Role of Wnt signaling in tissue fibrosis, lessons from skeletal muscle and kidney.

Several studies have provided clear evidence of the importance of Wnt signaling in the function of several tissues. Wnt signaling has been related to several cellular processes including pre-natal development, cell division, regeneration and stem cell generation. By contrast, deregulation of this pathway has been associated with several diseases such as cancer, Alzheimer's disease, diabetes and, in recent years, fibrotic diseases in tissues such as skeletal muscle and kidney. Fibrotic diseases are characterized by an increase in the production and accumulation of extracellular matrix (ECM) components leading to the loss of tissue architecture and function. In a classical view, several molecules are related to the establishment of the fibrotic condition, including angiotensin II, transforming growth factorß(TGF-ß) and the connective tissue growth factor (CTGF) and a crosstalk has been suggested between these signaling molecules and the Wnt pathway. Skeletal muscle fibrosis, the most common disease, is typical of muscle dystrophies, where deregulation of the regenerative process in postnatal muscle leads to fibrotic differentiation and eventually to the failure of skeletal muscle. The fibrotic condition is also present in kidney pathologies such as polycystic kidney disease (PKD), in which fibrosis leads to a loss of tubule architecture and to a loss of function, which in almost all cases requires kidney surgery. A new actor in the pro-fibrotic effect of Wnt signaling in the kidney has been described, the primary cilium, an organelle that plays an important role in the onset of fibrosis. The aim of this review is to discuss the pro-fibrotic effect of Wnt signaling in both skeletal muscle and kidney, and to try to understand how this pathway is associated with the TGF-ß, CTGF and angiotensin II pro-fibrotic pathway.

Hypokalemia induces renal injury and alterations in vasoactive mediators that favor salt sensitivity.

We investigated the hypothesis that hypokalemia might induce renal injury via a mechanism that involves subtle renal injury and alterations in local vasoactive mediators that would favor sodium retention. To test this hypothesis, we conducted studies in rats with diet-induced K+ deficiency. We also determined whether rats with hypokalemic nephropathy show salt sensitivity. Twelve weeks of hypokalemia resulted in a decrease in creatinine clearance, tubulointerstitial injury with macrophage infiltration, interstitial collagen type III deposition, and an increase in osteopontin expression (a tubular marker of injury). The renal injury was greatest in the outer medulla with radiation into the cortex, suggestive of an ischemic etiology. Consistent with this hypothesis, we found an increased uptake of a hypoxia marker, pimonidazole, in the cortex. The intrarenal injury was associated with increased cortical angiontensin-converting enzyme (ACE) expression and continued cortical angiotensin II generation despite systemic suppression of the renin-angiotensin system, an increase in renal endothelin-1, a decrease in renal kallikrein, and a decrease in urinary nitrite/nitrates and prostaglandin E(2) excretion. At 12 wk, hypokalemic rats were placed on a normal-K+ diet with either high (4%)- or low (0.01%)-NaCl content. Despite correction of hypokalemia and normalization of renal function, previously hypokalemic rats showed an elevated blood pressure in response to a high-salt diet compared with normokalemic controls. Hypokalemia is associated with alterations in vasoactive mediators that favor intrarenal vasoconstriction and an ischemic pattern of renal injury. These alterations may predispose the animals to salt-sensitive hypertension that manifests despite normalization of the serum K+.

Renal cyclooxygenase-2: evidence for recruitment of thick ascending limb of henle cells in microdissected nephron segments.

Prostaglandins participate in the regulation of sodium and water renal excretion. They are synthesized by cyclooxygenases (COX): the constitutive isoform and the enzyme regulated by physiological stimuli (COX-2). Our previous immunohistochemical studies have demonstrated the presence of COX-2 in a subset of thick ascending limb (TAL) of Henle cells and its induction during the postnatal period and after adrenalectomy. Previous results suggested that this induction phenomenon proceeds by recruitment of TAL cells from the cortex to the outer medulla. The present work aimed to specifically address these preliminary observations by using immunohistochemical techniques in single microdissected nephron segments. Normal adult rats, adrenalectomized rats, adrenalectomized rats on dexamethasone and 5, 10, and 15 days postnatal age were used (Sprague-Dawley rats, n= 5 each group). Glomeruli and different segments of nephron were microdissected from collagenase-treated kidney tissue. Tubules were immunostained with specific antibodies against COX-2. We confirmed that COX-2 was localized exclusively in TAL segments; it was induced after adrenalectomy and during postnatal age, peaking at 15 days after birth. We provided morphological evidence that the induction of COX-2 along TAL proceeded in a defined pattern by recruitment of cells from the cortical portion close to the glomeruli toward the outer medulla. No COX-2 was observed in the post-macula densa portion of the segments. Our results provide the anatomical basis for the contribution of COX-2 in physiological mechanisms such as renin secretion, tubuloglomerular feedback, and the interaction with neuronal NO synthase at the juxtaglomerular apparatus.

