cells labeled with aquaporin 1, cytokeratin 7, and α-smooth muscle actin. Thus, our data show the power of CODEX multiplexed immunofluorescence for surveying the cellular diversity of the human kidney and the potential for applications within pathology, histology, and building anatomical atlases.

Peritoneal dialysis involves diffusive and convective transports and osmosis through the highly vascularized peritoneal membrane. The capillary endothelium offers the rate-limiting hindrance for solute and water transport. It can be functionally described in terms of a three-pore model including transcellular, ultrasmall pores responsible for free-water transport during crystalloid osmosis. Several lines of evidence have demonstrated that the water channel aquaporin-1 (AQP1) corresponds to the ultrasmall pore located in endothelial cells. Studies in Aqp1 mice have shown that deletion of AQP1 is reflected by a 50% decrease in ultrafiltration and a disappearance of the sodium sieving. Haploinsufficiency in AQP1 is also reflected by a significant attenuation of water transport. Conversely, studies in a rat model and in PD patients have shown that the induction of AQP1 in peritoneal capillaries by corticosteroids is reflected by increased water transport and ultrafiltration, without affecting the osmotic gradient and small-solute transport. Recent data have demonstrated that a novel agonist of AQP1, predicted to stabilize the open-state conformation of the channel, modulates water transport and improves ultrafiltration. Whether increasing the expression of AQP1 or gating the already existing channels would be clinically useful in PD patients remains to be investigated.

Excessive intraperitoneal absorption of glucose during peritoneal dialysis has both local cytotoxic and systemic metabolic effects. Here we evaluate peritoneal dialysis solutions containing L-carnitine, an osmotically active compound that induces fluid flow across the peritoneum. In rats, L-carnitine in the peritoneal cavity had a dose-dependent osmotic effect similar to glucose. Analogous ultrafiltration and small solute transport characteristics were found for dialysates containing 3.86% glucose, equimolar L-carnitine, or combinations of both osmotic agents in mice. About half of the ultrafiltration generated by L-carnitine reflected facilitated water transport by aquaporin-1 (AQP1) water channels of endothelial cells. Nocturnal exchanges with 1.5% glucose and 0.25% L-carnitine in four patients receiving continuous ambulatory peritoneal dialysis were well tolerated and associated with higher net ultrafiltration than that achieved with 2.5% glucose solutions, despite the lower osmolarity of the carnitine-containing solution. Addition of L-carnitine to endothelial cells in culture increased the expression of AQP1, significantly improved viability, and prevented glucose-induced apoptosis. In a standard toxicity test, the addition of L-carnitine to peritoneal dialysis solution improved the viability of L929 fibroblasts. Thus, our studies support the use of L-carnitine as an alternative osmotic agent in peritoneal dialysis.

EPO and α-MSH prevent ischemia/reperfusion-induced down-regulation of AQPs and sodium transporters in rat kidney. Background Ischemia-induced acute renal failure (ARF) is known to be associated with significant impairment of urinary concentrating ability and down-regulation of renal aquaporins (AQPs) and sodium transporters in rats. We tested whether treatment with erythropoietin (EPO) or α-melanocyte-stimulating hormone (α-MSH) in combination with EPO reduces the renal ischemia/reperfusion (I/R) injury and prevents the down-regulation of renal AQPs and major sodium transporters. Methods I/R-induced ARF was established in rats by 40-minute temporary bilateral obstruction of renal arteries, and rats were kept in metabolic cages for urine measurements. After 2 or 4 days following EPO and/or α-MSH treatment, kidneys were removed to determine the expression levels of AQPs and sodium transporters by semiquantitative immunoblotting. Results Rats with ARF showed significant renal insufficiency, increased urine output, and high fractional excretion of urinary sodium. Consistent with this, immunoblotting and immunocytochemistry revealed that the kidney expression of AQPs (AQP-1, -2 and -3) and sodium transporters [Na,K-ATPase, rat type 1 bumetanide-sensitive Na-K-2Cl cotransporter (BSC-1), Na/H exchanger type 3 (NHE3), and thiazide-sensitive sodium chloride cotransporter (TSC)] in ARF rats was significantly decreased compared to sham-operated control rats. In contrast, EPO treatment at the time of ischemia of rats with ARF significantly prevented the ischemia-induced down-regulation of renal AQPs and sodium transporters and in parallel improved the urinary concentrating capability and renal sodium reabsorption. Importantly, similar effects were observed following the initiation of EPO or α-MSH treatment 4 hours after the onset of ischemia injury. Moreover, the combination of EPO with α-MSH potentiated the beneficial effects of single compound treatment. Conclusion EPO and/or α-MSH treatment significantly prevent I/R-induced injuries such as urinary-concentrating defects and down-regulation of renal AQPs and sodium transporters.

