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.

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.

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.

Immunoreactive kallikrein localization in the rat kidney: an immuno-electron-microscopic study.

The cellular and subcellular localization of immunoreactive kallikrein was studied in the rat kidney using the peroxidase-antiperoxidase (PAP) method for the electron microscope. The effect of various tissue-processing protocols on ultrastructural preservation and immunocytochemical staining was evaluated by fixing kidneys with four different mixtures. The tissues were immunostained and further stained with OsO4 or silver methenamine. The best ultrastructural and immunocytochemical staining was obtained with Zamboni's-glutaraldehyde fixative. The kallikrein-immunoreactive cell type was identified, according to its localization and ultrastructural features, as the connecting-tubule cell. Immunoreactive kallikrein was concentrated mainly in the upper one-third of the cell and at both sides of the nuclei, and to a less extent was associated with the plasma membranes and basolateral infoldings. The immunoreactivity was related to free polyribosomes, the rough endoplasmic reticulum (RER), and the Golgi complex, suggesting that kallikrein is actively synthesized in this particular type of cell.

Anatomical relationship between kallikrein-containing tubules and the juxtaglomerular apparatus in the human kidney.

Current evidence suggests a functional and biochemical link between the renin and the kallikrein systems. The purpose of this work was to study the localization of kallikrein along the human nephron to elucidate whether there exists an anatomical base for such interrelation. Serial sections of human kidney tissue were stained by immunocytochemical methods with antisera against kallikrein. Kallikrein immunostaining was observed exclusively in segments of the distal nephron lying in the cortical labyrinths and forming arcades in its distal portion. Consistently the tubules containing kallikrein established a close anatomical relationship with the afferent arteriole of the juxtaglomerular apparatus providing an anatomical base for an interaction between the renin and kallikrein systems in the human kidney.

Synthesis and expression of functional angiotensin II receptors in Xenopus oocytes injected with rat brain mRNA.

Xenopus laevis oocytes were injected with poly(A)+ mRNA isolated from rat brain and superfused in a medium containing either serotonin, angiotensin II or bradykinin. Applications of serotonin or angiotensin II to injected oocytes elicited, in a dose-dependent manner, changes in membrane potential. The angiotensin II receptor was desensitized fairly rapidly in the continued presence of the agonist. No response was obtained with bradykinin. The selectivity of the angiotensin II-induced response was demonstrated by the finding that the angiotensin II antagonist [( Sar1,Ala8]angiotensin II, saralasin) blocked the angiotensin II-induced response. It is concluded that an appropriate fraction of brain mRNA is capable of directing the synthesis and correct insertion of functional angiotensin II receptors in the Xenopus oocyte membrane.

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)

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.

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.

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 a stimulatory effect of high potassium diet on renal kallikrein.

Considerable evidence indicates that the connecting tubule cells, a type of cell of the distal nephron which seems to participate on potassium secretion, may be the place where renal kallikrein is synthetized. As potassium secretion and kallikrein synthesis may occur in the same cells, we studied the effect of high potassium diet on renal kallikrein production. The kallikrein containing cells from rats fed a normal and high potassium diet were evaluated using a combination of morphometric analysis, conventional electron microscopy, and ultrastructural immunocytochemistry. High potassium diet produced hypertrophy and hyperplasia of the kallikrein containing cells. Hyperplasia was sustained by an increased number of immunoreactive cells/mm2 (151 +/- 14 vs. 86.4 +/- 12, P less than 0.01), an increased number of binucleated immunoreactive cells/mm2 (11.90 +/- 2.1 vs. 3.77 +/- 0.17, P less than 0.005), and by the presence of mitosis. Cell hypertrophy was sustained by an increased cross-sectional area of immunoreactive cells (mu 2) (320.4 +/- 21 vs. 104.5 +/- 6.1, P less than 0.001), by an increased area of basal plasma membrane infoldings, by an hypertrophy of the components of the Golgi complex, hypertrophy of the components of the rough endoplasmic reticulum, and by a larger number of secretory-like vesicles containing kallikrein. The rats fed with high potassium diet had higher values on urinary kallikrein excretion-amidase activity (3.70 +/- 0.51 vs. 2.01 +/-0.37 units/day, P less than 0.02), higher values on potassium excretion (18.8 +/- 1.7 vs. 1.31 +/- 0.1 mmol/day, P less than 0.001), and higher urinary volume (51.5 +/- 5.3 vs. 12.2 +/- 1.6 ml/day, P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Subcellular localization of renal kallikrein by ultrastructural immunocytochemistry.

The subcellular distribution of immunoreactive kallikrein was described in the rat nephron using ultrastructural immunocytochemistry. The renal tissue was fixed with a mixture of buffered picric acid-paraformaldehyde-glutaraldehyde and immunostained with the peroxidase-antiperoxidase method for the electron microscope with the following steps: antikallikrein antiserum, anti-IgG serum, peroxidase-antiperoxidase complex, 3-3' diaminobenzidine-H2O2, and post-staining with osmium tetroxide. Preabsorption of the primary antiserum with purified rat urinary kallikrein and substitution with normal serum were used as controls. As we have described previously, kallikrein was present exclusively in the connecting tubule cell of the distal nephron. Subcellularly, kallikrein was distributed in luminal membranes, basal membranes, rough endoplasmic reticulum, Golgi apparatus, and vesicles. The immunoreactive vesicles were present in the proximity of the Golgi apparatus and in the cytoplasm in the way between the Golgi and the luminal and basal plasma membranes. No immunostaining was observed in other subcellular components of the connecting tubule cell or in the other type of cell. With the description of kallikrein in subcellular organelles involved in the synthesis, processing, and transport of glycoproteins, we have advanced an hypothetical intracellular processing pathway for renal kallikrein.