Thick Ascending Limb Research Articles

ROMK inhibitor actions in the nephron probed with diuretics.

Diuretics acting on specific nephron segments to inhibit Na+ reabsorption have been used clinically for decades; however, drug interactions, tolerance, and derangements in serum K+ complicate their use to achieve target blood pressure. ROMK is an attractive diuretic target, in part, because its inhibition is postulated to indirectly inhibit the bumetanide-sensitive Na+-K+-2Cl- cotransporter (NKCC2) and the amiloride- and benzamil-sensitive epithelial Na+ channel (ENaC). The development of small-molecule ROMK inhibitors has created opportunities for exploring the physiological responses to ROMK inhibition. The present study evaluated how inhibition of ROMK alone or in combination with NKCC2, ENaC, or the hydrochlorothiazide (HCTZ) target NCC alter fluid and electrolyte transport in the nephron. The ROMK inhibitor VU591 failed to induce diuresis when administered orally to rats. However, another ROMK inhibitor, termed compound A, induced a robust natriuretic diuresis without kaliuresis. Compound A produced additive effects on urine output and Na+ excretion when combined with HCTZ, amiloride, or benzamil, but not when coadministered with bumetanide, suggesting that the major diuretic target site is the thick ascending limb (TAL). Interestingly, compound A inhibited the kaliuretic response induced by bumetanide and HCTZ, an effect we attribute to inhibition of ROMK-mediated K+ secretion in the TAL and CD. Compound A had no effect on heterologously expressed flow-sensitive large-conductance Ca2+-activated K+ channels (Slo1/β1). In conclusion, compound A represents an important new pharmacological tool for investigating the renal consequences of ROMK inhibition and therapeutic potential of ROMK as a diuretic target.

Real-time monitoring of NKCC2 endocytosis by total internal reflection fluorescence (TIRF) microscopy.

The apical Na-K-2Cl cotransporter (NKCC2) mediates NaCl reabsorption by the thick ascending limb (TAL). The amount of NKCC2 at the apical membrane of TAL cells is determined by exocytic delivery, recycling, and endocytosis. Surface biotinylation allows measurement of NKCC2 endocytosis, but it has low time resolution and does not allow imaging of the dynamic process of endocytosis. We hypothesized that total internal reflection fluorescence (TIRF) microscopy imaging of labeled NKCC2 would allow monitoring of NKCC2 endocytosis in polarized Madin-Darby canine kidney (MDCK) and TAL cells. Thus we generated a NKCC2 construct containing a biotin acceptor domain (BAD) sequence between the transmembrane domains 5 and 6. Once expressed in polarized MDCK or TAL cells, surface NKCC2 was specifically biotinylated by exogenous biotin ligase (BirA). We also demonstrate that expression of a secretory form of BirA in TAL cells induces metabolic biotinylation of NKCC2. Labeling biotinylated surface NKCC2 with fluorescent streptavidin showed that most apical NKCC2 was located within small discrete domains or clusters referred to as "puncta" on the TIRF field. NKCC2 puncta were observed to disappear from the TIRF field, indicating an endocytic event which led to a decrease in the number of surface puncta at a rate of 1.18 ± 0.16%/min in MDCK cells, and a rate 1.09 ± 0.08%/min in TAL cells (n = 5). Treating cells with a cholesterol-chelating agent (methyl-β-cyclodextrin) completely blocked NKCC2 endocytosis. We conclude that TIRF microscopy of labeled NKCC2 allows the dynamic imaging of individual endocytic events at the apical membrane of TAL cells.

ADH-induced Stimulation of Na-activated K Channels is Responsible for Maintaining Basolateral K Conductance of the Thick Ascending Limb (TAL) in EAST/Sesame Syndrome

ADH-induced Stimulation of Na-activated K Channels is Responsible for Maintaining Basolateral K Conductance of the Thick Ascending Limb (TAL) in EAST/Sesame Syndrome

Alternative channels for urea in the inner medulla of the rat kidney.