Aristolochic acid nephropathy and the peritoneum: Functional, structural, and molecular studies.BackgroundAristolochic acid nephropathy (AAN) is a rapidly progressive interstitial nephropathy linked to the exposure to aristolochic acid (AA) and characterized by extensive fibrosis and urothelial atypia. Although the fibrotic process has been documented in extrarenal tissues, the involvement of the peritoneum, as well as the efficacy of peritoneal dialysis in AAN patients, remain uncertain.MethodsThe structure of the peritoneal membrane and the expression of basic fibroblast growth factor (bFGF), collagen type III, endothelial nitric oxide synthase (eNOS), and aquaporin-1 (AQP1) were investigated in peritoneal biopsies from an index AAN patient, four other AAN patients, four regular peritoneal dialysis patients, and two controls. Similar methods were used to investigate a rabbit model of AAN after intraperitoneal exposure to high-dose AA. AA-DNA adducts were screened by 32P-postlabeling analysis.ResultsThe AAN patients had renal failure, renal fibrosis, and urothelial atypia. The peritoneum of AAN patients had a normal structure, lacked cellular atypia, and, in comparison with regular peritoneal dialysis patients and controls, did not show abnormal regulation of fibrotic and endothelial markers. Furthermore, specific AA-DNA adducts were not identified in the peritoneum of AAN patients. In contrast, AA-DNA adducts were detected in peritoneal and kidney tissues of all exposed rabbits, and one of them developed a malignant mesothelioma.ConclusionThese data demonstrate the lack of fibrotic and vascular alterations and the absence of cellular atypia in the peritoneum from AAN patients. Thus, peritoneal dialysis should not be discouraged in these patients. Nevertheless, studies in a rabbit model of high-dose AA exposure may suggest a potential risk of peritoneal malignancy.

Acute renal failure leads to dysregulation of lung salt and water channels. Background Renal ischemia/reperfusion (I/R) injury and the acute respiratory distress syndrome (ARDS) frequently coexist in the intensive care setting, and this combination is associated with a high mortality. Recent experimental data demonstrate that renal I/R injury leads to an increase in pulmonary vascular permeability, similar to that observed in ARDS. However, the effects of renal I/R injury on alveolar fluid clearance—of potential importance in the setting of increased permeability—are unknown. We investigated the effects of renal I/R injury on pulmonary epithelial sodium channel (ENaC), Na,K-ATPase and aquaporin expression as a first step in addressing this question. Methods Sprague Dawley rats were subjected to four protocols: ( 1 ) surgery for bilateral I/R injury, ( 2 ) sham surgery, ( 3 ) surgery for unilateral I/R injury, or ( 4 ) bilateral nephrectomy. Lung tissue was examined for Na channel, Na,K-ATPase, aquaporin-1, and aquaporin-5 expression. Northern and Western blots were performed. Results Renal I/R injury and bilateral nephrectomy both led to marked down-regulation of pulmonary ENaC, Na,K-ATPase and aquaporin-5 but not aquaporin-1 compared to sham surgery. These changes were not influenced by the animals' volume status. In contrast, unilateral I/R with an intact contralateral kidney did not lead to down-regulation of channel down-regulation. Conclusions Ischemic acute renal failure leads to down regulation of pulmonary ENaC, Na,K-ATPase and aquaporin-5, but not aquaporin-1. Since bilateral nephrectomy but not single kidney I/R injury also leads to lung changes, these changes are likely mediated by systemic effects of acute renal failure (ARF), such as "uremic toxins," rather than reperfusion products. These changes may modulate lung dysfunction, susceptibility to lung injury, or both.

Expression of renal aquaporins 1, 2, and 3 in a rat model of cisplatin-induced polyuria.BackgroundCisplatin (CP)-induced polyuria in rats is attributed to decreased medullary hypertonicity and/or an end-organ resistance to vasopressin. However, the roles of renal aquaporins (AQPs) have not yet been explored.MethodsMale Sprague-Dawley rats (230 to 245 g) received either a single injection of CP (5 mg/kg, N = 4) or saline (N = 4) intraperitoneally five days before sacrifice. Urine, blood, and kidney samples were analyzed.ResultsPlatinum accumulated in the cortex and outer medulla of CP-treated rats (39.05 ± 7.50 and 36.48 ± 12.44 μg/g vs. 2.52 ± 0.43 and 1.87 ± 0.84 μg/g dry tissue in controls, respectively). Histologically, tubular damage and decreased AQP1 immunolabeling were detected in the S3 segment of proximal tubules. CP treatment caused 4.4- and 4.8-fold increases, respectively, in blood urea nitrogen and urine volume, and a 4.4-fold decrease in urine osmolality. Immunoblots showed that AQP2 and AQP3 were significantly reduced to 33 ± 10% (P < 0.001) and 69 ± 11% (P < 0.05), respectively, in the inner medulla of CP-treated rats. Immunocytochemical analysis showed a decrease in AQP2 labeling in the inner medulla of CP-treated rats. Northern hybridization revealed a 33 ± 11% (P < 0.002) decrease in AQP2 mRNA expression in the inner medulla of CP-treated rats. AQP1 protein expression levels were modestly (67 ± 7%, P = 0.057) and significantly (53 ± 13%, P < 0.007) decreased in outer and inner medullae, respectively, of CP-treated rats.ConclusionsCP-induced polyuria in rats is associated with a significant decrease in the expression of collecting duct (AQP2 and AQP3) and proximal nephron and microvascular (AQP1) water channels in the inner medulla.