Aquaporin-2, a regulated water channel, is expressed in apical membranes of rat distal colon epithelium.

Aquaporin-2 (AQP-2) is the vasopressin-regulated water channel expressed in the apical membrane of principal cells in the collecting duct and is involved in the urinary concentrating mechanism. In the rat distal colon, vasopressin stimulates water absorption through an unknown mechanism. With the hypothesis that AQP-2 could contribute to this vasopressin effect, we studied its presence in rat colonic epithelium. We used RT-PCR, in situ hybridization, immunoblotting, and immunocytochemistry to probe for AQP-2 expression. An AQP-2 amplicon was obtained through RT-PCR of colon epithelium RNA, and in situ hybridization revealed AQP-2 mRNA in colonic crypts and, to a lesser extent, in surface absorptive epithelial cells. AQP-2 protein was localized to the apical membrane of surface absorptive epithelial cells, where it colocalized with H(+)-K(+)-ATPase but not with Na(+)-K(+)-ATPase. AQP-2 was absent from the small intestine, stomach, and liver. Water deprivation increased the hybridization signal and the protein level (assessed by Western blot analysis) for AQP-2 in distal colon. This was accompanied by increased p-chloromercuriphenylsulfonic acid-sensitive water absorption. These results indicate that AQP-2 is present in the rat distal colon, where it might be involved in a water-sparing mechanism. In addition, these results support the idea that AQP-2, and probably other aquaporins, are involved in water absorption in the colon.

Renal phenotype of low kallikrein rats.

BACKGROUND: Renal kallikrein has been linked with inheritance of arterial hypertension and with sensitivity to drug nephrotoxicity. Identification of a cause--effect relationship between low kallikrein and intermediate phenotypes has been hampered by the lack of adequate animal models. METHODS: Kallikrein was measured in tissues obtained from rats inbred for low urinary kallikrein excretion (LKR) and wild-type controls. Blood pressure and indices of myocardial contractility were recorded via an intraventricular cannula connected to a transducer. The functional relevance of endogenous angiotensin II (Ang II) in LKR was explored by determining the effect of Ang II subtype 1 (AT(1)) receptor blockade on glomerular filtration rate, renal blood flow, and urinary sodium excretion. In addition, sensitivity to gentamycin-induced nephrotoxicity was evaluated. RESULTS: Kallikrein activity was reduced by 60% in the kidney of LKR (P < 0.01), whereas it was increased in the heart (P < 0.05) and was unaltered in the pancreas, liver, and salivary glands. Heart rate and myocardial contractility were reduced, and the mean blood pressure (MBP) was increased in LKR as compared with controls (P < 0.05). LKR exhibited polydipsia, polyuria, glomerular hyperfiltration, and reduced fractional sodium excretion under basal conditions and impaired renal vasodilation in response to volume expansion. These functional alterations were significantly attenuated by AT(1) receptor blockade. Gentamycin reduced the glomerular filtration rate in LKR, but not in controls. CONCLUSIONS: In LKR, unopposed activity of Ang II appears to be responsible for increased glomerular hydrostatic pressure and augmented tubular reabsorption. Balance between the kallikrein-kinin and renin-angiotensin systems is essential for normal renal function.

Renal and vascular injury induced by exogenous angiotensin II is AT1 receptor-dependent.

