An intact kidney slice model to investigate vasa recta properties and function in situ.

BACKGROUND: Medullary blood flow is via vasa recta capillaries, which possess contractile pericytes. In vitro studies using isolated descending vasa recta show that pericytes can constrict/dilate descending vasa recta when vasoactive substances are present. We describe a live kidney slice model in which pericyte-mediated vasa recta constriction/dilation can be visualized in situ. METHODS: Confocal microscopy was used to image calcein, propidium iodide and Hoechst labelling in 'live' kidney slices, to determine tubular and vascular cell viability and morphology. DIC video-imaging of live kidney slices was employed to investigate pericyte-mediated real-time changes in vasa recta diameter. RESULTS: Pericytes were identified on vasa recta and their morphology and density were characterized in the medulla. Pericyte-mediated changes in vasa recta diameter (10-30%) were evoked in response to bath application of vasoactive agents (norepinephrine, endothelin-1, angiotensin-II and prostaglandin E(2)) or by manipulating endogenous vasoactive signalling pathways (using tyramine, L-NAME, a cyclo-oxygenase (COX-1) inhibitor indomethacin, and ATP release). CONCLUSIONS: The live kidney slice model is a valid complementary technique for investigating vasa recta function in situ and the role of pericytes as regulators of vasa recta diameter. This technique may also be useful in exploring the role of tubulovascular crosstalk in regulation of medullary blood flow.

Potential role of serine proteases in modulating renal sodium transport in vivo.

The maintenance of sodium (Na+) homeostasis is an essential function of the kidney. It is achieved by a variety of transport processes localized all along the highly specialised segments of the nephron. Impairment of these transport mechanisms, and thereby Na+ handling, is associated with disturbed Na+ and water balance, leading to hypertension and oedema. This review focuses on the novel regulation of sodium reabsorption by serine proteases acting along the entire nephron.

Albumin uptake in OK cells exposed to rotenone: a model for studying the effects of mitochondrial dysfunction on endocytosis in the proximal tubule?

BACKGROUND: The renal proximal tubule (PT) is clinically vulnerable to mitochondrial dysfunction; sub-lethal injury can lead to the Fanconi syndrome, with elevated urinary excretion of low-molecular-weight proteins. As the mechanism that couples mitochondrial dysfunction to impaired PT low-molecular weight protein uptake is unknown, we investigated the effect of respiratory chain (RC) inhibitors on endocytosis of FITC-albumin in PT-derived OK cells. METHODS: Uptake of FITC-albumin was quantified using confocal microscopy. Cytosolic ATP levels were measured in real time using both luciferin/luciferase assays and measurements of free [Mg(2+)]. Reactive oxygen species production was measured using mitosox. RESULTS: RC blockade produced only a small decrease in cytosolic ATP levels and had minimal effect on FITC-albumin uptake. Inhibition of glycolysis caused a much bigger decrease in both cytosolic ATP levels and FITC-albumin endocytosis. Rotenone led to higher rates of reactive oxygen species production than other RC inhibitors. Rotenone also caused widespread structural damage on electron microscopy, which was mimicked by colchicine and prevented by taxol; consistent with inhibition of microtubule polymerisation as the underlying mechanism. CONCLUSIONS: Endocytosis of FITC-albumin is ATP-dependent in OK cells, but the cells are very glycolytic and therefore represent a poor metabolic model of the PT. Rotenone has toxic extra-mitochondrial structural effects.

Human hypertension caused by mutations in WNK kinases.

Hypertension is a major public health problem of largely unknown cause. Here, we identify two genes causing pseudohypoaldosteronism type II, a Mendelian trait featuring hypertension, increased renal salt reabsorption, and impaired K+ and H+ excretion. Both genes encode members of the WNK family of serine-threonine kinases. Disease-causing mutations in WNK1 are large intronic deletions that increase WNK1 expression. The mutations in WNK4 are missense, which cluster in a short, highly conserved segment of the encoded protein. Both proteins localize to the distal nephron, a kidney segment involved in salt, K+, and pH homeostasis. WNK1 is cytoplasmic, whereas WNK4 localizes to tight junctions. The WNK kinases and their associated signaling pathway(s) may offer new targets for the development of antihypertensive drugs.