Dysregulation of renal aquaporins and Na-Cl cotransporter in Hepatic cirrhosis is associated with defective regula- CCl4-induced cirrhosis. tion of water balance. At initial stages of decompensated Background. Severe hepatic cirrhosis is associated with ab- cirrhosis, water retention by the kidney is initially isos- normal renal water retention. motic and is thought to be secondary to excessive stimu- Methods. Semiquantitative immunoblotting was employed lation of renal tubule salt absorption. At least part of to investigate the abundance of the major renal aquaporins (water channels) and sodium-dependent cotransporters in kid- this isosmotic water retention is thought to be associated neys from control rats and rats with cirrhosis secondary to with a marked increase in proximal tubule fluid absorp- chronic CCl4 inhalation. tion documented by micropuncture studies in animal Results. The cirrhotic rats had ascites and manifested a water models (1, 2) and lithium clearance measurements in excretion defect detected by a standard water-loading test. cirrhotic patients (3, 4). With more severe hepatic cirrho- The abundance of aquaporin-1 (the major aquaporin in the proximal tubule) was increased, an effect markedly accentu- sis, free water retention can occur in excess of NaCl ated in high-density membrane fractions prepared by differen- retention leading to dilution of the extracellular fluid. tial centrifugation. Differential centrifugation studies demon- Excess free water retention presumably involves modu- strated a redistribution of aquaporin-2 from high-density to lation of water transport beyond the proximal tubule. low-density membranes, compatible with increased trafficking Both phases of water retention are reproduced in animal of aquaporin-2 to the plasma membrane. The abundance of aquaporin-3, but not aquaporin-2, was increased in collecting models of hepatic cirrhosis, including a model of cirrhosis ducts of rats with CCl4-induced cirrhosis. The Na-K-2Cl co- induced by repetitive CCl4 inhalation (5). In the present transporter of the thick ascending limb showed no change in study, we use the CCl4-inhalation model of hepatic cir- abundance. However, the abundance of the thiazide-sensitive rhosis to investigate the molecular basis of the water Na-Cl cotransporter of the distal convoluted tubule was mark- excretion abnormalities seen in hepatic cirrhosis. edly suppressed in cirrhotic rats, possibly contributing to a defect in urinary dilution. Physiological studies in aquaporin knockout mice (6, 7) Conclusions. In this model of cirrhosis, the development of and studies of patients with mutations of the aquaporin-2 a defect in urinary dilution may be multifactorial, with contribu- water channel (8) have made it clear that the major tions from at least four abnormalities in transporter regulation: fraction of water transport across renal tubule epithelia (1) an increase in the renal abundance of aquaporin-1, (2 )a is mediated by aquaporins. Thus, the abundance of indi- cellular redistribution of aquaporin-2 in the collecting duct vidual aquaporins in the plasma membranes of renal compatible with trafficking to the plasma membrane without an increase in total cellular aquaporin-2, (3) an increase in the epithelial cells is believed to be the major determinant renal abundance of aquaporin-3, and (4) a decrease in the of epithelial water permeability. Aquaporins are ex- abundance of the thiazide-sensitive cotransporter of the distal pressed in three main sites in the kidney, namely, the convoluted tubule. proximal tubule, the descending limb of Henle's loop, and the collecting duct (9). Aquaporin abundance at these sites can be readily assessed by quantitative immu

Concentrating defect in experimental nephrotic syndrome: Altered expression of aquaporins and thick ascending limb Na + transporters. Background Several pathophysiological states associated with deranged water balance are associated with altered expression and/or intracellular distribution of aquaporin water channels. The possible role of dysregulation of thick ascending limb NaCl transporters, which are responsible for countercurrent multiplication in the kidney, has not been evaluated. Methods Semiquantitative immunoblotting and immunocytochemistry were carried out in the kidneys of rat with adriamycin-induced nephrotic syndrome and in vehicle-injected control rats. Results Preliminary studies confirmed the presence of a severe concentrating defect. Semiquantitative immunoblotting of outer medullary homogenates demonstrated a marked decrease in the abundance of three thick ascending limb Na + transporters in nephrotic rats, namely the bumetanide-sensitive Na-K-2Cl cotransporter (BSC-1), the type 3 Na/H exchanger (NHE-3), and the α 1 -subunit of the Na-K-ATPase. These results are predictive of a decrease in the NaCl transport capacity of the medullary thick ascending limb and therefore a decrease in countercurrent multiplication. Immunocytochemistry of outer medullary thin sections demonstrated broad (but highly variable) suppression of BSC-1 expression in the outer medullas of adriamycin-nephrotic rats. There was also a large decrease in outer medullary expression of two collecting duct water channels (aquaporin-2 and -3) and the major water channel of the thin descending limb of Henle's loop (aquaporin-1). Conclusion The concentrating defect in adriamycin-induced nephrotic syndrome in rats is a consequence of multiple defects in water and solute transporter expression, which would alter both the generation of medullary interstitial hypertonicity and osmotic equilibration in the collecting duct. Whether a similar widespread defect in transporter expression is present in idiopathic nephrotic syndrome in humans is, at this point, untested.

Renal aquaporins. Aquaporins (AQPs) are a newly recognized family of transmembrane proteins that function as molecular water channels. At least four aquaporins are expressed in the kidney where they mediate rapid water transport across water-permeable epithelia and play critical roles in urinary concentrating and diluting processes. AQP1 is constitutively expressed at extremely high levels in the proximal tubule and descending limb of Henle's loop. AQP2, -3 and -4 are expressed predominantly in the collecting duct system. AQP2 is the predominant water channel in the apical plasma membrane and AQP3 and -4 are found in the basolateral plasma membrane. Short-term regulation of collecting duct water permeability by vasopressin is largely a consequence of regulated trafficking of AQP2-containing vesicles to and from the apical plasma membrane.