Angiotensin II (Ang II) infusion in rats augments vascular injury in balloon-injured carotid arteries and induces marked vascular and tubulointerstitial injury in kidneys. We examined how the AT1 receptor is modulated and whether blockade of the receptor with losartan could prevent the phenotypic and cellular changes. We also examined the role of the local renin-angiotensin system (RAS) by examining the expression of angiotensin-converting enzyme (ACE) and the effect of treatment with the ACE inhibitor, ramipril. Ang II infusion resulted in systemic hypertension and accelerated intimal and medial thickening in balloon-injured carotid arteries. Renal injury was manifested by proteinuria, glomerular phenotypic changes (mesangial expression of alpha-actin and podocyte expression of desmin), and tubulointerstitial injury with the tubular upregulation of the macrophage-adhesive protein, osteopontin, the interstitial accumulation of macrophages and myofibroblasts, and the deposition of collagen types III and IV. Ang II infusion decreased AT1 receptor number in the renal interstitium but not in glomeruli. Losartan completely blocked the Ang II-mediated hypertension, proteinuria, and injury to both carotid and kidney. Ang II infusion was also associated with an increase in ACE protein in both the proximal tubular brush border as well as at interstitial sites of injury, but despite evidence for activation of the local RAS, treatment with ramipril was without effect. These studies demonstrate that the renal and vascular injury induced by Ang II infusion is mediated by the AT1 receptor despite downregulation of the receptor in the interstitium. In addition, although there is evidence for local RAS activation, the injury appears to be mediated solely by the exogenous Ang II.

Postnatal development of cyclooxygenase-2 in the rat kidney.

Prostaglandins are local mediators/modulators of kinin effects in the kidney. The prostaglandin G2/H2 synthase (cyclooxygenase, COX) is the key regulatory enzyme of prostanoid synthesis pathway. Two COX isoenzymes (constitutive or COX-1 and inducible or COX-2) have been described in the rat kidney. We have demonstrated the presence of COX-2 in a subset of thick ascending limb of Henle (TAL) cells in normal adult rats [Vio, C.P., Cespedes, C., Gallardo, P., Masferrer, J.L., 1997. Renal identification of cyclooxygenase-2 in a subset of thick ascending limb cells. Hypertension 30, 687-692]. The present work was designed to evaluate COX-2 during the postnatal development of the rat kidney. Kidneys from Sprague-Dawley rats were studied during postnatal days 5, 10, 15 days and adult (60 days) (n = 8 each group). Renal tissue was immunostained with specific antibodies against COX-2. COX-2 was observed exclusively in TAL. A small number of COX-2 cells were observed during early postnatal life, increasing from day 5 to 15, and decreasing thereafter to reach adult levels. During maximal expression, near 20% of TAL were COX-2 positive whereas in early postnatal period and adults, only 2% of TAL cells contain COX-2. This transient induction of COX-2 during development suggest that the enzyme is necessary for the postnatal development of the kidney. This change in COX-2 seems to correspond to a derepression of COX-2 gene expression secondary to low levels of glucocorticoids.

Long-term nitric oxide synthase inhibition in rat pregnancy reduces renal kallikrein.

This study was performed to test the hypothesis that long-term nitric oxide synthase (NOS) inhibition during pregnancy may alter the predominance of the vasodilator kallikrein system. Sprague-Dawley rats were treated with the competitive inhibitor of NOS N(omega)-nitro-L-arginine (L-NNA, 50 mg. kg(-1). d(-1), dissolved in water) from days 7 to 21 of pregnancy. Rats were studied before treatment (day 5), at days 11, 17, and 21 of pregnancy (during treatment), and at postpartum days 7 and 21 (after the drug was withdrawn at delivery). Each group (n=5 to 8) had its corresponding control group (C) that received only vehicle. Additional rats were treated with N(G)-nitro-L-arginine methyl ester (L-NAME) alone or with an excess of L-arginine. At each study day, we measured blood pressure, collected urine overnight, obtained blood samples, and processed the kidneys for conventional histology and immunohistochemistry. In L-NNA rats, fetal and placental weights were reduced at days 17 and 21. Blood pressure was higher at days 17 and 21, returning to normal after L-NNA was removed. Urinary kallikrein activity was lower at days 11 and 17 (L-NNA=1147+/-213 and C=2317+/-146 nmol/16 h, P<0.001). Plasma renin activity was reduced at day 21 (L-NNA=9.6+/-2.1 and C=25.9+/-5 ng x mL(-1) x h(-1), P<0.05) and remained lower at postpartum day 7 x L-NNA rats exhibited glomerular lesions and tubular atrophy, particularly of connecting tubules that displayed reduced kallikrein staining. Tubulointerstitial infiltrating macrophages (ED1+) were also observed. Renal lesions were present as early as day 11 and persisted at day 7 postpartum. L-NAME rats exhibited similar alterations that were attenuated with an excess of L-arginine. We postulate that the reduction in renal kallikrein may contribute to the hemodynamic alterations described in this model.