Familial distal renal tubular acidosis is associated with mutations in the red cell anion exchanger (Band 3, AE1) gene.

All affected patients in four families with autosomal dominant familial renal tubular acidosis (dRTA) were heterozygous for mutations in their red cell HCO3-/Cl- exchanger, band 3 (AE1, SLC4A1) genes, and these mutations were not found in any of the nine normal family members studied. The mutation Arg589--> His was present in two families, while Arg589--> Cys and Ser613--> Phe changes were found in the other families. Linkage studies confirmed the co-segregation of the disease with a genetic marker close to AE1. The affected individuals with the Arg589 mutations had reduced red cell sulfate transport and altered glycosylation of the red cell band 3 N-glycan chain. The red cells of individuals with the Ser613--> Phe mutation had markedly increased red cell sulfate transport but almost normal red cell iodide transport. The erythroid and kidney isoforms of the mutant band 3 proteins were expressed in Xenopus oocytes and all showed significant chloride transport activity. We conclude that dominantly inherited dRTA is associated with mutations in band 3; but both the disease and its autosomal dominant inheritance are not related simply to the anion transport activity of the mutant proteins.

Renal tubular acidosis.

Renal tubular acidosis (RTA) is a form of metabolic acidosis due to abnormal alkali (bicarbonate) loss by the kidneys or their failure to excrete net acid. While the latter does occur in chronic renal failure, the term RTA is usually applied only when the glomerular filtration rate is normal or near normal. As well as a cause of metabolic acidosis, RTA often presents as renal stone disease with nephrocalcinosis, rickets/osteomalacia, and growth retardation in children. In this brief review, we have summarized the classification, clinical features and the underlying cell and molecular pathophysiology of RTA. However, despite significant advances in our understanding of the mechanisms of RTA, its treatment is still empirical and based largely on alkali replacement therapy; but its wider significance in renal stone and bone disease is becoming increasingly recognized.

Uric acid and the kidney: urate transport, stone disease and progressive renal failure.

In this brief review and update, we try to cover recent developments in our understanding of uric acid transport by the kidney, the contribution of uric acid to renal stone disease, its potential role in progressive renal failure and, most recently, the novel and as yet unexplained link between the urinary glycoprotein Tamm-Horsfall protein (uromodulin) and hyperuricaemia and two inherited forms of renal disease with chronic renal failure.

The P2X7 ATP receptor in the kidney: a matter of life or death?

P2X7 is an intriguing membrane receptor for the extracellular nucleotide ATP, which functions as a ligand-gated ion channel; it can activate cell membrane permeabilization and also has a wide range of downstream signaling pathways, including mediation of inflammatory responses and modulation of cell turnover. Despite recent identification of P2X7 receptor protein in the renal tract, the biological and potential pathological functions of this receptor and its signaling cascades in the kidney are not yet fully understood. P2X7 receptor protein is expressed in normal kidney development, predominantly in the condensing mesenchyme, and later in the maturing and adult derivatives of the ureteric bud. Glomerular expression of the molecule is scarce in normal kidney, but is upregulated in chronic and inflammatory conditions, suggesting a role in the inflammatory response or in repair and remodeling in these settings. P2X7 receptor expression in the adult collecting ducts of murine kidney, as well as the collecting duct cysts in autosomal recessive polycystic kidney disease, has been described and agonists of the receptor can modulate the development of renal cysts in an in vitro model of cyst formation derived from the cpk/cpk mouse. Further investigation of the function of the P2X7 receptor in normal and abnormal kidneys might lead to novel therapeutic targets in a wide range of renal diseases.

Renal P2 receptors and hypertension.

The regulation of extracellular fluid volume is a key component of blood pressure homeostasis. Long-term blood pressure is stabilized by the acute pressure natriuresis response by which changes in renal perfusion pressure evoke corresponding changes in renal sodium excretion. A wealth of experimental evidence suggests that a defect in the pressure natriuresis response contributes to the development and maintenance of hypertension. The mechanisms underlying the relationship between renal perfusion pressure and sodium excretion are incompletely understood. Increased blood flow through the vasa recta increases renal interstitial hydrostatic pressure, thereby reducing the driving force for transepithelial sodium reabsorption. Paracrine signalling also contributes to the overall natriuretic response by inhibiting tubular sodium reabsorption in several nephron segments. In this brief review, we discuss the role of purinergic signalling in the renal control of blood pressure. ATP is released from renal tubule and vascular cells in response to increased flow and can activate P2 receptor subtypes expressed in both epithelial and vascular endothelial/smooth muscle cells. In concert, these effects integrate the vascular and tubular responses to increased perfusion pressure and targeting P2 receptors, particularly P2X7, may prove beneficial for treatment of hypertension.