The ascending thin limbs (ATLs) and lower descending thin limbs (DTLs) of Henle's loop in the inner medulla of the rat are highly permeable to urea, and yet no urea transporters have been identified in these sections. We hypothesized that novel, yet-unidentified transporters in these tubule segments could explain the high urea permeability. cDNAs encoding for Na(+)-glucose transporter 1a (SGLT1a), Na(+)-glucose transporter 1 (NaGLT1), urea transporter (UT)-A2c, and UT-A2d were isolated and cloned from the Munich-Wistar rat inner medulla. SGLT1a is a novel NH2-terminal truncated variant of SGLT1. NaGLT1 is a Na(+)-dependent glucose transporter primarily located in the proximal tubules and not previously described in the thin limbs. UT-A2c and UT-A2d are novel variants of UT-A2. UT-A2c is truncated at the COOH terminus, and UT-A2d has one exon skipped. When rats underwent water restriction for 72 h, mRNA levels of SGLT1a increased in ATLs, NaGLT1 levels increased in both ATLs and DTLs, and UT-A2c increased in ATLs. [(14)C]urea uptake assays performed on Xenopus oocytes heterologously expressing these proteins revealed that despite having structural differences from their full-length versions, SGLT1a, UT-A2c, and UT-A2d enhanced urea uptake. NaGLT1 also facilitated urea uptake. Uptakes were Na(+) independent and inhibitable by phloretin and/or phloridzin. Our data indicate that there are several alternative channels for urea in the rat inner medulla that could potentially contribute to the high urea permeabilities in thin limb segments.

Distribution of epidermal growth factor receptor, bone morphogenetic protein-2, and p53 in kidney of healthy newborn, adult and old highland-plateau yaks.

Kidney has long been thought to be a body's largest organ of elimination for maintaining acid-base balance. In recent years, the research on kidneys has mainly focused on the structural characteristics of the kidney of single age group animals. In this paper we used histological and immunohistochemical methods to observe and compare the structure characteristics of yak kidney and the expression of epidermal growth factor receptor (EGFR), bone morphogenetic protein-2 (BMP-2) and p53 in the kidney of yaks of three different age groups. The aim of the study was to investigate histological characteristics of age-related chan- ges in the kidney of yak and expression and localisation of kidney-related factors. Fifteen healthy male and female yaks from highland plateaus (three groups: newborn, adult and old yaks, n = 5 per group). Histo- logical methods were used to compare the relevant characteristics of the kidney of yaks. The immunohistochemistry method was used to observe the expression and localisation of EGFR, BMP-2, and p53 of the kidney of different ages, and the optical density value was measured and analysed by using image analysis software. This is an overall observation of the kidney tissue section, which includes the surface of the renal capsule and the internal parenchyma. In the renal parenchyma, there are renal corpuscles, renal tubules. The internal substance included cortex and medulla, which were bounded by the arched artery. In the cortex, there were renal corpuscles, convoluted part of renal tubules (proximal convoluted tubule and distal convoluted tubule) and collecting tubules. The medulla included straight parts of renal tubules (proximal straight tubule and distal straight tubule), thin segments and collecting tubules. It was observed that the organisational structure of the kidney of yaks did not change with age, but the degree of development of the internal structure (glomeruli, renal tubules and collecting tubules) of the kidney changed with age. Immunohistochemical results demonstrated that EGFR and BMP-2-positive reaction in the newborn group was mainly distributed in the proximal tubule epithelial cells, and widely distributed in the adult and old groups. However, the p53-positive reaction was widely distributed in the newborn, adult and old groups. The results revealed that the kidney structure tended to be com- pleted with age, and the function of the kidney gradually improved. EGFR and BMP-2 had the effect of promoting kidney development. However, p53 had been widely distributed in the newborn kidney of the yaks. It is suggested that p53 had been involved in cell migration and metabolic differentiation and self-renewal in the new stage.

Rab-GAP TBC1D4 (AS160) is dispensable for the renal control of sodium and water homeostasis but regulates GLUT4 in mouse kidney.

