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Background: There is mounting anecdotal and empirical evidence that gardening and art-making afford therapeutic benefits., Objectives: This randomly controlled pilot study tested the hypothesis that participation in group-based indoor gardening or art-making activities for one hour twice a week for four weeks would provide quantifiably different therapeutic benefits to a population of healthy women ages 26-49., Methods: A population of 42 volunteers was randomly assigned to parallel gardening or art-making treatment groups. A total of 36 participants initiated the treatment protocol and 32 (Gardening n = 15 and Art n = 17) received the interventions and completed all assessments. Treatments included eight one-hour group-based gardening or art intervention sessions. Self-report psychometric assessments were conducted for anxiety, depression symptomatology, mood disturbance, stress, satisfaction with discretionary social activities, and quality of life measures. Cardiac physiological data were also collected. Outcomes were measured at baseline, during, and post-intervention., Results: Engaging in both gardening and art-making activities resulted in apparent therapeutic improvements for self-reported total mood disturbance, depression symptomatology, and perceived stress with different effect sizes following eight one-hour treatment sessions. Gardening also resulted in improvements for indications of trait anxiety. Based on time-course evidence, dosage responses were observed for total mood disturbance, perceived stress, and depression symptomatology for both gardening and art-making. However, gardening or art-making did not have an apparent influence on heart rate or blood pressure or result in marked improvement for satisfaction with discretionary leisure activities., Conclusion: The data did not support the hypothesis of differential therapeutic benefits of gardening and art-making for healthy women. When taken together, group-based gardening or art-making can provide quantitatively measurable improvements in healthy women's psychosocial health status that imply potentially important public health benefits., Trial Registration: ClinicalTrials.gov NCT03266120., Competing Interests: The authors have declared that no competing interests exist.

We recently showed an association between strict BP control and lower mortality risk during two decades of follow-up of prior participants in the Modification of Diet in Renal Disease (MDRD) trial. Here, we determined the risk of ESRD and mortality during extended follow-up of the African American Study of Kidney Disease and Hypertension (AASK) trial. We linked 1067 former AASK participants with CKD previously randomized to strict or usual BP control (mean arterial pressure ≤92 mmHg or 102-107 mmHg, respectively) to the US Renal Data System and Social Security Death Index; 397 patients had ESRD and 475 deaths occurred during a median follow-up of 14.4 years from 1995 to 2012. Compared with the usual BP arm, the strict BP arm had unadjusted and adjusted relative risks of ESRD of 0.92 (95% confidence interval [95% CI], 0.75 to 1.12) and 0.95 (95% CI, 0.78 to 1.16; P=0.64), respectively, and unadjusted and adjusted relative risks of death of 0.92 (95% CI, 0.77 to 1.10) and 0.81 (95% CI, 0.68 to 0.98; P=0.03), respectively. In meta-analyses of individual-level data from the MDRD and the AASK trials, unadjusted relative risk of ESRD was 0.88 (95% CI, 0.78 to 1.00) and unadjusted relative risk of death was 0.87 (95% CI, 0.76 to 0.99) for strict versus usual BP arms. Our findings suggest that, during long-term follow-up, strict BP control does not delay the onset of ESRD but may reduce the relative risk of death in CKD., (Copyright © 2017 by the American Society of Nephrology.)