A London experience 1995-2012: demographic, dietary and biochemical characteristics of a large adult cohort of patients with renal stone disease.

BACKGROUND: Kidney stone disease has an estimated prevalence of around 10%. Genetic as well as environmental factors are thought to play an important role in the pathogenesis of renal stones. AIM: The aim of our study was to analyse and report the main characteristics of patients with kidney stones attending a large UK metabolic stone clinic in London between 1995 and 2012. DESIGN: A cross-sectional study. METHODS: Analysis of data from stone formers attending the University College and Royal Free Hospitals' metabolic stone clinic from 1995 to 2012. Demographic, clinical, dietary and biochemical characteristics have been summarized and analysed for men and women separately; trends over time have also been analysed. RESULTS: Of the 2861 patients included in the analysis, 2016 (70%) were men with an average age of 47 years (range 18-87 years) and median duration of disease of 6 years (range 0-60 years). The prevalence of low urine volume, hypercalciuria, hyperoxaluria, hyperuricosuria and hypocitraturia was 5.6%, 38%, 7.9%, 18% and 23%, respectively. The prevalence of several risk factors for stones increased over time. The majority of stones were mixed, with around 90% composed of calcium salts in varying proportion. CONCLUSION: Our findings in a large cohort of patients attending a London-based stone clinic over the past 20 years show differences in distributions of risk factors for stones for men and women, as well as metabolic profiles and stone composition. The impact of most risk factors for stones appeared to change over time.

Neurotensin and antinatriuresis in the conscious rabbit.

The mechanism of alteration in renal sodium excretion in response to dietary changes is complex and poorly understood. A gut 'sensor' might exist which regulates the renal response and this may involve one or more of the now ubiquitous gastrointestinal peptides. Several of these gut peptides, including neurotensin, have been found within the kidney. Plasma levels of neurotensin, which is both a circulating hormone and putative neurotransmitter, rise promptly on feeding. When infused into the conscious rabbit, neurotensin produces a dose-related fall in renal sodium excretion.

Renal effects of gastrin C-terminal tetrapeptide (as pentagastrin) and cholecystokinin octapeptide in conscious rabbit and man.

Pentagastrin and cholecystokinin octapeptide (CCK8) were infused i.v. at three different doses in two sets of 4 conscious rabbits following a repeated measurements design (130, 1,300 and 13,000 pmol kg-1 min-1 pentagastrin; 5, 50 and 450 pmol kg-1 min-1 CCK8). In man, two different doses of pentagastrin (13 and 65 pmol kg-1 min-1) were infused in two groups of 6 subjects, and CCK8 (2 pmol kg-1 min-1) in a third group. According to published human postprandial levels, plasma CCK8-like immunoreactivity concentrations were supraphysiological at all doses infused. In the rabbit, pentagastrin produced a dose-related fall in urine flow and free water clearance, but no significant change in systemic and renal haemodynamics, electrolyte excretion and measured plasma constituents; however, in human subjects, pentagastrin increased renal sodium excretion and reduced potassium excretion but did not change glomerular filtration rate. In the rabbit, CCK8 produced a dose-related fall in plasma renin activity, plasma calcium concentration and mean arterial blood pressure; dose-dependent increases in effective renal plasma flow, glomerular filtration rate and renal sodium excretion. In man, changes in sodium and potassium excretion similar to pentagastrin were observed; there were no significant changes in plasma renin activity, plasma calcium concentration, blood pressure, effective renal plasma flow or glomerular filtration rate. The pharmacological renal effects of pentagastrin in conscious water-loaded rabbits resemble vasopressin. In contrast, CCK8's most striking effect was vasodilatation and was unusual in inhibiting rather than stimulating renin release. In man the net changes in urine composition found during infusion of these peptides are similar to those produced by the potassium-sparing diuretics, amiloride and triamterene. However the generally weak renal effects observed, even at pharmacological doses, indicate that these peptides are unlikely to influence renal function under normal physiological conditions.