The Rab GTPase-activating protein TBC1D4 (AS160) controls trafficking of the glucose transporter GLUT4 in adipocytes and skeletal muscle cells. TBC1D4 is also highly abundant in the renal distal tubule, although its role in this tubule is so far unknown. In vitro studies suggest that it is involved in the regulation of renal transporters and channels such as the epithelial sodium channel (ENaC), aquaporin-2 (AQP2), and the Na+-K+-ATPase. To assess the physiological role of TBC1D4 in the kidney, wild-type (TBC1D4+/+) and TBC1D4-deficient (TBC1D4-/-) mice were studied. Unexpectedly, neither under standard nor under challenging conditions (low Na+/high K+, water restriction) did TBC1D4-/- mice show any difference in urinary Na+ and K+ excretion, urine osmolarity, plasma ion and aldosterone levels, and blood pressure compared with TBC1D4+/+ mice. Also, immunoblotting did not reveal any change in the abundance of major renal sodium- and water-transporting proteins [Na-K-2Cl cotransporter (NKCC2) NKCC2, NaCl cotransporter (NCC), ENaC, AQP2, and the Na+-K+-ATPase]. However, the abundance of GLUT4, which colocalizes with TBC1D4 along the distal nephron of TBC1D4+/+ mice, was lower in whole kidney lysates of TBC1D4-/- mice than in TBC1D4+/+ mice. Likewise, primary thick ascending limb (TAL) cells isolated from TBC1D4-/- mice showed an increased basal glucose uptake and an abrogated insulin response compared with TAL cells from TBC1D4+/+ mice. Thus, TBC1D4 is dispensable for the regulation of renal Na+ and water transport, but may play a role for GLUT4-mediated basolateral glucose uptake in distal tubules. The latter may contribute to the known anaerobic glycolytic capacity of distal tubules during renal ischemia.

Abstract P103: Cellular Stretch Induces Higher Intracellular Calcium Increases in Dahl Salt-Sensitive Than Salt-Resistant Rat Thick Ascending Limbs: Role of Transient Receptor Potential Vanilloid 4

The thick ascending limb (TAL) reabsorbs 30 % of the filtered NaCl playing a pivotal role in the regulation of salt homeostasis. Abnormal reabsorption of NaCl in this segment causes salt-sensitive hypertension. We showed that flow-induced cellular stretch stimulates superoxide production in epithelial cells from TALs. Dahl salt-sensitive rat (DSS) TALs produce more superoxide than those from salt-resistant rats (DSR). Mechanical stimulation exerted by cellular stretch increases intracellular calcium (Cai) in several types of cells. We have shown that luminal flow-induced increases in Cai is mediated by Transient Receptor Potential Vanilloid (TRPV4). Therefore, we hypothesized that cellular stretch increases Cai in TALs from DSS rats more than those from DSR rats due to elevated TRPV4 activity. We measured Cai using the calcium-sensitive dye Fura-2 in isolated, perfused DSR and DSS TALs. Cellular stretch increased Cai by 243 ± 51 nM in DSS TALs (n=9; p<0.0008 vs. no stretch conditions) and by 124 ± 27 nM in DSR TALs (n=10; p<0.0005 vs no stretch conditions). Cellular stretch-induced Cai increases were significantly higher in the DSS group (p<0.05 vs DSR). When these animals were fed a high-salt diet (4% NaCl) similar responses were obtained. Cellular stretch increased Cai by 236 ± 58 nM in DSS TALs (n=7; p<0.004 vs no stretch conditions) and by 92 ± 15 nM in DSR TALs (n=6; p<0.0008 vs no stretch conditions). To study whether DSS and DSR TALs express different amounts of TRPV4 we performed Western blots. We found that TRPV4 protein expression was similar in TALs lysates from both strains fed either a control standard or a high-salt diet. We then tested the effects of transfecting DSS TALs with TRPV4-small hairpin (sh) RNA on stretch-induced Cai increases. Under these circumstances, the difference in stretch-induced Cai increases between DSS and DSR TALs was not significant (75 ± 15 nM in DSS vs 56 ± 28 nM vs DSR, n=4 for each group) . We conclude that DSS TALs are more sensitive to increases in cellular stretch than DSR TALs possibly due to enhanced TRPV4 activity.

Abstract 104: Targeting the Alms1 Gene in Rats Causes Hypertension, Obesity and Enhanced NKCC2 Trafficking