The cardioprotective inducible enzyme heme oxygenase-1 (HO-1) degrades prooxidant heme into equimolar quantities of carbon monoxide, biliverdin, and iron. We hypothesized that HO-1 mediates cardiac protection, at least in part, by regulating mitochondrial quality control. We treated WT and HO-1 transgenic mice with the known mitochondrial toxin, doxorubicin (DOX). Relative to WT mice, mice globally overexpressing human HO-1 were protected from DOX-induced dilated cardiomyopathy, cardiac cytoarchitectural derangement, and infiltration of CD11b+ mononuclear phagocytes. Cardiac-specific overexpression of HO-1 ameliorated DOX-mediated dilation of the sarcoplasmic reticulum as well as mitochondrial disorganization in the form of mitochondrial fragmentation and increased numbers of damaged mitochondria in autophagic vacuoles. HO-1 overexpression promotes mitochondrial biogenesis by upregulating protein expression of NRF1, PGC1α, and TFAM, which was inhibited in WT animals treated with DOX. Concomitantly, HO-1 overexpression inhibited the upregulation of the mitochondrial fission mediator Fis1 and resulted in increased expression of the fusion mediators, Mfn1 and Mfn2. It also prevented dynamic changes in the levels of key mediators of the mitophagy pathway, PINK1 and parkin. Therefore, these findings suggest that HO-1 has a novel role in protecting the heart from oxidative injury by regulating mitochondrial quality control.

Renal ischemia-reperfusion injury is mediated by a complex cascade of events, including the immune response, that occur secondary to injury to renal epithelial cells. We tested the hypothesis that heme oxygenase-1 (HO-1) expression, which is protective in ischemia-reperfusion injury, regulates trafficking of myeloid-derived immune cells in the kidney. Age-matched male wild-type (HO-1(+/+)), HO-1-knockout (HO-1(-/-)), and humanized HO-1-overexpressing (HBAC) mice underwent bilateral renal ischemia for 10 minutes. Ischemia-reperfusion injury resulted in significantly worse renal structure and function and increased mortality in HO-1(-/-) mice. In addition, there were more macrophages (CD45(+) CD11b(hi)F4/80(lo)) and neutrophils (CD45(+) CD11b(hi) MHCII(-) Gr-1(hi)) in HO-1(-/-) kidneys than in sham and HO-1(+/+) control kidneys subjected to ischemia-reperfusion. However, ischemic injury resulted in a significant decrease in the intrarenal resident dendritic cell (DC; CD45(+)MHCII(+)CD11b(lo)F4/80(hi)) population in HO-1(-/-) kidneys compared with controls. Syngeneic transplant experiments utilizing green fluorescent protein-positive HO-1(+/+) or HO-1(-/-) donor kidneys and green fluorescent protein-negative HO-1(+/+) recipients confirmed increased migration of the resident DC population from HO-1(-/-) donor kidneys, compared to HO-1(+/+) donor kidneys, to the peripheral lymphoid organs. This effect on renal DC migration was corroborated in myeloid-specific HO-1(-/-) mice subjected to bilateral ischemia. These mice also displayed impaired renal recovery and increased fibrosis at day 7 after injury. These results highlight an important role for HO-1 in orchestrating the trafficking of myeloid cells in AKI, which may represent a key pathway for therapeutic intervention., (Copyright © 2015 by the American Society of Nephrology.)

Interleukin-10 (IL-10) is a pleiotropic cytokine that plays a pivotal role in the regulation of immune responses. Hence, we evaluated the effects of a recombinant adeno-associated viral vector 1 (rAAV1) encoding rat IL-10 (rAAV1-IL-10) in a rat model of kidney allograft rejection. Dark Agouti rat kidneys were transplanted into Wistar-Furth (WF) rats 8 weeks following a single intramuscular administration of either rAAV1-IL-10 or rAAV1-green fluorescence protein (GFP). Isografts (WF-WF) served as an additional experimental control. Both allograft and isograft recipients received daily cyclosporine (10 mg/kg) for 14 days after transplantation. Serum IL-10 levels increased at 8, 12 and 16 weeks following vector administration in rAAV1-IL-10-treated animals, but not in rAAV1-GFP and isograft groups. rAAV1-IL-10 treatment resulted in lower BUN and creatinine levels (p<0.001), as well as increased allograft survival rates from 22% to 90%. Allograft histological abnormalities were significantly attenuated in the rAAV1-IL-10-treated rats compared with those of rAAV1-GFP controls. Serum levels of proinflammatory cytokines such as growth-related oncogene were also significantly higher in the rAAV1-GFP group than in the rAAV1-IL-10 group. These data suggest delivery of IL-10 using a rAAV1 vector improves renal function and prolongs graft survival in a rat model of kidney transplant rejection.