Response to atrial natriuretic peptide, endopeptidase 24.11 inhibitor and C-ANP receptor ligand in the rat.

1. The present studies compared the renal and hypotensive response to (a) exogenous atrial natriuretic peptide (ANP) (99-126), (b) an endopeptidase-24.11 inhibitor (candoxatrilat) and (c) an antagonist of ANP clearance receptors (SC 46542) in conscious rats. 2. Infusion of low-dose-ANP (100 ng kg-1 min-1) produced a gradual increase in urinary sodium and guanosine 3':5'-cyclic monophosphate (cyclic GMP) excretion without significant change in glomerular filtration rate (GFR) or fractional lithium clearance (FeLi). There was a significant fall in blood pressure. 3. Infusion of high-dose ANP (300 ng kg-1 min-1) produced a brisk, 3 fold increase in urinary sodium and cyclic GMP excretion along with a rise in GFR, but had no significant effect on FeLi compared to the control group. The renal response was accompanied by a pronounced fall in blood pressure. 4. Candoxatrilat or SC 46542, alone, had no significant effect on sodium excretion compared to control animals. Both compounds enhanced the natriuretic and cyclic GMP responses to a low-dose ANP infusion (100 ng kg-1 min-1) to levels similar to, or greater than, those observed with the high-dose ANP (300 ng kg-1 min-1). However, unlike high-dose ANP, these renal effects were not accompanied by a significant change in GFR and neither compound potentiated the hypotensive effect of the low-dose ANP infusion. Only candoxatrilat when given with ANP produced a marked rise in FeLi.5. Similarly, combined administration of candoxatrilat and SC 46542 (without exogenous ANP) induced an increase in sodium and cyclic GMP excretion comparable to high-dose ANP but did so without a significant increase in GFR and with a significantly smaller fall in blood pressure. Interestingly, there was no increase in FeLi with the combination of the two compounds, suggesting that the major contribution to sodium excretion came from SC 46542.6. Both candoxatrilat and SC 46542 increased sodium and cyclic GMP excretion in the rat A-V fistula model of heart failure, a model hyporesponsive to infusions of ANP, without significant change in blood pressure.7. These data show that candoxatrilat and SC 46542 do not simply reproduce the effects of an ANP infusion but preferentially enhance the natriuretic response to ANP. Inhibition of E-24.11 may potentiate a tubule action of ANP while the renal mechanism of action of the C-ANP receptor ligand needs further study. Both manipulations are of potential value in the management of heart failure.

Vasoactive intestinal peptide as a bronchodilator in asthmatic subjects.

Vasoactive intestinal peptide (V.I.P.) caused bronchodilatation in 7 asthmatic volunteers when given intravenously at the rate of 6 pmol kg-1 min-1 for 15 minutes during a double blind study. Mean baseline FEV1 was 2.8 (+/- 0.3 S.E.) which was 81% of predicted and increased by 0.21 (range 0.1-0.45) l after 15 minutes infusion (p greater than 0.02). Tachycardia and cutaneous flushing were also observed during the infusion. Subsequent induced bronchoconstriction with a predetermined dose of histamine was ameliorated at 180 seconds following challenge when compared with placebo. Mean fall in FEV1 0.26 compared with 0.741 when pre-infusion FEV1 was taken on baseline. Mean fall in FEV1 0.49 l compared with 0.75 l when the FEV1 immediately preceding challenge was used on baseline (p greater than 0.02). The demonstration that V.I.P. is a bronchodilator in asthmatics and ameliorates histamine induced bronchoconstriction has important implications for the pharmacology of asthma.

Renal function during vasoactive intestinal peptide (VIP) infusions in normal man and patients with liver disease.

VIP containing nerves are present in the kidney and plasma VIP levels are elevated in cardiac failure and severe liver disease. We studied the effects of intravenous VIP; 6 pmol kg-1 min-1 on 6 normal subjects and 3 patients with liver disease. In normal subjects VIP produced flushing and significant rises in heart rate and pulse pressure but the clearance rates of paraaminohippurate and creatinine did not change significantly. Urine flow fell to about 1/3 and the rate of excretion of electrolytes (except phosphate) fell to about a half of control values. Plasma renin activity rose about 3-fold and there were significant rises in haematocrit and the plasma concentrations of solids, calcium and phosphate. The patients with liver disease responded similarly. Elevated plasma VIP could contribute to salt and water retention in disease states.