Enhanced NaCl reabsorption by the thick ascending limb (TAL) is associated with salt sensitive hypertension in rodents and humans. NaCl absorption by the TAL depends on the apical Na/K/2Cl cotransporter - NKCC2. NKCC2 activity is regulated in part by protein-protein interactions with its carboxyl terminus that controls its trafficking to the apical membrane. We hypothesized that the proteins binding to this region of NKCC2 may be involved in NKCC2 regulation and trafficking. To identify new TAL proteins that bind NKCC2, we performed a proteomics screen between the NKCC2 carboxyl terminus and TAL proteins. We identified ALMS1 (Alstrom syndrome 1) as a specific interacting partner for this NKCC2 region and confirmed that ALMS1 is expressed in TALs. Little is known about ALMS1 in renal function. Mutation of this gene causes severe metabolic syndrome in humans and is associated with hypertension. To study ALMS1 function we obtained ALMS1 knockout (KO) rats in collaboration with the rat genome editing consortium. We found that ALMS1 KO rats fed a normal salt diet have higher systolic blood pressure (151± 5 mmHg) compared to wild type littermates (125± 4 mmHg, p< 0.02). We also observed an increase in body weight in ALMS1 KO at all ages (WT: 344± 4 g vs KO: 429±11 g, at 11 weeks, p<0.05). ALMS1 KO rats showed large intra-abdominal and sub-cutaneous fat deposits. We then obtained TALs and measured surface and total NKCC2 expression. In TALs from ALMS1 KO, the percentage of total NKCC2 at the surface was higher compared to WT (27±4 vs 14±2 %, p<0.05, n=6). Total NKCC2 expression was not significantly different between ALMS1 KO and WT rats. We conclude that ALMS1 is important for blood pressure regulation. The mechanism for hypertension in ALMS1 KO is unclear but may involve an increase in NKCC2 trafficking to the apical membrane and activity. ALMS1 has been associated with poor kidney function, hypertension, and type 2 diabetes in humans. Understanding the function of ALMS1 could develop new avenues for research into these diseases.

Abstract 142: A Fructose-but Not a Glucose-enriched Diet, Induces a Salt-dependent Increase in Blood Pressure and Enhances NKCC2 Phosphorylation in Normal Rats

Human consumption of fructose as a sweetener has increased in the past 30 years. High fructose intake has been implicated in the development of hypertension, diabetes, and obesity. In the US, the upper 10th percentile of the population consumes up to 40% of their caloric intake from added sugars, in which fructose represents half of these. Fructose metabolism is strikingly different from that of glucose. Yet, the effect of a fructose or glucose-enriched diet in salt handling by the kidney, affecting blood pressure, and its interaction with high salt intake has been poorly studied. In genetic models of salt-sensitive hypertension, the activity of the Na + /K + /2Cl - cotransporter (NKCC2) in the thick ascending limb (TAL) is abnormally enhanced. We hypothesized that chronic fructose in drinking water induces a salt-dependent increase in blood pressure and stimulates NKCC2 during high salt intake in normal rats. Sprague-Dawley rats were given 20% fructose or 20% glucose in drinking water for 1 week after which a high salt (HS) diet (4% Na + in chow) was started for 3 weeks. When we measured systolic blood pressure (SBP) by tail cuff plethysmography in fructose-fed and glucose-fed rats on a HS diet, only the fructose-fed rats had an increased SBP from 120±10 to 132±6 mmHg on day 7 of HS (p<0.01). SBP continued to increase up to 144±18 mmHg after 3 weeks (p<0.01 vs glucose). Fructose or glucose alone did not increase SBP after 4 weeks. We then repeated the protocol using radiotelemetry to monitor the blood pressure (BP). In rats fed fructose, by day 5 of HS the SBP increased by 12±3 mmHg (p<0.02) and SBP remained elevated for 3 weeks (delta: 10±2.5 mmHg, n=3). In rats fed glucose, a HS diet did not significantly change SBP for 3 weeks (n=5). Moreover, NKCC2 activity in the TAL is enhanced by phosphorylation at Thr96, 101. We found that NKCC2 phosphorylation was higher in rats fed fructose plus HS (p<0.02) but not in rats fed glucose plus HS for 3 weeks (HS: 100, fructose+HS: 250±40%, glucose+HS: 95±10%). Therefore, we conclude that a high fructose (but not a glucose) diet in normal rats induces a salt-dependent increase in BP independently from caloric intake. Thus, the increase in BP may in part be due to the stimulation of NKCC2 phosphorylation in the TAL by fructose.

Vasopressin-induced stimulation of the Na+-activated K+ channels is responsible for maintaining the basolateral K+ conductance of the thick ascending limb (TAL) in EAST/SeSAME syndrome

