Macula Densa NOS1ß Modulates Renal Hemodynamics and Blood Pressure during Pregnancy: Role in Gestational Hypertension.

BACKGROUND: Regulation of renal hemodynamics and BP via tubuloglomerular feedback (TGF) may be an important adaptive mechanism during pregnancy. Because the ß-splice variant of nitric oxide synthase 1 (NOS1ß) in the macula densa is a primary modulator of TGF, we evaluated its role in normal pregnancy and gestational hypertension in a mouse model. We hypothesized that pregnancy upregulates NOS1ß in the macula densa, thus blunting TGF, allowing the GFR to increase and BP to decrease. METHODS: We used sophisticated techniques, including microperfusion of juxtaglomerular apparatus in vitro, micropuncture of renal tubules in vivo, clearance kinetics of plasma FITC-sinistrin, and radiotelemetry BP monitoring, to determine the effects of normal pregnancy or reduced uterine perfusion pressure (RUPP) on macula densa NOS1ß/NO levels, TGF responsiveness, GFR, and BP in wild-type and macula densa-specific NOS1 knockout (MD-NOS1KO) mice. RESULTS: Macula densa NOS1ß was upregulated during pregnancy, resulting in blunted TGF, increased GFR, and decreased BP. These pregnancy-induced changes in TGF and GFR were largely diminished, with a significant rise in BP, in MD-NOS1KO mice. In addition, RUPP resulted in a downregulation in macula densa NOS1ß, enhanced TGF, decreased GFR, and hypertension. The superimposition of RUPP into MD-NOS1KO mice only caused a modest further alteration in TGF and its associated changes in GFR and BP. Finally, in African green monkeys, renal cortical NOS1ß expression increased in normotensive pregnancies, but decreased in spontaneous gestational hypertensive pregnancies. CONCLUSIONS: Macula densa NOS1ß plays a critical role in the control of renal hemodynamics and BP during pregnancy.

NaCl cotransporter activity and Mg2+ handling by the distal convoluted tubule.

The genetic disease Gitelman syndrome, knockout mice, and pharmacological blockade with thiazide diuretics have revealed that reduced activity of the NaCl cotransporter (NCC) promotes renal Mg2+ wasting. NCC is expressed along the distal convoluted tubule (DCT), and its activity determines Mg2+ entry into DCT cells through transient receptor potential channel subfamily M member 6 (TRPM6). Several other genetic forms of hypomagnesemia lower the drive for Mg2+ entry by inhibiting activity of basolateral Na+-K+-ATPase, and reduced NCC activity may do the same. Lower intracellular Mg2+ may promote further Mg2+ loss by directly decreasing activity of Na+-K+-ATPase. Lower intracellular Mg2+ may also lower Na+-K+-ATPase indirectly by downregulating NCC. Lower NCC activity also induces atrophy of DCT cells, decreasing the available number of TRPM6 channels. Conversely, a mouse model with increased NCC activity was recently shown to display normal Mg2+ handling. Moreover, recent studies have identified calcineurin and uromodulin (UMOD) as regulators of both NCC and Mg2+ handling by the DCT. Calcineurin inhibitors paradoxically cause hypomagnesemia in a state of NCC activation, but this may be related to direct effects on TRPM6 gene expression. In Umod-/- mice, the cause of hypomagnesemia may be partly due to both decreased NCC expression and lower TRPM6 expression on the cell surface. This mini-review discusses these new findings and the possible role of altered Na+ flux through NCC and ultimately Na+-K+-ATPase in Mg2+ reabsorption by the DCT.

Macula Densa SGLT1-NOS1-Tubuloglomerular Feedback Pathway, a New Mechanism for Glomerular Hyperfiltration during Hyperglycemia.