Renal function during bovine neurotensin infusion in man.

The peptide hormone neurotensin (NT) is found mainly in gut endocrine cells of the ileum, but has also been identified as a putative neurotransmitter in the central and peripheral nervous systems. It may have a dual role as a circulating gastrointestinal hormone and peripheral neurotransmitter. Its predominant effects are to reduce oesophageal sphincter tone, inhibit gastric secretion and emptying and inhibit intestinal motility, but stimulate intestinal and pancreatic exocrine secretion; NT-like immunoreactivity has been found in kidney and therefore NT may influence renal function. When infused i.v. in rabbits it causes antinatriuresis. We have studied its renal effects in 11 healthy males by i.v. infusion under conditions of altered dietary sodium. Postprandial circulating neurotensin levels were reproduced by infusion. There were no consistent systemic or renal haemodynamic effects. Plasma electrolytes and renin did not change. Only renal chloride excretion changed significantly, falling by ca. 30%, and recovering after infusion. There is no evidence for a specific renal tubular chloride transport mechanism, but coupled cotransport, Na+:K+:2CI-, may be hormonally regulated. NT might stimulate this process and contribute to the renal response to changes in dietary composition, especially sodium intake.

An overview of divalent cation and citrate handling by the kidney.

Urinary calcium, magnesium and citrate levels are important in promoting or inhibiting renal stone formation. Here we review current information on the tubular handling of these ions. Most filtered calcium is reabsorbed in the proximal tubule and the thick ascending limb (TAL) of the loop of Henle, largely paracellularly; most of the remainder is reabsorbed in the distal tubule, transcellularly. Calcium reabsorption in the TAL and distal tubule is stimulated by parathyroid hormone and vitamin D; other factors influencing its renal handling include extracellular volume status and acid-base balance. Little filtered magnesium is reabsorbed in the proximal tubule; the bulk is reabsorbed paracellularly in the TAL, while most of the remainder is reabsorbed transcellularly in the distal tubule. Dietary intake, peptide hormones and chronic potassium depletion can all influence magnesium reabsorption in the TAL and distal tubule. Most filtered citrate is taken up across the apical membrane of the proximal tubule via a sodium-dicarboxylate co-transporter (NaDC-1). It also enters proximal tubular cells across the basolateral membrane; citrate contributes to the cells' oxidative metabolism. Citrate excretion is affected by acid-base balance, acetazolamide treatment, chronic potassium depletion and urinary excretion of calcium and magnesium. Where possible, we have indicated the mechanisms of these complex interactions.

P2Y receptors present in the native and isolated rat glomerulus.

Extracellular ATP can mobilize intracellular calcium in rat glomeruli by interacting with P2Y receptors. However, the identity of the receptor subtypes involved is not known. In the present study, we have used RT-PCR to identify mRNAs for specific P2Y receptor subtypes expressed in the rat glomerulus: mRNA for P2Y1, P2Y2, P2Y4 and P2Y6 receptors was detected. Functional expression of P2Y1 and P2Y2/P2Y4, but not P2Y6, receptors in intact glomeruli was confirmed by measuring the relative stimulation of the inositol phosphate pathway induced by selective agonists of a particular receptor subtype. Finally, we have used available polyclonal antibodies to confirm the expression of P2Y1 and P2Y2 in the glomerulus, in mesangial cells and glomerular epithelial cells (podocytes), respectively; but we could not demonstrate P2Y4 or P2Y6 receptor expression by this means. In a separate series of experiments, we have examined the possibility that intra-renal sympathetic nerve terminals are a source of extracellular ATP and that this would be supported, though not excluded, by supersensitivity to ATP following denervation. Nucleotide-induced stimulation of the inositol phosphate pathway was measured in both control rats and rats that had been sympathectomized by intraperitoneal injection of 6-hydroxydopamine. The response to norepinephrine was measured as a positive control. In the sympathectomized rats, the effect of norepinephrine was significantly enhanced, whereas ATP-induced inositol phosphate production was unaffected, being similar in both groups of animals.