The renal phenotype of EAST syndrome, a disease caused by the loss-of-function-mutations of Kcnj10 (Kir4.1), is a reminiscence of Gitelman's syndrome characterized by the defective function in the distal convoluted tubule (DCT). The aim of the present study is to test whether antidiuretic hormone (vasopressin)-induced stimulation of the Na+-activated 80–150pS K+ channel is responsible for compensating the lost function of Kcnj10 in the thick ascending limb (TAL) of subjects with EAST syndrome. Immunostaining and western blot showed that the expression of aquaporin 2 (AQP2) was significantly higher in Kcnj10−/− mice than those of WT littermates, suggesting that the disruption of Kcnj10 stimulates vasopressin response in the kidney. The role of vasopressin in stimulating the basolateral K+ conductance of the TAL was strongly indicated by the finding that the application of arginine-vasopressin (AVP) hyperpolarized the membrane in the TAL of Kcnj10−/− mice. Application of AVP significantly stimulated the 80–150pS K+ channel in the TAL and this effect was blocked by tolvaptan (V2 receptor antagonist) or by inhibiting PKA. Moreover, the water restriction for 24h significantly increased the probability of finding the 80–150pS K+ channel and the K+ channel open probability in the TAL. The application of a membrane permeable cAMP analog also mimicked the effect of AVP and activated this K+ channel, suggesting that cAMP–PKA pathway stimulates the 80–150pS K+ channels. The role of the basolateral K+ conductance in maintaining transcellular Cl− transport is further suggested by the finding that the inhibition of basolateral K+ channels significantly diminished the AVP-induced stimulation of the basolateral 10pS Cl− channels. We conclude that vasopressin stimulates the 80–150pS K+ channel in the TAL via a cAMP-dependent mechanism. The vasopressin-induced stimulation of K+ channels is responsible for compensating lost function of Kcnj10 thereby rescuing the basolateral K+ conductance which is essential for the transport function in the TAL.

Architecture of the human renal inner medulla and functional implications.

The architecture of the inner stripe of the outer medulla of the human kidney has long been known to exhibit distinctive configurations; however, inner medullary architecture remains poorly defined. Using immunohistochemistry with segment-specific antibodies for membrane fluid and solute transporters and other proteins, we identified a number of distinctive functional features of human inner medulla. In the outer inner medulla, aquaporin-1 (AQP1)-positive long-loop descending thin limbs (DTLs) lie alongside descending and ascending vasa recta (DVR, AVR) within vascular bundles. These vascular bundles are continuations of outer medullary vascular bundles. Bundles containing DTLs and vasa recta lie at the margins of coalescing collecting duct (CD) clusters, thereby forming two regions, the vascular bundle region and the CD cluster region. Although AQP1 and urea transporter UT-B are abundantly expressed in long-loop DTLs and DVR, respectively, their expression declines with depth below the outer medulla. Transcellular water and urea fluxes likely decline in these segments at progressively deeper levels. Smooth muscle myosin heavy chain protein is also expressed in DVR of the inner stripe and the upper inner medulla, but is sparsely expressed at deeper inner medullary levels. In rodent inner medulla, fenestrated capillaries abut CDs along their entire length, paralleling ascending thin limbs (ATLs), forming distinct compartments (interstitial nodal spaces; INSs); however, in humans this architecture rarely occurs. Thus INSs are relatively infrequent in the human inner medulla, unlike in the rodent where they are abundant. UT-B is expressed within the papillary epithelium of the lower inner medulla, indicating a transcellular pathway for urea across this epithelium.

Furosemide-induced urinary acidification is caused by pronounced H+ secretion in the thick ascending limb.

The loop diuretic furosemide inhibits NaCl reabsorption in the thick ascending limb (TAL). In addition, furosemide acidifies the urine, which is traditionally explained by increased Na+ loading to the distal tubule causing an activation of H+ secretion via H+-ATPase in α-intercalated cells. The inability to acidify urine in response to furosemide serves to diagnose distal renal tubular acidosis (dysfunction of α-intercalated cells). Since the TAL is important for acid/base regulation, we speculated that it is involved in furosemide-induced urinary acidification. Luminal furosemide (100 μM) caused major, stable, and reversible intracellular alkalization (7.27 ± 0.06 to 7.6 ± 0.04) in isolated perfused murine medullary TAL and pronounced H+ secretion. This H+ secretion was fully inhibited with luminal amiloride (1 mM) and the Na+/H+ exchanger (NHE)3-specific antagonist #4167 (1 μM). Moreover, furosemide triggered a substantial drop of intracellular Na+ concentration in the medullary TAL. These results suggest that the furosemide-induced H+ secretion is a consequence of a drop in intracellular Na+ concentration, increasing the driving force for NHE3. Intriguingly, in whole animal experiments, furosemide-induced urinary acidification and net acid excretion were markedly reduced by specific NHE3 inhibition. Furthermore, the furosemide-induced urinary acidification was partially preserved during epithelial Na+ channel inhibition with benzamil. These results provide new insights in the mechanism of furosemide-induced urinary acidification and emphasize the role of the TAL in renal acid/base handling.