BACKGROUND: Glomerular hyperfiltration is common in early diabetes and is considered a risk factor for later diabetic nephropathy. We propose that sodium-glucose cotransporter 1 (SGLT1) senses increases in luminal glucose at the macula densa, enhancing generation of neuronal nitric oxide synthase 1 (NOS1)-dependent nitric oxide (NO) in the macula densa and blunting the tubuloglomerular feedback (TGF) response, thereby promoting the rise in GFR. METHODS: We used microperfusion, micropuncture, and renal clearance of FITC-inulin to examine the effects of tubular glucose on NO generation at the macula densa, TGF, and GFR in wild-type and macula densa-specific NOS1 knockout mice. RESULTS: Acute intravenous injection of glucose induced hyperglycemia and glucosuria with increased GFR in mice. We found that tubular glucose blunts the TGF response in vivo and in vitro and stimulates NO generation at the macula densa. We also showed that SGLT1 is expressed at the macula densa; in the presence of tubular glucose, SGLT1 inhibits TGF and NO generation, but this action is blocked when the SGLT1 inhibitor KGA-2727 is present. In addition, we demonstrated that glucose increases NOS1 expression and NOS1 phosphorylation at Ser1417 in mouse renal cortex and cultured human kidney tissue. In macula densa-specific NOS1 knockout mice, glucose had no effect on NO generation, TGF, and GFR. CONCLUSIONS: We identified a novel mechanism of acute hyperglycemia-induced hyperfiltration wherein increases in luminal glucose at the macula densa upregulate the expression and activity of NOS1 via SGLT1, blunting the TGF response and promoting glomerular hyperfiltration.

Renal Na-handling defect associated with PER1-dependent nondipping hypertension in male mice.

Many physiological functions have a circadian rhythm, including blood pressure (BP). BP is highest during the active phase, whereas during the rest period, BP dips 10-20%. Patients that do not experience this dip at night are termed "nondippers." Nondipping hypertension is associated with increased risk of cardiovascular disease. The mechanisms underlying nondipping hypertension are not understood. Without the circadian clock gene Per1, C57BL/6J mice develop nondipping hypertension on a high-salt diet plus mineralocorticoid treatment (HS/DOCP). Our laboratory has shown that PER1 regulates expression of several genes related to sodium (Na) transport in the kidney, including epithelial Na channel (ENaC) and Na chloride cotransporter (NCC). Urinary Na excretion also demonstrates a circadian pattern with a peak during active periods. We hypothesized that PER1 contributes to circadian regulation of BP via a renal Na-handling-dependent mechanism. Na-handling genes from the distal nephron were inappropriately regulated in KO mice on HS/DOCP. Additionally, the night/day ratio of Na urinary excretion by Per1 KO mice is decreased compared with WT (4 × vs. 7×, P < 0.001, n = 6 per group). Distal nephron-specific Per1 KO mice also show an inappropriate increase in expression of Na transporter genes αENaC and NCC. These results support the hypothesis that PER1 mediates control of circadian BP rhythms via the regulation of distal nephron Na transport genes. These findings have implications for the understanding of the etiology of nondipping hypertension and the subsequent development of novel therapies for this dangerous pathophysiological condition.

Deletion of claudin-10 rescues claudin-16-deficient mice from hypomagnesemia and hypercalciuria.

The tight junction proteins claudin-10 and -16 are crucial for the paracellular reabsorption of cations along the thick ascending limb of Henle's loop in the kidney. In patients, mutations in CLDN16 cause familial hypomagnesemia with hypercalciuria and nephrocalcinosis, while mutations in CLDN10 impair kidney function. Mice lacking claudin-16 display magnesium and calcium wasting, whereas absence of claudin-10 results in hypermagnesemia and interstitial nephrocalcinosis. In order to study the functional interdependence of claudin-10 and -16 we generated double-deficient mice. These mice had normal serum magnesium and urinary excretion of magnesium and calcium and showed polyuria and sodium retention at the expense of increased renal potassium excretion, but no nephrocalcinosis. Isolated thick ascending limb tubules of double mutants displayed a complete loss of paracellular cation selectivity and functionality. Mice lacking both claudin-10 and -16 in the thick ascending limb recruited downstream compensatory mechanisms and showed hypertrophic distal convoluted tubules with changes in gene expression and phosphorylation of ion transporters in this segment, presumably triggered by the mild decrease in serum potassium. Thus, severe individual phenotypes in claudin-10 and claudin-16 knockout mice are corrected by the additional deletion of the other claudin.