Structure and function of kidney water channels. There is now firm evidence that water transporting proteins are expressed in renal and extrarenal tissues. In the kidney, proximal-type (CHIP28) and collecting duct (WCH-CD) water channels have been identified. We have cloned three kidney cDNAs with homology to the water channel (aquaporin) family, including a mercurial-insensitive water channel (MIWC), and a glycerol-transporting protein (GLIP) in collecting duct basolateral membrane. To elucidate water transporting mechanisms, a series of molecular and spectroscopic studies were carried out on purified CHIP28 protein and expressed chimeric and mutated CHIP28 cDNAs. The results indicate that CHIP28 transports water selectively, that CHIP28 monomers are assembled in membranes as tetramers, but that individual monomers function independently. Monomers contain multiple membrane-spanning helical domains. Based on these data and recent electron crystallography results, a model for water transport is proposed in which water moves through narrow pores located within individual CHIP28 monomers.

The article by Ni et al. solidifies the important role of aquaporin-1 in the process of fluid removal from anephric patients treated with peritoneal dialysis. The presence of the water-only channel in the subperitoneal endothelia provides the mechanism for solute-free ultrafiltrate observed early in dialysis and accounts for approximately half of all the filtration observed in dialysis.

Impaired aquaporin and urea transporter expression in rats with adriamycin-induced nephrotic syndrome. Nephrotic syndrome is associated with abnormal regulation of renal water excretion. To investigate the role of collecting duct water channels and solute transporters in this process, we have carried out semiquantitative immunoblotting of kidney tissues from rats with adriamycin-induced nephrotic syndrome. These experiments demonstrated that adriamycin-induced nephrotic syndrome is associated with marked decreases in expression of aquaporin-2, aquaporin-3, aquaporin-4, and the vasopressin-regulated urea transporter in renal inner medulla, indicative of a suppression of the capacity for water and urea absorption by the inner medullary collecting duct. In contrast, expression of the α 1 -subunit of the Na,K-ATPase in the inner medulla was unaltered. Light and electron microscopy of perfusion-fixed kidneys demonstrated that the collecting ducts are morphologically normal and unobstructed. Inner medullary expression of the descending limb water channel, aquaporin-1, was not significantly altered, pointing to a selective effect on the collecting duct. Aquaporin-2 and aquaporin-3 expression was also markedly diminished in the renal cortex, indicating that the effect is not limited to the inner medullary collecting duct. Differential centrifugation studies and immunocytochemistry in inner medullary thin sections demonstrated increased targeting of aquaporin-2 to the plasma membrane, consistent with the expected short-term action of vasopressin on aquaporin-2 trafficking. The extensive down-regulation of aquaporin and urea transporter expression may represent an appropriate renal response to the extracellular volume expansion observed in nephrotic syndrome, but may occur at the expense of decreased urinary concentrating and diluting capacity.

We thank Dr Liakopoulos and co-workers1 for their comment about the interpretation of sodium sieving in the aquaporin-1 (AQP1)-knockout (Aqp1-/-) mice. Sodium sieving is a clinically useful, but indirect parameter to assess free-water transport across the peritoneal membrane. Our data show a complete lack of sodium sieving in the Aqp1-/- mice, and intermediate values in the heterozygous Aqp1-/+ mice vs wild-type littermates.2 The correction of sodium sieving for the diffusion of sodium from the circulation, based on the mass transfer area coefficient of urate or creatinine, may indeed be important in clinical situations associated with an increased transport of small solutes.3 However, this correction has not been validated in rodent models and is unlikely to have significant effect on the absence of sodium sieving observed in Aqp1-/- mice, as the transport of small solutes, the plasma sodium (at 0 and 30 min), and the osmotic gradient (dialysate/plasma osmolality at 30 min) are unchanged vs wild-type littermates.2 Furthermore, our computed simulations of sodium profiles and volume curves over the first 60 min of the dwell were very close to the measured data in AQP1 mice, confirming the total lack of sodium sieving in Aqp1-/- mice. Our conclusions about the essential role of AQP1 in water permeability during peritoneal dialysis are based on (i) determination of initial ultrafiltration rate, a direct indicator of solute-free water transport; (ii) measurement of net ultrafiltration across the membrane; and (iii) analysis of the dose–effect observed in heterozygous Aqp1-/+ mice.2 Dr Liakopoulos also mentions preliminary data showing that inhibition of AQP1 by HgCl2 in isolated parietal sheep peritoneum is associated with modifications of the transmesothelial resistance, suggesting an interaction between AQP1 and other solute transporters. However, such data should be interpreted with caution because the use of Ussing chambers on leaky membranes such as the peritoneum is prone to many artifacts and because the application of HgCl2in vitro is associated with toxic and nonspecific effects including increased membrane leakiness.4 Also, the fact that sodium channels are upregulated in the renal tubular cells of Aqp1-/- mice, probably reflecting the severe concentrating defect,5 should not be generalized. The transport of water and solutes across the peritoneum is completely passive, and only challenged when an osmotic gradient is created in the peritoneal cavity. This is different from the situation in kidney tubules, in which solute transport occurs by active or secondary active processes.