Role of Prox1 in the Transforming Ascending Thin Limb of Henle's Loop during Mouse Kidney Development.

The homeobox transcription factor Prox1 is critical to the development of many embryonic organs and tissues, although current understanding of its expression in the developing renal medulla is limited. We examined the functional role of Prox1 during mouse kidney development with particular emphasis on the developing loop of Henle. Our data show that Prox1 is expressed in the transdifferentiating region from the NKCC2-positive thick ascending limb, into the CLC-K1-positive ascending thin limb of Henle’s loop beginning at embryonic day 18. From 1 to 14 days of age, Prox1-positive cells gradually disappeared from the papillary tip, and remained in the initial part of inner medulla after 21 days. In this transforming area, no Prox1 was observed in cells undergoing apoptosis but was expressed strongly in the remaining cells, which differentiated into ascending thin limb epithelial cells. In vitro and in vivo approaches showed that Prox1 expression increases where the osmolality is near optimal range, but decreases at below- or above-optimal ranges. Renal hypoosmolality induced by furosemide (NKCC2 inhibitor) inhibited Prox1 expression and delayed maturation of the ascending limb of Henle’s loop. Together, these studies suggest that Prox1 appears to be a critical stage specific regulator of specifying ascending thin limb cell fate and that its expression is regulated by osmolality.

Calcineurin and Sorting-Related Receptor with A-Type Repeats Interact to Regulate the Renal Na⁺-K⁺-2Cl⁻ Cotransporter.

The furosemide-sensitive Na(+)-K(+)-2Cl(-)-cotransporter (NKCC2) is crucial for NaCl reabsorption in kidney thick ascending limb (TAL) and drives the urine concentrating mechanism. NKCC2 activity is modulated by N-terminal phosphorylation and dephosphorylation. Serine-threonine kinases that activate NKCC2 have been identified, but less is known about phosphatases that deactivate NKCC2. Inhibition of calcineurin phosphatase has been shown to stimulate transport in the TAL and the distal convoluted tubule. Here, we identified NKCC2 as a target of the calcineurin Aβ isoform. Short-term cyclosporine administration in mice augmented the abundance of phospho-NKCC2, and treatment of isolated TAL with cyclosporine increased the chloride affinity and transport activity of NKCC2. Because sorting-related receptor with A-type repeats (SORLA) may affect NKCC2 phosphoregulation, we used SORLA-knockout mice to test whether SORLA is involved in calcineurin-dependent modulation of NKCC2. SORLA-deficient mice showed more calcineurin Aβ in the apical region of TAL cells and less NKCC2 phosphorylation and activity compared with littermate controls. In contrast, overexpression of SORLA in cultured cells reduced the abundance of endogenous calcineurin Aβ. Cyclosporine administration rapidly normalized the abundance of phospho-NKCC2 in SORLA-deficient mice, and a functional interaction between calcineurin Aβ and SORLA was further corroborated by binding assays in rat kidney extracts. In summary, we have shown that calcineurin Aβ and SORLA are key components in the phosphoregulation of NKCC2. These results may have clinical implications for immunosuppressive therapy using calcineurin inhibitors.

Tubular cross talk in acute kidney injury: a story of sense and sensibility.

The mammalian kidney is an organ composed of numerous functional units or nephrons. Beyond the filtering glomerulus of each nephron, various tubular segments with distinct populations of epithelial cells sequentially span the kidney from cortex to medulla. The highly organized folding of the tubules results in a spatial distribution that allows intimate contact between various tubular subsegments. This unique arrangement can promote a newly recognized type of horizontal epithelial-to-epithelial cross talk. In this review, we discuss the importance of this tubular cross talk in shaping the response of the kidney to acute injury in a sense and sensibility model. We propose that injury-resistant tubules such as S1 proximal segments and thick ascending limbs (TAL) can act as "sensors" and thus modulate the responsiveness or "sensibility" of the S2-S3 proximal segments to injury. We also discuss new findings that highlight the importance of tubular cross talk in regulating homeostasis and inflammation not only in the kidney, but also systemically.