The role of VEGF in vascular disease is complicated. Vascular endothelial growth factor (VEGF) expression can be deleterious in diabetic vasculopathy, especially in kidney and retina. In contrast, VEGF seems to be renoprotective in nondiabetic renal disease. VEGF exerts it biologic effects in association with nitric oxide (NO), yet it is known that NO bioavailability is reduced in diabetes. Thus, it was hypothesized that this diverse biologic effect of VEGF on diabetic vasculopathy is due to uncoupling of VEGF with NO. VEGF stimulated NO production in a dose-dependent manner in bovine aortic endothelial cells (BAEC), and this was inhibited by either high glucose or Nomega-nitro-l-arginine methyl ester (L-NAME) treatment. Endothelial NO synthase phosphorylation by VEGF was also inhibited by high glucose. It is interesting that both high glucose and L-NAME enhanced the proliferative response of endothelial cells, which was prevented by an NO donor. Furthermore, high glucose as well as L-NAME stimulated VEGF and kinase-insert domain receptor (KDR) (VEGF receptor 2) mRNA expression in BAEC. These data suggest that the uncoupling of VEGF with NO enhances endothelial cell proliferation via the KDR pathway. Compatible with these findings, a KDR antagonist blocked this response. In addition, a VEGF mutant, which binds only KDR, induced extracellular signal-regulated kinase (ERK) activation, and inhibition of ERK completely blocked endothelial cell proliferation under this condition, suggesting a role of the KDR-ERK1/2 pathway on endothelial cell proliferation. In conclusion, high glucose causes an uncoupling of VEGF with NO, which enhances endothelial cell proliferation via activation of the KDR-ERK1/2 pathway. These results may provide new insights into the understanding of the mechanism of diabetic vascular disease.

Interleukin 10 (IL-10) is a pleiotropic cytokine with well known antiinflammatory, immunosuppressive, and immunostimulatory properties. Chronic allograft rejection, characterized by vascular neointimal proliferation, is a major cause of organ transplant loss, particularly in heart and kidney transplant recipients. In a Dark Agouti to Lewis rat model of aortic transplantation, we evaluated the effects of a single intramuscular injection of a recombinant adeno-associated viral vector (serotype 1) encoding IL-10 (rAAV1-IL-10) on neointimal proliferation and inflammation. rAAV1-IL-10 treatment resulted in a significant reduction of neointimal proliferation and graft infiltration with macrophages and T and B lymphocytes. The mechanism underlying the protective effects of IL-10 in aortic allografts involved heme oxygenase 1 (HO-1) because inhibition of HO activity reversed not only neointimal proliferation but also inflammatory cell infiltration. Our results indicate that IL-10 attenuates neointimal proliferation and inflammatory infiltration and strongly imply that HO-1 is an important intermediary through which IL-10 regulates the inflammatory responses associated with chronic vascular rejection.