Potassium intake modulates the thiazide-sensitive sodium-chloride cotransporter (NCC) activity via the Kir4.1 potassium channel.

Kir4.1 in the distal convoluted tubule plays a key role in sensing plasma potassium and in modulating the thiazide-sensitive sodium-chloride cotransporter (NCC). Here we tested whether dietary potassium intake modulates Kir4.1 and whether this is essential for mediating the effect of potassium diet on NCC. High potassium intake inhibited the basolateral 40 pS potassium channel (a Kir4.1/5.1 heterotetramer) in the distal convoluted tubule, decreased basolateral potassium conductance, and depolarized the distal convoluted tubule membrane in Kcnj10flox/flox mice, herein referred to as control mice. In contrast, low potassium intake activated Kir4.1, increased potassium currents, and hyperpolarized the distal convoluted tubule membrane. These effects of dietary potassium intake on the basolateral potassium conductance and membrane potential in the distal convoluted tubule were completely absent in inducible kidney-specific Kir4.1 knockout mice. Furthermore, high potassium intake decreased, whereas low potassium intake increased the abundance of NCC expression only in the control but not in kidney-specific Kir4.1 knockout mice. Renal clearance studies demonstrated that low potassium augmented, while high potassium diminished, hydrochlorothiazide-induced natriuresis in control mice. Disruption of Kir4.1 significantly increased basal urinary sodium excretion but it abolished the natriuretic effect of hydrochlorothiazide. Finally, hypokalemia and metabolic alkalosis in kidney-specific Kir4.1 knockout mice were exacerbated by potassium restriction and only partially corrected by a high-potassium diet. Thus, Kir4.1 plays an essential role in mediating the effect of dietary potassium intake on NCC activity and potassium homeostasis.

Calcineurin inhibitor cyclosporine A activates renal Na-K-Cl cotransporters via local and systemic mechanisms.

Calcineurin dephosphorylates nuclear factor of activated T cells transcription factors, thereby facilitating T cell-mediated immune responses. Calcineurin inhibitors are instrumental for immunosuppression after organ transplantation but may cause side effects, including hypertension and electrolyte disorders. Kidneys were recently shown to display activation of the furosemide-sensitive Na-K-2Cl cotransporter (NKCC2) of the thick ascending limb and the thiazide-sensitive Na-Cl cotransporter (NCC) of the distal convoluted tubule upon calcineurin inhibition using cyclosporin A (CsA). An involvement of major hormones like angiotensin II or arginine vasopressin (AVP) has been proposed. To resolve this issue, the effects of CsA treatment in normal Wistar rats, AVP-deficient Brattleboro rats, and cultured renal epithelial cells endogenously expressing either NKCC2 or NCC were studied. Acute administration of CsA to Wistar rats rapidly augmented phosphorylation levels of NKCC2, NCC, and their activating kinases suggesting intraepithelial activating effects. Chronic CsA administration caused salt retention and hypertension, along with stimulation of renin and suppression of renal cyclooxygenase 2, pointing to a contribution of endocrine and paracrine mechanisms at long term. In Brattleboro rats, CsA induced activation of NCC, but not NKCC2, and parallel effects were obtained in cultured cells in the absence of AVP. Stimulation of cultured thick ascending limb cells with AVP agonist restored their responsiveness to CsA. Our results suggest that the direct epithelial action of calcineurin inhibition is sufficient for the activation of NCC, whereas its effect on NKCC2 is more complex and requires concomitant stimulation by AVP.

Role of Collecting Duct Renin in the Pathogenesis of Hypertension.