Flow‐Stimulated NO Production is Altered in Thick Ascending Limbs of Dahl Salt‐Sensitive Rats Fed Either normal or High‐Salt Diets

Thick ascending limbs (TALs) of the loop of Henle reabsorb 30% of the filtered NaCl, regulating extracellular fluid volume and electrolyte excretion, thereby affecting blood pressure. NO produced by NO synthase 3 (NOS3) inhibits NaCl transport by TALs and luminal flow increases NO production. Reductions in TAL NO are associated with salt‐sensitive hypertension. In TALs of Sprague‐Dawley rats, dietary salt increases NOS3 expression by day 7, and animals don't become hypertensive. We hypothesized that Dahl salt‐sensitive rat (SS) TALs produce less NO in response to luminal flow than those from Dahl‐salt resistant rats (SR) due to altered NOS3 expression. We used SS or SR fed either normal or high‐salt diets for 7 days. We measured flow‐induced NO in isolated perfused TALs and performed Western blots of renal medullary lysates. On normal‐salt, flow‐induced NO was 32 ± 2 AFU/min in SR TALs and 12 ± 2 AFU/min in SS TALs (Δ 267 ± 63 %; p<0.03; n=6). In the presence of the superoxide (O2‐)scavenger TEMPOL, flow‐induced NO was 35 ± 5 AFU/min in SR TALs and 19 ± 2 AFU/min (p<0.05; n=9) in SS TALs, indicating that NO scavenging by O2‐ plays a minor role in this difference. NOS3 expression was not different between strains fed normal salt. When animals were given a high‐salt diet, flow‐induced NO was 44 ± 10 AFU/min (n = 11) in SR TALs and 9 ± 2 AFU/min (n=8) in SS TALs (Δ 389 ± 110 %; p<0.01). NOS3 expression was 69 ± 15 % (p<0.02; n=5) higher in TALs from SR compared to SS. We conclude that on a normal‐salt diet SR TALs produce more NO in response to luminal flow than those from SS, and that factors other than NOS3 expression or increased O2‐ are involved. On a high‐salt diet NO production is further affected in SS TALs by differences in NOS3 expression.

AVP‐mediated regulation of the paracellular permeability in the thick ascending limb (TAL)

The TAL drives an important part of the reabsorption of divalent cations. This occurs through the cation selective paracellular pathway. Arginine vasopressin (AVP) stimulates the transepithelial transport in the TAL. We wanted to study how AVP stimulation affects paracellular permeability in the TAL.TALs were mechanically dissected for microperfusion from 8‐10 weeks old mice who were kept for 4 days on water‐restricted (WR) (0.26ml/gBM/d) or water‐loaded (WL) (0.78ml/gBM/d) conditions, and from 5‐10 week old mice to be stimulated acutely with AVP.We measured transepithelial resistance and voltage (Rte, Vte), and calculated the equivalent short circuit current (Isc). From diffusion voltages, in presence of furosemide, we calculated the paracellular permeability (P) for Na+, Cl‐, Mg2+ and Ca2+. Measurements were performed at 37°C with continuous perfusion at a constant pressure.Freshly isolated TAL from the WR group (n=20) were transporting more actively compared to the WL group tubules (n=15). Vte and Isc were a 26% and a 48% higher, respectively, while Rte was 27% lower. Diffusion voltages were also increased in the WR group by 21%, indicating a 30% higher PNa/Cl, and a 27% higher PNa compared to WL.Acute TAL stimulation ex‐vivo for 12 minutes with AVP (n=6), in paired experiments, increased transepithelial transport. Isc and Vte increased by 23% and 25% respectively, while in the time control group Isc and Vte decreased by 28% and 37%. At the same time, AVP induced 20% more pronounced diffusion voltages compared to control, with a 25% increase in PNa/PCl.ConclusionWater restriction and AVP increase paracellular selectivity and permeability for cations in the TAL.

Nitric Oxide Decreases the Permselectivity of the Paracellular Pathway in Thick Ascending Limbs by Elevating cGMP Levels and Activating PKG