Recombinant adeno-associated virus (rAAV) has become an attractive tool for gene therapy because of its ability to transduce both dividing and nondividing cells, elicit a limited immune response, and the capacity for imparting long-term transgene expression. Previous studies have utilized rAAV serotype 2 predominantly and found that transduction of vascular cells is relatively inefficient. The purpose of the present study was to evaluate the transduction efficiency of rAAV serotypes 1 through 5 in human and rat aortic endothelial cells (HAEC and RAEC). rAAV vectors with AAV2 inverted terminal repeats containing the human alpha1-antitrypsin (hAAT) gene were transcapsidated using helper plasmids to provide viral capsids for the AAV1 through 5 serotypes. True type rAAV2 and 5 vectors encoding beta-galactosidase or green fluorescence protein were also studied. Infection with rAAV1 resulted in the most efficient transduction in both HAEC and RAEC compared to other serotypes (p < 0.001) at 7 days posttransduction. Interestingly, expression was increased in cells transduced with rAAV5 to levels surpassing rAAV1 by day 14 and 21. Transduction with rAAV1 was completely inhibited by removal of sialic acid with sialidase, while heparin had no effect. These studies are the first demonstration that sialic acid residues are required for rAAV1 transduction in endothelial cells. Transduction of rat aortic segments ex vivo and in vivo demonstrated significant transgene expression in endothelial and smooth muscle cells with rAAV1 and 5 serotype vectors, in comparison to rAAV2. These results suggest the unique potential of rAAV1 and rAAV5-based vectors for vascular-targeted gene-based therapeutic strategies.

Gene therapy has the potential to provide a therapeutic strategy for numerous renal diseases such as diabetic nephropathy, chronic rejection, Alport syndrome, polycystic kidney disease, and inherited tubular disorders. In previous studies using cationic liposomes or adenoviral or retroviral vectors to deliver genes into the kidney, transgene expression has been transient and often associated with adverse host immune responses, particularly with the use of adenoviral vectors. The unique properties of recombinant adeno-associated viral (rAAV) vectors permit long-term stable transgene expression with a relatively low host immune response. The purpose of the present study was to evaluate gene expression in the rat kidney after intrarenal arterial infusion of a rAAV (serotype 2) vector encoding green fluorescence protein (GFP) induced by a cytomegalovirus-chicken beta-actin hybrid promoter. The left kidney of experimental animals was treated with either saline or transduced with rAAV2-GFP (0.125 ml/100 g body wt, 1 x 10(10)/ml infectious units) through the renal artery. A time-dependent expression of GFP was observed in all kidneys injected with rAAV2-GFP, with maximal expression observed at 6 wk posttransduction. The expression of GFP was restricted to cells in the S(3) segment of the proximal tubule and intercalated cells in the collecting duct, the latter identified by co-localization with H(+)-ATPase. No transduction was observed in the glomeruli or the intrarenal vasculature. These studies demonstrate successful transgene expression in tubular epithelial cells, specifically in the S(3) segment of the proximal tubule and intercalated cells, after intrarenal administration of a rAAV vector and provide the impetus for further studies to exploit its use as a tool for gene therapy in the kidney.

Recent studies have demonstrated that a novel anion exchanger, pendrin, is expressed in the apical domain of type B intercalated cells in the mammalian collecting duct. The purpose of this study was 1) to determine the expression and distribution of pendrin along the collecting duct and connecting tubule of mouse and rat kidney and establish whether pendrin is expressed in the non-A-non-B intercalated cells and 2) to determine the intracellular localization of pendrin in the different populations of intercalated cells by immunoelectron microscopy. A peptide-derived affinity-purified antibody was generated that specifically recognized pendrin in immunoblots of rat and mouse kidney. Immunohistochemistry and confocal laser scanning microscopy demonstrated the presence of pendrin in apical domains of all type B intercalated cells in mouse and rat connecting tubule and collecting duct. In addition, strong pendrin immunostaining was observed in non-A-non-B intercalated cells. There was no labeling of type A intercalated cells. Immunoelectron microscopy demonstrated that pendrin was located in the apical plasma membrane and intracellular vesicles of both type B intercalated cells and non-A-non-B cells; the latter was identified by the presence of H(+)-ATPase in the apical plasma membrane. The results of this study demonstrate that both pendrin and H(+)-ATPase are expressed in the apical plasma membrane of non-A-non-B intercalated cells, suggesting that these cells are capable of both HCO and proton secretion. Furthermore, the presence of pendrin in both the apical plasma membrane and the apical intracellular vesicles of type B and non-A-non-B intercalated cells suggests that HCO secretion may be regulated by trafficking of pendrin between the two membrane compartments.