The longstanding puzzle of membrane water-permeability was advanced by discovery of a new class of proteins known as the "aquaporins" (AQPs). First identified in red blood cells, AQP1 was shown to function as a water channel when expressed in Xenopus oocytes or when pure AQP1 protein was reconstituted into synthetic membranes. Analysis of the primary sequence revealed that the two halves of the AQP1 polypeptide are tandem repeats; site directed mutagenesis studies indicate that the repeats may fold into an obversely symmetric structure which resembles an hourglass. Electron crystallography elucidated the tetrameric organization of AQP1, and functional studies suggest that each tetramer contains multiple functionally independent aqueous pores. AQP1 is abundant in the apical and basolateral membranes of renal proximal tubules and descending thin limbs, and is also present in multiple extra renal tissues. AQP2 is expressed only in the principal cells of renal collecting duct where it is the predominant vasopressin (ADH, antidiuretic hormone) regulated water channel. AQP2 is localized in the apical membrane and in intracellular vesicles which are targeted to the apical plasma membranes when stimulated by ADH. Humans with mutations in genes encoding AQP1 and AQP2 exhibit contrasting clinical phenotypes. AQP3 resides in the basolateral membranes of renal collecting duct principal cells providing an exit pathway for water; AQP4 is abundant in brain where it may function as the hypothalamic osmoreceptor responsible for secretion of ADH. Continued analysis of the aquaporins is providing detailed molecular insight into the fundamental physiological problems of water balance and disorders of water balance.

The ability to concentrate the urine was an essential step in the adaptation of animals to life in a terrestrial environment. Only mammals and birds produce a urine which is hypertonic to plasma. A concentrated urine is formed in the kidney collecting duct in response to vasopressin by the massive reabsorption of water from luminal fluid. The most prominent feature of water transport in the collecting duct is the rapid and large change in water permeability induced by vasopressin. In isolated perfused rabbit collecting duct segments, for example, the lag time between the addition of vasopressin and the appearance of an increase of water permeability is less than 30 seconds [1], and more than 10-fold increases in water permeability have been reported [2, 3]. The mechanisms responsible for this drastic change in water permeability have been studied extensively over the past three decades. Current models, as reviewed by Jo and Harris in this issue, hold that the vasopressin-induced changes in water permeability are due to the insertion and retrieval of “water channel” proteins to and from the apical membrane of collecting duct cells. There is now abundant functional and morphological evidence supporting the presence of water channels in the apical membrane of collecting duct. Following the discovery of AQP-CHIP as the first identified water channel protein [4, 5], which is present in many water transporting tissues including the kidney proximal tubules and thin descending limb of Henle [6–8], we succeeded in cloning another water channel, AQP–CD, which is expressed in collecting ducts [9]. Accumulating evidence indicates that AQP–CD is the vasopressin-regulated water channel of collecting duct. Very recently we have also cloned an additional water channel protein, named AQP3, which is expressed at the basolateral membrane of collecting ducts of the kidney [10]. This review will focus mainly on these collecting duct specific water channels.

Ischemic acute renal failure (ARF) is the most common form of ARF in the adult population. The molecular mechanisms of tubular regeneration after ischemic renal injury remain largely unknown. An understanding of the mechanisms that lead to renal cell proliferation and regeneration will be necessary for the exploration of novel therapeutic strategies for the treatment of ARF. It has been suggested that regeneration processes may recapitulate developmental processes in order to restore organ or tissue function. The adult tubular epithelial cells have a potent ability of regenerate after cellular damage. We examined functional role of two developmental genes, Wnt-4 and Ets-1, in renal tubular regeneration in ARF. The Wnt-β-catenin pathway plays key roles in embryogenesis. Wnt-4 is known to be expressed in the mesonephric duct in the embryonic development. To clarify the significance of the Wnt-4-β-catenin pathway in ARF, we used a rat ARF model in vivo and LLC-PK1 cells as an in vitro model. After clamping left rat renal artery for 1 hour, we examined whole kidney homogenate and total RNA extracted at 3, 6, 12, 24, 48, and 72 hours after reperfusion by Western blot analysis and real-time reverse transcription-polymerase chain reaction (RT-PCR). Wnt-4 mRNA and protein expression were strongly increased at 3 to 12 hours and 6 to 24 hours after ischemia, respectively. In immunohistologic examination, Wnt-4 was expressed in the proximal tubules and coexpressed with aquaporin 1 and proliferating cell nuclear antigen (PCNA). Cyclin D1 and cyclin A were expressed at 12 to 48 hours after reperfusion. Furthermore, overexpression of Wnt-4 and β-catenin promoted the cell cycle and increased the promoter activity and protein expression of cyclin D1 and cyclin A in LLC-PK1 cells. These data suggest that the Wnt-4-β-catenin pathway plays a key role in the cell cycle progression of renal tubules in ARF. The Ets family of transcription factors is defined by a conserved DNA-binding Ets domain that forms a winged helix-turn-helix structure motif. The Ets family is involved in a diverse array of biologic functions, including cellular growth, migration, and differentiation. To clarify the significance of Ets-1 in ARF, we used a rat ARF model in vivo and LLC-PK1 cells as an in vitro model. Ets-1 mRNA and protein expression were strongly increased at 3 to 12 hours and 6 to 24 hours after the ischemia, respectively. In the immunohistologic examination, Ets-1 was expressed in the proximal tubules and coexpressed with PCNA. Furthermore, overexpression of Ets-1 promoted the cell cycle and increased the promoter activity and protein expression of cyclin D1 in LLC-PK1 cells. Ets-1 promoter activity increased between 3 hours and 6 hours in hypoxia, and hypoxia also induced changes in the Ets-1 protein level in LLC-PK1 cells. Taken together, these data suggest that Ets-1 plays a key role in the cell cycle progression of renal tubules in ARF. Our data suggest that Wnt-4-β-catenin and Ets-1 pathways regulate the transcription of cyclin D1 and control the regeneration of renal tubules in ARF. These developmental genes may play key roles in dedifferentiation and regeneration of the renal tubular cells after ARF.