The presence of renin production by the principal cells of the collecting duct has opened new perspectives for the regulation of intrarenal angiotensin II (Ang II). Angiotensinogen (AGT) and angiotensin-converting enzyme (ACE) are present in the tubular fluid coming from the proximal tubule and collecting duct. All the components needed for Ang II formation are present along the nephron, and much is known about the mechanisms regulating renin in juxtaglomerular cells (JG); however, those in the collecting duct remain unclear. Ang II suppresses renin via protein kinase C (PKC) and calcium (Ca2+) in JG cells, but in the principal cells, Ang II increases renin synthesis and release through a pathophysiological mechanism that increases further intratubular Ang II de novo formation to enhance distal Na + reabsorption. Transgenic mice overexpressing renin in the collecting duct demonstrate the role of collecting duct renin in the development of hypertension. The story became even more interesting after the discovery of a specific receptor for renin and prorenin: the prorenin receptor ((P)RR), which enhances renin activity and fully activates prorenin. The interactions between (P)RR and prorenin/renin may further increase intratubular Ang II levels. In addition to Ang II, other mechanisms have been described in the regulation of renin in the collecting duct, including vasopressin (AVP), bradykinin (BK), and prostaglandins. Current active investigations are aimed at elucidating the mechanisms regulating renin in the distal nephron segments and understand its role in the pathogenesis of hypertension.

Pathophysiology and clinical presentations of salt-losing tubulopathies.

At least three renal tubular segments are involved in the pathophysiology of salt-losing tubulopathies (SLTs). Whether the pathogenesis starts either in the thick ascending limb of the loop of Henle (TAL) or in the distal convoluted tubule (DCT), it is the function of the downstream-localized aldosterone sensitive distal tubule (ASDT) to contribute to the adaptation process. In isolated TAL defects (loop disorders) ASDT adaptation is supported by upregulation of DCT, whereas in DCT disorders the ASDT is complemented by upregulation of TAL function. This upregulation has a major impact on the clinical presentation of SLT patients. Taking into account both the symptoms and signs of primary tubular defect and of the secondary reactions of adaptation, a clinical diagnosis can be made that eventually leads to an appropriate therapy. In addition to salt wasting, as occurs in all SLTs, characteristic features of loop disorders are hypo- or isosthenuric polyuria and hypercalciuria, whereas characteristics of DCT disorders are hypokalemia and (symptomatic) hypomagnesemia. In both SLT categories, replacement of urinary losses is the primary goal of treatment. In loop disorders COX inhibitors are also recommended to mitigate polyuria, and in DCT disorders magnesium supplementation is essential for effective treatment. Of note, the combination of a salt- and potassium-rich diet together with an adequate fluid intake is always the basis of long-term treatment in all SLTs.

Sex differences in proximal and distal nephron function contribute to the mechanism of idiopathic hypercalcuria in calcium stone formers.

Idiopathic hypercalciuria (IH) is a common familial trait among patients with calcium nephrolithiasis. Previously, we have demonstrated that hypercalciuria is primarily due to reduced renal proximal and distal tubule calcium reabsorption. Here, using measurements of the clearances of sodium, calcium, and endogenous lithium taken from the General Clinical Research Center, we test the hypothesis that patterns of segmental nephron tubule calcium reabsorption differ between the sexes in IH and normal subjects. When the sexes are compared, we reconfirm the reduced proximal and distal calcium reabsorption. In IH women, distal nephron calcium reabsorption is decreased compared to normal women. In IH men, proximal tubule calcium reabsorption falls significantly, with a more modest reduction in distal calcium reabsorption compared to normal men. Additionally, we demonstrate that male IH patients have lower systolic blood pressures than normal males. We conclude that women and men differ in the way they produce the hypercalciuria of IH, with females reducing distal reabsorption and males primarily reducing proximal tubule function.

[EVALUATION OF RENAL FUNCTION IN PATIENTS WITH SPINAL CORD INJURY].