Context: Incidence of end-stage renal disease due to hypertension has increased in recent decades, but the optimal strategy for treatment of hypertension to prevent renal failure is unknown, especially among African Americans., Objective: To compare the effects of an angiotensin-converting enzyme (ACE) inhibitor (ramipril), a dihydropyridine calcium channel blocker (amlodipine), and a beta-blocker (metoprolol) on hypertensive renal disease progression., Design, Setting, and Participants: Interim analysis of a randomized, double-blind, 3 x 2 factorial trial conducted in 1094 African Americans aged 18 to 70 years with hypertensive renal disease (glomerular filtration rate [GFR] of 20-65 mL/min per 1.73 m(2)) enrolled between February 1995 and September 1998. This report compares the ramipril and amlodipine groups following discontinuation of the amlodipine intervention in September 2000., Interventions: Participants were randomly assigned to receive amlodipine, 5 to 10 mg/d (n = 217), ramipril, 2.5 to 10 mg/d (n = 436), or metoprolol, 50 to 200 mg/d (n = 441), with other agents added to achieve 1 of 2 blood pressure goals., Main Outcome Measures: The primary outcome measure was the rate of change in GFR; the main secondary outcome was a composite index of the clinical end points of reduction in GFR of more than 50% or 25 mL/min per 1.73 m(2), end-stage renal disease, or death., Results: Among participants with a urinary protein to creatinine ratio of >0.22 (corresponding approximately to proteinuria of more than 300 mg/d), the ramipril group had a 36% (2.02 [SE, 0.74] mL/min per 1.73 m(2)/y) slower mean decline in GFR over 3 years (P =.006) and a 48% reduced risk of the clinical end points vs the amlodipine group (95% confidence interval [CI], 20%-66%). In the entire cohort, there was no significant difference in mean GFR decline from baseline to 3 years between treatment groups (P =.38). However, compared with the amlodipine group, after adjustment for baseline covariates the ramipril group had a 38% reduced risk of clinical end points (95% CI, 13%-56%), a 36% slower mean decline in GFR after 3 months (P =.002), and less proteinuria (P<.001)., Conclusion: Ramipril, compared with amlodipine, retards renal disease progression in patients with hypertensive renal disease and proteinuria and may offer benefit to patients without proteinuria.

In this study, a nitric oxide (NO) sensor was used to examine the ability of angiotensin II (AngII), AngIV, and bradykinin (Bk) to stimulate NO release from porcine pulmonary artery (PPAE) and porcine aortic endothelial (PAE) cells and to explore the mechanism of the AngII-stimulated NO release. Physiologic concentrations of AngII, but not Bk, caused release of NO from PPAE cells. In contrast, Bk, but not AngII, stimulated NO release from PAE cells. AngIII-stimulated NO release from PPAE cells required extracellular L-arginine and was inhibited by L-nitro-arginine methyl ester. AT1 and AT2 receptor inhibition had no affect on AngII-mediated NO release or activation of NO synthase (NOS). AngIV, an AngII metabolite with binding sites that are pharmacologically distinct from the classic AngII receptors, stimulated considerably greater NO release and greater endothelial-type constitutive NOS activity than the same amount of AngII. The AngIV receptor antagonist, divalinal AngIV, blocked both AngII- and AngIV-mediated NO release as well as NOS activation. The results demonstrate that AngIV and the AngIV receptor are responsible, at least in part, for AngII-stimulated NO release and the associated endothelium-dependent vasorelaxation. Furthermore, these results suggest that differences exist in both AngII- and Bk-mediated NO release between PPAE and PAE cells, which may reflect important differences in response to these hormones between vascular beds.