"Avian-type" renal medullary tubule organization causes immaturity of urine-concentrating ability in neonates. Background While neonatal kidneys are not powerful in concentrating urine, they already dilute urine as efficiently as adult kidneys. To elucidate the basis for this paradoxical immaturity in urine-concentrating ability, we investigated the function of Henle's loop and collecting ducts (IMCDs) in the inner medulla of neonatal rat kidneys. Methods Analyses of individual renal tubules in the inner medulla of neonatal and adult rat kidneys were performed by measuring mRNA expression of membrane transporters, transepithelial voltages, and isotopic water and ion fluxes. Immunofluorescent identification of the rCCC2 and rCLC-K1 using polyclonal antibodies was also performed in neonatal and adult kidney slices. Results On day 1, the transepithelial voltages (V Ts ) in the thin ascending limbs (tALs) and IMCDs were 14.6 ± 1.1mV ( N = 27) and -42.7 ± 6.1mV ( N = 14), respectively. The V Ts in the thin descending limbs (tDLs) were zero on day 1. The V Ts in the tALs were strongly inhibited by luminal bumetanide or basolateral ouabain, suggesting the presence of a NaCl reabsorption mechanism similar to that in the thick ascending limb (TAL). The diffusional voltage (V D ) of the tAL due to transepithelial NaCl gradient was almost insensitive to a chloride channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB). The V Ts in the IMCDs were strongly inhibited by luminal amiloride. On day 1, both the tDL and tAL were impermeable to water, indicating the water impermeability of the entire loop. Diffusional water permeability (P dw ) and urea permeabilities (P urea ) in the IMCDs indicated virtual impermeability to water and urea on day 1. Stimulation by vasopressin (1nmol/L) revealed that only P dw was sensitive to vasopressin by day 14. A partial isoosmolar replacement of luminal urea by NaCl evoked negligible water flux across the neonatal IMCDs, indicating the absence of urea-dependent volume flux in the neonatal IMCD. These transport characteristics in each neonatal tubule are similar to those in quail kidneys. Identification of mRNAs and immunofluorescent studies for specific transporters, including rAQP-1, rCCC2, rCLC-K1, rENaC β subunit, rAQP-2, and rUT-A1, supported these findings. Conclusion We hypothesize that the renal medullary tubule organization of neonatal rats shares a tremendous similarity with avian renal medulla. The qualitative changes in the organization of medullary tubules may be primarily responsible for the immature urine-concentrating ability in mammalian neonates.

The water channel aquaporin-1 (AQP1) is considered as the molecular counterpart of the ultrasmall pore predicted by the three-pore model of fluid transport across the peritoneal membrane. However, the definitive proof of the implication of AQP1 in solute-free water transport, sodium sieving, and ultrafiltration (UF) during peritoneal dialysis (PD) is lacking, and the effects of its deletion on the structure of the membrane are unknown. Using real-time reverse transcriptase-polymerase chain reaction and immunogold electron microscopy, we showed that AQP1 is the most abundant member of the AQP gene family expressed in the mouse peritoneum, and the only one located in the capillary endothelium. Transport studies during a 2-h dwell demonstrated that, in comparison with Aqp1(+/+) littermates, Aqp1(-/-) mice had no sodium sieving; an approximately 70% decrease in the initial, solute-free UF; and an approximately 50% decrease in cumulative UF. These modifications occurred despite unchanged osmotic gradient and transport of small solutes in the Aqp1(-/-) mice. Heterozygous Aqp1(+/-) mice showed intermediate values in sodium sieving and initial UF, whereas cumulative UF was similar to Aqp1(+/+) mice. The deletion of AQP1 had no effect on the expression of other AQPs and on the density, structure, or diameter of peritoneal capillaries. These data provide direct evidence for the role of AQP1 during PD. They validate essential predictions of the three-pore model: (i) the ultrasmall pores account for the sodium sieving, and (ii) they mediate 50% of UF during a hypertonic dwell.