A comprehensive evaluation of renal function in 59 patients with spinal cord injury was performed. Excretory urography, biochemical and ion-selective analysis of serum and daily urine were used. Excretory urography had showed a reduction in the cumulative and excretory renal functions, which was accompanied by an increase in glomerular filtration rate, regardless of the level of damage and the period of traumatic disease. Excretory renal function has undergone a number of changes: 1) decrease in clearance of nitrogenous components; 2) an increase in clearance of mineral components in acute phase; 3) decrease in the rate of calcium excretion. Spinal cord injury contributed to an increase in the stimulating effect of aldosterone on the distal tubules of nephrons function of the kidneys, manifested in the form of increased excretion of potassium in the urine. In case of injury of the cervical and thoracic spine, significant increase in the K/Na coefficient was determined in acute phase, in patients with lumbar spine injury - in the late period.

Decreased urinary calbindin 1 levels in proteinuric rats and humans with distal nephron segment injuries.

BACKGROUND: Several proteins have been proposed as new urinary biomarkers of kidney injuries, but they are not always capable of identifying the kidney nephron segment that has been injured. Since calbindin 1 protein is exclusively localized in the kidney distal nephron segment, it is presumed that its expression is altered during distal nephron segment injuries, resulting in changes in its urinary excretion. METHODS: Calbindin 1 expression in normal rat kidneys was compared with that in the kidneys of rats that had suffered distal nephron segment injuries (unilateral ureteral obstruction [UUO] or anti-glomerular basement membrane glomerulonephritis [anti-GBM GN]) using immunohistochemical examinations and real-time polymerase chain reaction. The urinary calbindin 1 protein concentration of normal rats was also compared with that of anti-GBM GN rats and of cisplatin nephropathy rats using Western blotting. We also compared the kidney and urinary calbindin 1 protein concentrations of normal human subjects with those of proteinuric patients [immunoglobulin (Ig)A nephropathy; IgAN] with distal nephron segment injuries. RESULTS: Calbindin 1 mRNA expression in the renal cortices and calbindin 1 protein expression in the kidney distal nephron segments were decreased in the UUO and anti-GBM GN rat kidneys. The urinary calbindin 1 protein levels of the anti-GBM GN rats were also markedly decreased, whereas those of the cisplatin nephropathy rats were slightly decreased. The human IgAN patients displayed decreased renal calbindin 1 protein expression in their dilated distal tubules, and some patients displayed decreased urinary calbindin 1 levels. CONCLUSION: Since it has been demonstrated that decreased urinary calbindin 1 levels are indicative of decreased calbindin 1 kidney expression due to distal nephron segment injuries, calbindin 1 might be a useful urinary biomarker for identifying distal nephron segment injuries.

Cdc42 deficiency causes ciliary abnormalities and cystic kidneys.

Ciliogenesis and cystogenesis require the exocyst, a conserved eight-protein trafficking complex that traffics ciliary proteins. In culture, the small GTPase Cdc42 co-localizes with the exocyst at primary cilia and interacts with the exocyst component Sec10. The role of Cdc42 in vivo, however, is not well understood. Here, knockdown of cdc42 in zebrafish produced a phenotype similar to sec10 knockdown, including tail curvature, glomerular expansion, and mitogen-activated protein kinase (MAPK) activation, suggesting that cdc42 and sec10 cooperate in ciliogenesis. In addition, cdc42 knockdown led to hydrocephalus and loss of photoreceptor cilia. Furthermore, there was a synergistic genetic interaction between zebrafish cdc42 and sec10, suggesting that cdc42 and sec10 function in the same pathway. Mice lacking Cdc42 specifically in kidney tubular epithelial cells died of renal failure within weeks of birth. Histology revealed cystogenesis in distal tubules and collecting ducts, decreased ciliogenesis in cyst cells, increased tubular cell proliferation, increased apoptosis, increased fibrosis, and led to MAPK activation, all of which are features of polycystic kidney disease, especially nephronophthisis. Taken together, these results suggest that Cdc42 localizes the exocyst to primary cilia, whereupon the exocyst targets and docks vesicles carrying ciliary proteins. Abnormalities in this pathway result in deranged ciliogenesis and polycystic kidney disease.