At least two populations of intercalated cells, type A and type B, exist in the connecting tubule (CNT), initial collecting tubule (ICT), and cortical collecting duct (CCD). Type A intercalated cells secrete protons via an apical H+-ATPase and reabsorb bicarbonate by a band 3-like Cl-/HCO3-exchanger, AE1, located in the basolateral plasma membrane. Type B intercalated cells secrete bicarbonate by an apical Cl-/HCO3- exchanger that is distinct from AE1 and remains to be identified. They express H+-ATPase in the basolateral plasma membrane and in vesicles throughout the cytoplasm. A third type of intercalated cell with apical H+-ATPase, but no AE1, has been described in the CNT and CCD of both rat and mouse. The prevalence of the third cell type is not known. The aim of this study was to characterize and quantify intercalated cell subtypes, including the newly described third non A-non B cell, in the CNT, ICT, and CCD of the rat and mouse. A triple immunolabeling procedure was developed in which antibodies to H+-ATPase and band 3 protein were used to identify subpopulations of intercalated cells, and segment-specific antibodies were used to identify distal tubule and collecting duct segments. In both rat and mouse, intercalated cells constituted approximately 40% of the cells in the CNT, ICT, and CCD. Type A, type B, and non A-non B intercalated cells were observed in all of the three segments, with type A cells being the most prevalent in both species. In the mouse, however, non A-non B cells constituted more than half of the intercalated cells in the CNT, 39% in the ICT, and 22% in the CCD, compared with 14, 7, and 5%, respectively, in the rat. In contrast, type B intercalated cells accounted for only 8 to 16% of the intercalated cells in the three segments in the mouse compared with 26 to 39% in the rat. It is concluded that striking differences exist in the prevalence and distribution of the different types of intercalated cells in the CNT, ICT, and CCD of rat and mouse. In the rat, the non A-non B cells are fairly rare, whereas in the mouse, they constitute a major fraction of the intercalated cells, primarily at the expense of the type B intercalated cells.

Recent studies have suggested that less than 10% of intercalated cells in the rabbit outer cortical collecting duct (CCD) [1, 2] and less than 3% in the connecting segment (CNT) [3] are identifiable by functional criteria as acid-secreting (type A or alpha) intercalated cells. Other studies, using peanut lectin-binding and the absence of apical endocytic activity to identify bicarbonate-secreting (type B or beta) intercalated cells, have suggested that acid-loading increases the percentage of alpha intercalated cells in the CCD. Because our preliminary observations of band 3 immunoreactivity suggest that the percentages of alpha intercalated cells in the rabbit outer CCD and the CNT are underestimated by physiologic studies and are not altered by chronic acid-loading, we quantified the percentage of alpha intercalated cells in various segments of the collecting duct using light microscopic immunohistochemistry in kidneys of rabbits receiving tap water (control) or 75 mM NH4Cl for 12 days plus 8 daily gavages of 2 to 6 mEq NH4Cl/kg body wt. Mean urine pH values were 5.96 in acid-loaded animals versus 8.47 in controls. Kidneys were preserved by in vivo perfusion with periodatelysine-paraformaldehyde fixation and processed for immunohistochemical colocalization using sequential labeling with monoclonal antibodies and peanut lectin, followed by immunoperoxidase detection. Colocalization of band 3 and carbonic anhydrase II immunoreactivity revealed the following percentages of band 3-positive intercalated cells in control versus NH4Cl rabbits: CNT, 49.0 versus 52.8; initial collecting tubule (ICT), 27.2 versus 34.5; outer CCD, 33.5 versus 30.3; inner CCD, 38.2 versus 41.8; corticomedullary CD, 67.9 versus 58.8. There were no differences between the groups for all comparisons. Similar results were obtained using band 3 protein immunoreactivity and peanut lectin-binding to identify intercalated cell subtypes. However, in NH4Cl-loaded rabbits, peanut lectin-binding was observed in band 3 positive intercalated cells in the outer medullary CD. We conclude that: (1) the percentage of alpha intercalated cells in rabbit CCD subsegments are approximately 50% in the CNT, 30% in the ICT and the outer CCD, 40% in the inner CCD, and 60% in the corticomedullary CD; (2) the percentage of alpha intercalated cells is not altered by chronic NH4Cl-loading; (3) peanut lectin is not a specific marker of beta intercalated cells.