Cellular and functional aspects of the renal kallikrein system in health and disease.

The kallikrein kinin system is a tissue-derived system with potent renal and cardiovascular effects. Within the kidney, the components of the kallikrein kinin system (kallikrein, kininogen, kinins, kininases, kinin receptors and mediators/modulators) originate from or are located in discrete segments of the nephron in highly specialized cells which determine its physiological effects. The kallikrein system acts on the kidney in a paracrine fashion in two anatomical microenvironments where the system regulates glomerular function, renal hemodynamics, and salt and water excretion. Impairment of the renal kallikrein system contributes to the development of hypertension, in particular to the salt-sensitive hypertension, and other pathologies like diabetes. There are several links between the vasodepressor kallikrein system and the vasopressor renin system which are relevant to normal renal function and to the pathophysiology of hypertension and renal diseases. Local induction of kininase II or angiotensin converting enzyme in the kidney could be a novel mechanism contributing to the renal damage in hypertension and other renal diseases. This review evaluates cellular and functional aspects of the renal kallikrein system with emphasis placed on the cellular localization of its components along the nephron, the links to other vasoactive systems, and the contribution of the system to the pathogenesis of hypertension.

Cellular distribution of exogenous aprotinin in the rat kidney.

Aprotinin, an inhibitor of the enzymatic activity of kallikrein in vitro, has been used to study the possible contributions of the kallikrein-kinin systems to physiological and pathological conditions. Pharmacokinetic studies indicate that aprotinin is concentrated in the kidney; however, there is little information with regard to its cellular distribution. The purpose of the present work was to study the cellular distribution of aprotinin, which would be valuable for a better understanding of its intrarenal effects. Sprague-Dawley rats (200-250g, n = 36) received aprotinin (50000 KIU/rat) and were killed at different intervals after its administration. The kidneys were examined histologically and the cellular distribution of aprotinin was studied by immunohistochemistry. Aprotinin was localized at 30 min concentrated within vesicles in the apical border of the proximal tubule cells. Later (2 h) it was observed distributed over the cytoplasm, where it remained for the 24 h studied. Aprotinin was also detected in connecting tubule cells colocalized with kallikrein, and in the basal portion of collecting tubule cells. No evidence of endogenous aprotinin was observed. The binding of aprotinin to the connecting tubule cells and collecting ducts offers a partial explanation of its renal effects.

Renal identification of cyclooxygenase-2 in a subset of thick ascending limb cells.

The prostaglandin G2/H2 synthase (cyclooxygenase, COX) is a key regulatory enzyme of prostanoid synthesis pathway. The message-encoding COX isoenzymes (constitutive COX-1 and inducible COX-2) have been described in the rat kidney. However, there is scarce information on the localization of COX-2 in the kidney, although it has been recently reported to be localized in the macula densa. The present study was designed to evaluate the localization of COX-2 in adult rat kidneys. Normal rat kidneys (n=10) were fixed in Bouin and were immunostained with specific antibodies against COX-2 by the peroxidase method. The cellular origin of COX-2 was assessed by the immunostaining of serial consecutive sections with antibodies against Na-K-ATPase, Tamm-Horsfall glycoprotein, H-K-ATPase, kallikrein, and macrophages. COX-2 was consistently observed in a subset of tubular cells located in the cortex and in the outer medulla. The staining of serial sections showed that the COX-2+ cells contained both Na-K-ATPase and Tamm-Horsfall, indicating that they corresponded to thick ascending limb (TAL) cells. They were observed at a considerable distance from the corresponding macula densa, although occasionally they were observed close to glomeruli. The COX-2 staining in the TAL cells was not abolished by dexamethasone treatment (1 to 20 mg/kg), suggesting its constitutive expression in normal kidneys. The presence of COX-2 in TAL (a tubular segment postulated to be devoid of COX-1) may contribute to the handling of ions through local production of prostaglandins.

Postnatal maturation of tissue kallikrein-producing cells (connecting tubule cells) in the rat kidney: a morphometric and immunohistochemical study.