In vivo multiphoton imaging of mitochondrial structure and function during acute kidney injury.

Mitochondrial dysfunction has been implicated in the pathogenesis of acute kidney injury due to ischemia and toxic drugs. Methods for imaging mitochondrial function in cells using confocal microscopy are well established; more recently, it was shown that these techniques can be utilized in ex vivo kidney tissue using multiphoton microscopy. We extended this approach in vivo and found that kidney mitochondrial structure and function can be imaged in anesthetized rodents using multiphoton excitation of endogenous and exogenous fluorophores. Mitochondrial nicotinamide adenine dinucleotide increased markedly in rat kidneys in response to ischemia. Following intravenous injection, the mitochondrial membrane potential-dependent dye TMRM was taken up by proximal tubules; in response to ischemia, the membrane potential dissipated rapidly and mitochondria became shortened and fragmented in proximal tubules. In contrast, the mitochondrial membrane potential and structure were better maintained in distal tubules. Changes in mitochondrial structure, nicotinamide adenine dinucleotide, and membrane potential were found in the proximal, but not distal, tubules after gentamicin exposure. These changes were sporadic, highly variable among animals, and were preceded by changes in non-mitochondrial structures. Thus, real-time changes in mitochondrial structure and function can be imaged in rodent kidneys in vivo using multiphoton excitation of endogenous and exogenous fluorophores in response to ischemia-reperfusion injury or drug toxicity.

Intrinsic control of sodium excretion in the distal nephron by inhibitory purinergic regulation of the epithelial Na(+) channel.

PURPOSE OF REVIEW: This review summarizes the new evidence for an intrinsic control system in the aldosterone-sensitive distal nephron in which purinergic signaling regulates sodium transport and governs renal sodium excretion. RECENT FINDINGS: Electrophysiological studies identify epithelial Na(+) channels (ENaC) as final effectors of purinergic signaling via P2Y(2) receptors in the distal nephron. Inhibition of ENaC by autocrine/paracrine purinergic signaling reduces sodium reabsorption allowing an appropriately graded pressure-natriuresis response when delivery of sodium to the distal nephron is high. Disruption of this intrinsic control mechanism decreases sodium excretion and therefore has a prohypertensive effect. Because purinergic inhibition of ENaC is tonic yet submaximal, its enhancement increases sodium excretion and therefore has an antihypertensive action. SUMMARY: Purinergic inhibitory regulation of ENaC is a key component of an intrinsic control system that enables the distal nephron to respond appropriately to the delivered load of sodium. This control system is physiologically important and functions in parallel with extrinsic control by the renin-angiotensin-aldosterone system, enabling sodium excretion to keep pace with sodium intake, especially when intake is high, and thereby maintaining arterial blood pressure. Disruption of intrinsic control of sodium transport by the distal nephron likely contributes to diseases such as arterial hypertension.

The role of the kidney in salt-sensitive hypertension.

Primary hypertension is one of the leading risk factors for cardiovascular disease. Although the pathogenesis is not completely understood, an imbalance of sodium and chloride homeostasis seems to be relevant both in the induction and in the maintenance of salt-sensitive hypertension. Besides individual renal phenotypes, salt intake is one of the most important environmental determinants of this condition. The Milan hypertensive strain (MHS) of rats is an interesting model to investigate the molecular mechanisms underling the development of salt-sensitive hypertension. In young MHS rats, hypertension is anticipated by a phase of increased salt reabsorption localized along the medullary thick ascending limb associated with the up-regulation of the apical sodium-potassium-chloride cotransporter (NKCC2). Later, the frank hypertensive status of adult MHS rats is accompanied by the activation of the luminal and basal lateral transporters of sodium chloride (NaCl) in the distal convoluted tubule (DCT). Several lines of evidence have proven the key role of DCT in the maintenance of hypertension in MHS rats; more importantly, hypertensive patients carrying a mutation of α-adducin (resembling the MHS model) have a high sensitivity to thiazides, suggesting that the Na(+)-Cl(-) cotransporter also plays a pivotal role in humans.