The thick ascending limb (TAL) reabsorbs 25‐30% of the filtered load of NaCl. About 70% is reabsorbed via the transcellular pathway and 30% is reabsorbed through the Na‐selective paracellular pathway. Nitric oxide (NO) inhibits net transepithelial and transcellular Na reabsorption, but its effects on the paracellular pathway are unknown. We hypothesized that NO decreases the permselectivity of the paracellular pathway for Na in TALs via cGMP and cGMP‐dependent protein kinase (PKG). To assess relative Na/Cl permeability ratios (PNa/PCl), we perfused TALs from Sprague Dawley rats and measured dilution potentials with and without NO donors (spermine NONOate: SPM, nitroglycerin: NTG) and dibutyryl cGMP (db‐cGMP). Dilution potentials were generated by reducing the bath NaCl concentration from 141 to 32 mM and PNa/PCl was calculated using the Goldman‐Hodgkin‐Katz equation. During the control period, the dilution potential was ‐11.1 ± 2.1 mV and PNa/PCl was 2.2 ± 0.4. After SPM (200 μM) treatment, they were ‐6.5 ± 1.6 mV (n = 9; p < 0.04) and 1.5 ± 0.2 (p < 0.03), respectively. NTG produced a similar effect on both dilution potential and PNa/PCl. Time controls showed no significant changes. Db‐cGMP changed the dilution potential from ‐13.4 ± 2.9 to ‐7.5 ± 1.8 mV (n = 6; p < 0.01). In the presence of PKG inhibition with KT5823 (4 μM), SPM had no significant effect on dilution potentials (KT5823 alone: ‐8.6 ± 0.1 mV vs KT5823 + SPM: ‐7.1 ± 0.1 mV, N.S.; n = 6). KT5823 alone had no effect on dilution potentials. Phosphodiesterase 2 inhibition with BAY607550 (10 μM) did not block the effect of NO. We conclude that NO reduces the permselectivity of the paracellular pathway in TALs via cGMP and PKG. Funded by NIH HL28982.

Calcineurin Inhibition Affects Na(K)Cl Cotransporter Activity in Kidney Distal Nephron

The calcineurin inhibitors (CNI) tacrolimus and cyclosporine A (CsA) are widely used for immunosuppression. However, side effects such as hypertension and electrolyte disorders commonly occur. Our previous data suggest that tacrolimus exerts local effects in kidney distal convoluted tubule (DCT), leading to activation of the Na+, Cl- cotransporter (NCC), salt retention and hypertension. This study aims to provide the molecular basis for the renal action of CsA. Calcineurin Aa and Ab isoforms were ubiquitously detected along mouse isolated nephron segments by immunoblotting and qPCR. Calcineurin Aa was preferentially colocalized with NCC in DCT subapical region, whereas calcineurin Ab was enriched together with Na+, K+, Cl- cotransporter (NKCC2) in the thick ascending limb (TAL). Immunophilins, mediating the inhibitory effects of CsA, were detected in both TAL and DCT of mice and rats using immunofluorescence and in situ hybridization. Acute as well as chronic administration of CsA in rats induced strong increases of NKCC2 and NCC phosphorylation without changes in their abundance, suggesting that activation of both cotransporters occurred at the posttranslational level. Analysis of the WNK-SPAK/OSR1 kinase cascade, controlling phosphorylation of the transporters, suggested enhanced activity in both distal segments. In sum, our data identify local components of calcineurin signalling in TAL and DCT which are likely involved in the activation of NKCC2 and NCC upon CNI. These results may have implications for antihypertensive therapy during immune suppression.

Calcineurin homologous protein 1 interferes with calcineurin‐dependent regulation of the renal Na‐K‐2Cl‐cotransporter

The Na‐K‐2Cl‐cotransporter (NKCC2) of kidney thick ascending limb (TAL) is crucial for renal salt handling. Its activity tightly regulated by phosphorylation. Our previous data suggested that calcineurin is involved in dephosphorylation and thus, deactivation of NKCC2. Further results suggested calcineurin activity may be modulated by members of the calcineurin homologous protein family (CHP). Among these, CHP1 has been implicated in the regulation of several membrane proteins including ion transporters. The role of CHP1 in calcineurin‐dependent regulation of NKCC2 activity has therefore been studied.CHP1 was co‐localized with NKCC2 in rat TAL by immunofluorescence. Both products co‐immunoprecipitated, suggesting their interaction. GST pull down assays with recombinant N‐terminal NKCC2 mutants, mimicking its (de)phosphorylation at functionally relevant residues (T96, T101, T114, and S126), suggested competitive interactions of CHP1 and calcineurin with phosphorylated NKCC2. Overexpression of CHP1 in cultured TAL cells substantially increased the phosphorylation of endogenous NKCC2 at baseline and upon low‐chloride stimulation. Acute stimulation of NKCC2 by administration of desmopressin in rats facilitated NKCC2 interaction with CHP1 while attenuating its interaction with calcineurin.In sum, our data provide evidence that CHP1 facilitates NKCC2 phosphorylation, likely by interfering with calcineurin function in TAL.