The mature, fully differentiated connecting tubule (CNT) cell plays an important role in the regulation of serum potassium levels and synthesizes the enzyme tissue kallikrein, a main component of a renal vasoactive system, the kallikrein-kinin system. To characterize the growth of CNT cells (tissue kallikrein-producing cells), we studied the rat kidney at three different time points of postnatal development: at day 5, day 15, and day 30. The CNT cells were identified on tissue sections by a standardized immunohistochemical procedure. The tissue kallikrein content was determined by radioimmunoassay and the activity of the enzyme in kidney homogenates was measured using a selective synthetic substrate. The number of immunolabeled CNT and CNT cells per cortex area gradually increased from day 5 to day 30. A similar rise in the content and activity of tissue kallikrein was observed when the enzyme levels were determined by radioimmunoassay or by the enzymatic method. In addition, the morphometric analysis showed that the distal end of CNT had larger cells that displayed a more intense tissue kallikrein staining than those present in the proximal end, suggesting that the postnatal development of CNT is induced from its juxtamedullary portion. Our results show that tissue kallikrein expression is very low in the newborn rat, increasing gradually with age to reach adult levels at day 30. This finding, together with the morphometric data, suggests immaturity of CNT cells in newborn rats, a fact that could contribute to explaining the high serum potassium levels reported at this stage. In addition, the contrasting behavior of kallikrein and renin in the postnatal development (kallikrein increasing and renin decreasing) could explain the gradual decrease in renal vascular resistance and increase in renal blood flow observed after birth.

Cellular mechanisms of estrogen- and dopamine-induced control of glandular kallikrein in the anterior pituitary of the rat.

Glandular kallikrein (GK, a trypsin-like serine protease) exhibits estrogen induction and dopamine repression in rat pituitary lactotrophs. Steroid induction may reflect primary actions to increase selectively the synthesis of specific proteins, or may be part of broad cellular responses secondary to steroid-induced phenotype transitions. This study examined the cellular mechanisms underlying estrogen and dopaminergic control of lactotroph GK using a quantified immunocytochemical approach. Pituitaries from ovariectomized rats exhibited little GK staining. Estradiol treatment for 10 days produced dose-dependent increases in pituitary mass, the percentage of lactotrophs (indicating lactotroph proliferation) and the percentage of GK-positive cells. Also, GK staining intensity was dependent upon estradiol dose, increasing 4-fold between 5 micrograms and 50 micrograms/48 h. Dopamine receptor blockade with haloperidol (2.5 mg/kg/24 h) elicited weak GK immunostaining in 46% of the lactotrophs in the absence of estradiol, and markedly potentiated GK staining intensity elicited with low but not high doses of estradiol. The results suggest that GK induction is a primary estrogen effect, and is not secondary to a phenotype transition: the induction is enhanced by estrogen-induced lactotroph proliferation. Dopaminergic systems strongly inhibit GK induction by low estradiol levels. This dopaminergic modulation may shift the induction of lactotroph GK to physiological events associated with high estradiol levels or low dopaminergic tone.

Localization of immunoreactive tissue kallikrein in human trachea.

Tissue kallikrein is the major kininogenase detected in bronchoalveolar lavage fluids from asthmatics and may play a particularly important role in kinin generation during asthma. The present study was undertaken to determine the source of tissue kallikrein in the human lower airways. Specific antisera to human tissue kallikrein were used to localize this enzyme by immunocytochemistry in human trachea. Immunoreactive tissue kallikrein was localized in submucosal glands of the lamina propria but was not detected in epithelial cells or goblet cells. Specific staining for tissue kallikrein was not detected in all cells of the submucosal glands but was restricted to cells forming demilunes in the distal portions of the glands. When consecutive serial sections of submucosal glands were alternately stained using antiserum to tissue kallikrein and a periodic acid Schiff stain (to detect mucus), it was revealed that immunoreactive tissue kallikrein was present only in serous cells and not in mucus cells. The localization of tissue kallikrein to the serous cells of submucosal glands should facilitate studies to regulate the release of this enzyme. Regulation of tissue kallikrein release may provide a mechanism to reduce kinin generation during asthma.

Evidence for renal kinins as mediators of amino acid-induced hyperperfusion and hyperfiltration in the rat.