Life and death of the distal nephron: WNK4 and NCC as major players.

Missense mutations in the WNK4 gene lead to the development of familial hyperkalemic hypertension, a rare form of human hypertension. It was shown in vitro that WNK4 regulates the surface expression and activity of a number of ion channels and transporters. The in vivo analysis of wild-type and mutant WNK4 overexpression in transgenic mice models demonstrated that this serine-threonine kinase controls ion handling in the kidney mainly, and probably exclusively, through the regulation of the Na-Cl contransporter NCC activity.

The salt-wasting phenotype of EAST syndrome, a disease with multifaceted symptoms linked to the KCNJ10 K+ channel.

Mutations in the K+ channel gene KCNJ10 (Kir4.1) cause the autosomal recessive EAST syndrome which is characterized by epilepsy, ataxia, sensorineural deafness, and a salt-wasting tubulopathy. The renal salt-wasting pathology of EAST syndrome is caused by transport defects in the distal convoluted tubule where KCNJ10 plays a pivotal role as a basolateral K+ channel. This review on EAST syndrome outlines the molecular aspects of the physiology and pathophysiology of KCNJ10 in the distal convoluted tubule.

Rapamycin ameliorates PKD resulting from conditional inactivation of Pkd1.

Aberrant activation of the mammalian target of rapamycin (mTOR) pathway occurs in polycystic kidney disease (PKD). mTOR inhibitors, such as rapamycin, are highly effective in several rodent models of PKD, but these models result from mutations in genes other than Pkd1 and Pkd2, which are the primary genes responsible for human autosomal dominant PKD. To address this limitation, we tested the efficacy of rapamycin in a mouse model that results from conditional inactivation of Pkd1. Mosaic deletion of Pkd1 resulted in PKD and replicated characteristic features of human PKD including aberrant mTOR activation, epithelial proliferation and apoptosis, and progressive fibrosis. Treatment with rapamycin was highly effective: It reduced cyst growth, preserved renal function, inhibited epithelial cell proliferation, increased apoptosis of cyst-lining cells, and inhibited fibrosis. These data provide in vivo evidence that rapamycin is effective in a human-orthologous mouse model of PKD.

Short-term stimulation of the thiazide-sensitive Na+-Cl- cotransporter by vasopressin involves phosphorylation and membrane translocation.

Vasopressin influences salt and water transport in renal epithelia. This is coordinated by the combined action of V2 receptor-mediated effects along distinct nephron segments. Modulation of NaCl reabsorption by vasopressin has been established in the loop of Henle, but its role in the distal convoluted tubule (DCT), an effective site for fine regulation of urinary electrolyte composition and the target for thiazide diuretics, is largely unknown. The Na+-Cl- cotransporter (NCC) of DCT is activated by luminal trafficking and phosphorylation at conserved NH2-terminal residues. Here, we demonstrate the effects of short-term vasopressin administration (30 min) on NCC activation in Brattleboro rats with central diabetes insipidus (DI) using the V2 receptor agonist desmopressin (dDAVP). The fraction of NCC abundance in the luminal plasma membrane was significantly increased upon dDAVP as shown by confocal microscopy, immunogold cytochemistry, and Western blot, suggesting increased apical trafficking of the transporter. Changes were paralleled by augmented phosphorylation of NCC as detected by antibodies against phospho-threonine and phospho-serine residues (2.5-fold increase at Thr53 and 1.4-fold increase at Ser71). dDAVP-induced phosphorylation of NCC, studied in tubular suspensions in the absence of systemic effects, was enhanced as well (1.7-fold increase at Ser71), which points to the direct mode of action of vasopressin in DCT. Changes were more pronounced in early (DCT1) than in late DCT as distinguished by the distribution of 11beta-hydroxysteroid dehydrogenase 2 in DCT2. These results suggest that the vasopressin-V(2) receptor-NCC signaling cascade is a novel effector system to adjust transepithelial NaCl reabsorption in DCT.