Resistance of mTAL Na + -dependent transporters and collecting duct aquaporins to dehydration in 7-month-old rats. Background Aging is associated with a defect in urinary concentration in both human and experimental animals. The purpose of these studies was to examine the urinary concentrating ability, the expression of kidney water channels [aquaporins (AQP1 to AQP3)], and medullary thick ascending limb (mTAL) Na + -dependent transporters in old but not senescent versus young animals in response to water deprivation. Methods Two-month-old and 7-month-old rats were placed in metabolic cages and deprived of water for 72hours. Kidney tissues were isolated and examined for the expression of AQP1 to AQP3 and mTAL, peptide-derived polyclonal antibody specific to kidney apical Na + -K + -2Cl - cotransporter (BSC1), Na + /H + exchanger isoform 3 (NHE3), and Na + pump using semiquantitative immunoblotting and Northern hybridization. Results After 72hours of water deprivation, urine osmolality increased from 1269 to 3830 mOsm/kg H 2 O in 2-month-old rats, but only from 1027 to 2588 mOsm/kg H 2 O in 7-month-old rats. In response to water deprivation, AQP2 and AQP3 expression increased significantly in the cortex and medulla of 2-month-old rats but remained unchanged in the medulla or slightly increase in the cortex of 7-month-old animals. AQP1 expression was not altered by dehydration in both groups. The protein abundance of mTAL BSC1, NHE3, and Na + pump increased significantly in young but remained unchanged in 7-month-old rats subjected to water deprivation. Conclusion Age-related decrease in urinary concentrating ability is an early event, developed before the onset of senescence. This defect results from reduced responsiveness of cortical AQP2 and AQP3 and a blunted response of medullary AQP2 and mTAL BSC1, NHE3, and Na + pump to dehydration in aging kidneys.

Aquaporin-1 (AQP1) water channels are expressed widely in microvascular endothelia outside of the central nervous system, including renal vasa recta and tumor microvessels, as well as in non-vascular endothelia in pleura, peritoneum, cornea, and lymphatics. In kidney, AQP1-facilitated water transport in outer medullary descending vasa recta is likely an important component of the urinary concentrating mechanism. However, in most vascular endothelia outside of kidney, it remains uncertain whether AQP1 expression and high water permeability are physiologically important. AQP1 in non-vascular endothelia at the inner corneal surface is involved in the maintenance of corneal transparency. Recently, a new role of AQP1 in endothelial cell migration was discovered in analyzing the cause of defective tumor angiogenesis in AQP1-deficient mice. AQP1 facilitates endothelial cell migration by a mechanism that may involve facilitated water transport across cell protrusions (lamellipodia). AQP1 inhibitors may thus have aquaretic and antiangiogenic activity.

Functional and molecular characterization of a peritoneal dialysis model in the C57BL/6J mouse.BackgroundAnimal models are important for understanding the physiology and pathophysiology of peritoneal transport during peritoneal dialysis (PD). Mechanistic investigations of rat and rabbit models of PD are mostly based on intervention studies using pharmacologic agents or blocking antibodies. These models may be limited by the time-course, lack of specificity, or side effects of such interventions. Genetically modified mice could provide an attractive alternative to the above models. In this study, we have characterized PD parameters and tested the effect of gender and dialysate volume and/or osmolality in the C57BL/6J mouse.MethodsMice were submitted to a 2-hour peritoneal equilibration test in order to obtain permeability parameters. The expression of the water channel aquaporin-1 (AQP1) and endothelial NO synthase (eNOS) was investigated at the protein (immunoblotting, immunostaining) and mRNA [real-time reverse-transcription-polymerase chain reaction (RT-PCR)] levels. The potential effect of gender on these parameters was also studied.ResultsExposure of mice to 2 mL of 3.86% glucose dialysate yielded equilibration curves for urea and glucose, a sodium sieving, and a net ultrafiltration (UF) that were remarkably similar to those obtained in rats. The increase in dialysate volume (from 2 mL to 3 mL and 6 mL) resulted in a higher ultrafiltration and, for the highest volume, an increase in the diffusive mass transport coefficient (MTAC) for urea. The increase in dialysate glucose concentration (from 1.36% to 3.86% and 7%) resulted in increased sodium sieving and higher UF, whereas the MTAC for urea was unchanged. In comparison with males, females had a similar peritoneal transport rate for small solutes but a significantly lower sodium sieving, reflecting a lower AQP1 mRNA and protein expression in the peritoneum.ConclusionThese data demonstrate the structural and functional similarity between mouse and rat models of PD, and further emphasize the relevance of mouse models to understand PD in humans. They also suggest that gender may influence water transport and AQP1 expression in the peritoneum.

Classic laboratory studies performed in the 1950's and 1960's focused on the structures and biochemical actions of multiple neurohypophysial peptides that possessed profound effects on the overall water balance of many vertebrates [reviewed in 1]. These data emphasized the importance of antidiuretic hormones (ADH) in the evolution of vertebrates and provided a framework for our current understanding of their roles in renal function. In a similar fashion, the recent discovery of the family of aquaporin water channel proteins has supplied new tools to examine the molecular aspects of ADH-elicited water transport in epithelial physiology. Data concerning the structure and distribution of aquaporin water channels have been further advanced by recent research defining several of the most basic aspects of hormonally regulated transport in many cell types. Together, these findings have provided many new insights into the apical membrane events set in motion by the binding of ADH to the basolateral membrane of epithelial cells. The purpose of this review is to highlight the role of the ADH-elicited water permeability response in the terrestrial adaptation of amphibians and their evolutionary offspring, mammals including humans. To accomplish this, we have divided the review into three sections. The first section summarizes published data from studies in comparative physiology involving the evolution and distribution of the ADH-elicited water permeability response in various amphibian species. The second section provides an overview of the vesicle mediated water permeability response in toad bladder and mammals. In this selective review, we have emphasized several common features present in ADH-mediated insertion and removal of water channel containing vesicles and the insulin-induced vesicular translocation of a facilitative glucose transport protein. The third section re-examines two important questions about the evolution of the ADH-elicited water permeability response in light of recent data reviewed in the prior section. Taken together, these data illustrate that future research in this area will provide new insights into the origins of the ADH-elicited water permeability response through use of probes specific for various aquaporin water channel proteins.