At birth, the rat renal papilla has the structural composition of the mature inner stripe of the outer medulla. All loops of Henle have the configuration of short loops, and there are no ascending thin limbs. This study examines the role of apoptosis in the differentiation of the loop of Henle and the development of the ascending thin limb in the rat kidney. Kidneys of 20-day-old fetuses and 1-, 3-, 5-, 7-, 14-, and 21-day-old pups were preserved for immunohistochemistry and electron microscopy. Using a preembedding immunoperoxidase method, we identified thick ascending limbs by labeling with antibodies to the serotonin receptor, 5-HT1A, and descending thin limbs were identified by labeling with antibodies to aquaporin-1. Three methods were used to identify apoptotic cells as follows: 1) in situ nick end labeling using the ApopTag kit, 2) toluidine blue staining on plastic sections followed by etching, and 3) transmission electron microscopy. At birth, tubules with 5-HT1A immunoreactivity were present throughout the renal papilla, and there were no ascending thin limbs. From 1 to 14 days of age, staining for apoptosis was observed in numerous cells in the 5-HT1A-positive epithelium, beginning at the papillary tip and ascending to the border between outer and inner medulla. This was associated with transformation from a cuboidal to a squamous epithelium and subsequent disappearance of 5-HT1A immunostaining from the transformed cells. Electron microscopy confirmed the presence of apoptotic cells and phagocytosed apoptotic bodies in the thick ascending limb in the renal papilla. We conclude that the ascending thin limb is derived from the 5-HT1A-positive thick ascending limb by apoptotic deletion of thick ascending limb cells and transformation of the remaining tubule cells into the 5-HT1A-negative ascending thin limb.

The response of the glomerular mesangial cell to reactive oxygen species (ROS) has been implicated in the pathogenesis of several forms of inflammatory renal disease. As a model of glomerular inflammation, we studied the effects of heat-aggregated immunoglobulin G (HAG), an experimental immune complex, and interleukin-1 (IL-1) on the expression of the antioxidant enzyme, manganese superoxide dismutase (MnSOD), in primary cultures of rat mesangial cells. Following exposure to either HAG or IL-1 beta, increased steady-state MnSOD mRNA levels were observed and an additive effect was seen following co-treatment with HAG and IL-1 beta. No change was observed in steady-state levels of copper/zinc superoxide dismutase mRNA with HAG or IL-1 beta exposure. RNA protein synthesis inhibitor experiments demonstrated that the HAG- and IL-1 beta-mediated increase in MnSOD mRNA was dependent on new transcription, but was independent of de novo protein synthesis. Our data on the induction of the MnSOD gene by immune complexes and IL-1 beta could indicate an important role for MnSOD in the cellular defense against ROS toxicity in glomerular inflammation.