This study examined the role of tissue kallikrein and kinins in renal vasodilation produced by infusion of amino acids (AA). In rats fed a 9% protein diet for 2 wk, intravenous infusion of a 10% AA solution over 60-90 min reduced total renal vascular resistance and increased glomerular filtration rate (GFR) by 25-40% and renal plasma flow (RPF) by 23-30% from baseline. This was associated with a two- to threefold increase in urinary kinin excretion rate. Acute treatment of rats with aprotinin, a kallikrein inhibitor, resulted in deposition of immunoreactive aprotinin in kallikrein-containing connecting tubule cells and inhibited renal kallikrein activity by 90%. A protinin pretreatment abolished the rise in urinary kinins and prevented significant increases in GFR and RPF in response to AA. In a second group of rats pretreated with a B2 kinin receptor antagonist, [DArg Hyp3, Thi5,8 D Phe7]bradykinin, AA infusion raised urinary kinins identically as in untreated controls, but GFR and RPF responses were absent. Aprotinin or the kinin antagonist produced no consistent change in renal function in rats that were not infused with AA.AA-induced increases in kinins were not associated with an increase in renal kallikrein activity. Notably, tissue active kallikrein level fell 50% in AA-infused rats. These studies provide evidence that kinins generated in the kidney participate in mediating renal vasodilation during acute infusion of AA.

Hormonal regulation of pituitary glandular kallikrein: a morphometric study.

We have previously identified the lactotrophs as the Glandular Kallikrein (GK) containing cells in the rat anterior pituitary using immunocytochemistry, this localization has been independently confirmed with similar methods by other groups. The purpose of the present work was to evaluate the estrogen and dopaminergic control of the GK-containing cells using a morphometric analysis. Female (200-250 g) ovariectomized rats (n = 40) were treated with estradiol (5, 10, 50 micrograms/rat) in the presence or absence of haloperidol (2.5 mg/Kg). The pituitaries were fixed by perfusion with Bouin's and immunostained for prolactin (PRL) or GK. The number of cells and the intensity of the staining were determined by morphometric analysis. Little GK staining was observed in pituitaries from ovariectomized rats, whereas estradiol treatment produced a marked increase in GK staining; GK-positive lactotrophs increased from 4% in control to 75% with 5 micrograms of estradiol, higher doses produced little further increase. However, GK staining intensity in lactotrophs was markedly dependent upon estradiol dose increasing 4-fold between 5 micrograms and 50 micrograms. Haloperidol (2.5 mg/Kg) elicited weak GK staining in 46% of the lactotrophs in the absence of estradiol, and potentiated GK staining intensity elicited with low doses of estradiol. Estradiol also produced a dose-dependent increase in pituitary mass and % lactotrophs indicating lactotroph proliferation. Estradiol produced a dose dependent increase in pituitary wet weight, % PRL-positive cells and % GK-positive cells. Pituitary weight was correlated with % lactotrophs (r = 0.992), and % GK cells (r = 0.874), and % lactotrophs was correlated with % GK cells (r = 0.978).(ABSTRACT TRUNCATED AT 250 WORDS)

Potassium supplementation lowers blood pressure and increases urinary kallikrein in essential hypertensives.

In order to eludicate possible mechanism(s) involved in the blood pressure reduction induced by potassium (K) supplementation, we studied the changes of BP and of some of its regulatory systems, including levels of urinary kallikrein (UKal)--an index of renal kallikrein production. Twenty-four untreated essential hypertensives, with a basal BP of 147/96 +/- 13/7 mmHg and normal renal function, received in crossover, double-blind, randomised fashion, 64 mmol KCl or placebo during two periods of 4 weeks each. At the 4th week of potassium supplementation systolic, diastolic and mean BPs decreased by 6.3 +/- 2 (P less than 0.01), 3.0 +/- 2 and 4.1 +/- 2 (P less than 0.05) mmHg respectively for the supine position, and 5.0 +/- 2, 4.0 +/- 2 (P less than 0.05) and 4.0 +/- 1 (P less than 0.05) mmHg for the standing position. Urinary potassium (K) increased from 55 +/- 4 to 123 +/- 6 mmol/24 hours (P less than 0.001) and UKal from 692 +/- 69 to 1052 +/- 141 mU/24 hours (P less than 0.01). Serum K rose from 3.8 +/- 0.1 mEq/l to 4.1 +/- 0.1 mmol/l (P less than 0.001) and PRA from 0.77 +/- 0.12 to 0.99 +/- 0.14 ng/ml/h (P less than 0.05). Correlations were observed between UKal and urinary K (r = 0.44, P less than 0.0001); between differences in UKal and urinary K and in UKal and urinary Na (r = 0.50, P less than 0.0005 and r = 0.48, P less than 0.001 respectively).(ABSTRACT TRUNCATED AT 250 WORDS)