Aquaporin gene delivery to kidney. Background Several aquaporin- (AQP) type water channels are expressed in kidney tubules and microvessels, including AQP1 in proximal tubule, thin descending limb of Henle and vasa recta, AQP2 in collecting duct apical membrane, and AQP3 and AQP4 in collecting duct basolateral membrane. Mice deficient in these aquaporins have distinct phenotypic abnormalities. AQP1 null mice are polyuria and unable to generate a concentrated urine after water deprivation. AQP2-T126M mutant mice and AQP3 null mice manifest nephrogenic diabetes insipidus (NDI) with severe polyuria, whereas AQP4 null mice have only a mild defect in maximal urinary concentrating ability. We reasoned that these mice could serve as useful models for gene replacement because of their predictable and unambiguous phenotypes. Methods In an initial feasibility study, an adenovirus directing the expression of AQP1 was introduced into AQP1 null mice by intravenous infusion. Results At 1 week after adenovirus infusion, AQP1 was seen in many proximal tubules and microvessels. Compared with untreated null mice, the treated mice were able to partially concentrate their urine and lost less weight after water deprivation. However, AQP1 transgene expression and functional correction were lost over 3–5 weeks. Conclusion Although there remain many technical problems to overcome, aquaporin gene replacement has potential applications in hereditary and acquired NDI, and in the transient modulation of renal fluid conservation.

Comparative ontogeny, processing, and segmental distribution of the renal chloride channel, ClC-5.BackgroundThe renal chloride channel ClC-5, which is responsible for Dent's disease, is coexpressed with the vacuolar H+-ATPase in proximal tubules (PT) and α-type intercalated cells (IC) of the mature kidney. Neonatal cases of Dent's disease suggest that ClC-5 distribution must be acquired before birth. However, the ontogeny of ClC-5, and its processing and segmental distribution with respect to related proteins during nephrogenesis remain unknown.MethodsImmunoblotting, real-time polymerase chain reaction (RT-PCR), immunostaining, and deglycosylation studies were used to investigate the expression, distribution, and maturation of ClC-5 during mouse and human nephrogenesis, in comparison with H+-ATPase, type II carbonic anhydrase (CAII), and aquaporin-1 (AQP1).ResultsAn early induction (E13.5-E14.5) of ClC-5 was observed in mouse kidney, with persistence at high levels through late nephrogenesis. This pattern contrasted with the progressive expression of H+-ATPase and AQP1, and the postnatal upregulation of CAII. Immunostaining showed expression of ClC-5 in ureteric buds and, from E14.5, its location in developing PT. From E15.5, ClC-5 codistributed with H+-ATPase in PT cells and α-type IC. In the human kidney, ClC-5 was detected from 12 gestation weeks; its distribution was similar to that observed in mouse, except for a later detection in IC. Although mouse and human ClC-5 proteins are glycosylated, biochemical differences between fetal and adult proteins were observed in both species.ConclusionThe segmental expression of ClC-5 and H+-ATPase is essentially achieved during early nephrogenesis, in parallel with the onset of glomerular filtration. These data give insight into PT and IC maturation, and explain early phenotypic variants of Dent's disease.

Immunocytochemistry performed on paraffin or cryosections is often hampered by poor morphology. Epoxy sections, in contrast, generally retain well-preserved tissue architecture. Immunocytochemistry, however, on epoxy-embedded sections is difficult due in part to the plastic itself and to the fixation conditions. Here, we present a technique for visualization of membrane proteins by immunocytochemistry on epoxy sections of kidneys fixed with glutaraldehyde without or with osmium post-fixation. Semithin sections were obtained from Epon 812-embedded mouse and rat kidney blocks. Before immunoperoxidase or immunofluorescence labeling, the sections were etched with the epoxy solvent, methanolic potassium hydroxide, followed by antigen retrieval using microwave heating. The sections were then treated with the primary antibody followed by secondary antibodies as usual. The distribution and expression patterns of a variety of membrane proteins, such as aquaporin (AQP)-1, AQP-2, and megalin, were identical to those observed by traditional immunocytochemical procedures on paraffin or cryosections. The advantages of our novel method include not only enhanced morphological quality but also the feasibility for investigators to visualize antigens of interest using archival specimens in Epon blocks.

To the Editor: We read with great interest the article by Ni et al.1 regarding the pivotal role of aquaporin-1 (AQP1) in water removal and ultrafiltration during peritoneal dialysis. Their important findings validate the essential predictions of the three-pore model theory.