In the developing rat kidney, both type A and type B intercalated cells are present throughout the medullary collecting duct (MCD), as well as the papillary surface epithelium. After birth, intercalated cells gradually disappear from the papillary surface epithelium and the terminal MCD, and type B cells disappear from the entire MCD. The purpose of this study was to establish the mechanism(s) by which intercalated cells are deleted from the MCD during development. Kidneys from 14-, 16-, 18-, and 20-day-old fetuses and 1-, 3-, 7-, and 14-day-old pups were preserved for light microscopic immunohistochemistry and electron microscopy. Intercalated cells were identified by immunostaining for H(+)-adenosinetriphosphatase (H(+)-ATPase) and band 3 protein. Apoptosis was identified by nick end labeling of DNA fragments, staining with the vital dye toluidine blue, and transmission electron microscopy. Two distinct mechanisms of elimination of intercalated cells were detected. Cells with apical labeling for H(+)-ATPase and basolateral labeling for band 3 protein protruded into the lumen of the MCD as if they were being extruded from the epithelium, and many had lost contact with the basement membrane. Extrusion of the cells with basolateral H(+)-ATPase or with no labeling for H(+)-ATPase was never observed. Apoptosis was observed in the MCD from shortly before birth to 7 days after birth, gradually progressing from the papillary tip toward the outer medulla. Staining for apoptosis was present in H(+)-ATPase-positive apoptotic bodies, located in cells that were negative for H(+)-ATPase. Staining was also occasionally observed in apoptotic cells with basolateral H(+)-ATPase but never in cells with apical H(+)-ATPase. Electron microscopy confirmed the presence of apoptotic intercalated cells in the MCD and demonstrated that apoptotic bodies were located in inner medullary collecting duct (IMCD) cells and principal cells. These results demonstrate that intercalated cells are deleted from the MCD by two distinct mechanisms, one involving apoptosis and subsequent phagocytosis by neighboring principal cells or IMCD cells. Elimination by extrusion affects only type A intercalated cells, whereas deletion by apoptosis appears to occur only in type B intercalated cells.

Structurally and functionally distinct populations of intercalated cells have been described in the collecting duct of both rat and rabbit. However, little is known about these cells in the mouse kidney. The study presented here examines ultrastructural and immunological characteristics of different types of intercalated cells in the mouse. Kidneys of two strains of normal female mice, C57BL/6 and IBR, were preserved by in vivo perfusion with 1% glutaraldehyde or paraformaldehyde-picric acid fixatives and processed for morphological evaluation or light and electron microscopic immunohistochemistry, respectively. The avidin-biotin-horseradish peroxidase procedure was performed on was sections using antibodies against carbonic anhydrase II, H+ -ATPase and Band 3 protein. Immunogold cytochemistry was performed on Lowicryl sections using antibodies to H+ -ATPase and Band 3 protein. Colocalization of H+ -ATPase and Band 3 protein was performed by double labeling using an immunogold technique with silver enhancement. Intercalated cells identified by positive staining for H+ -ATPase and carbonic anhydrase II constituted 35% to 40% of all cells in the connecting tubule (CNT), cortical collecting duct (CCD), and outer medullary collecting duct (OMCD). Type A intercalated cells identified by positive Band 3 staining constituted 16%, 24%, and 33% of the total cell population in the CNT, CCD, and OMCD, respectively. Electron microscopy and immunogold cytochemistry demonstrated three distinct populations of intercalated cells. Type A intercalated cells with apical H+ -ATPase and basolateral Band 3 immunoreactivity were present in all segments examined, and had prominent apical microprojections and characteristic tubulovesicular structures beneath the apical surface, both coated with studs on the cytoplasmic face. Type B intercalated cells with basolateral and cytoplasmic H+-ATPase and no Band 3 immunoreactivity were most frequently observed in the initial collecting tubule, but were present also in the CNT and early CCD. Type B intercalated cells had a fairly smooth apical surface, a gray zone free of organelles beneath the apical plasma membrane, and small cytoplasmic vesicles without studs throughout the cell. A third type of intercalated cell with apical and cytoplasmic H+-ATPase, but no basolateral Band 3 protein, was observed exclusively in the CNT and the initial collecting tubule. This type of cell was large, with numerous mitochondria, and vesicles coated with studs were present throughout the cell. It resembled a third type of intercalated cell described previously in the rat. It is concluded that three morphologically and immunologically distinct types of intercalated cells are present in the mouse kidney.

Angiotensin II (ANG II) regulates whole kidney ion transport, yet its effects in the collecting duct are unknown. The purpose of these studies was to determine whether ANG II regulates luminal alkalinization and acidification in the rabbit cortical collecting duct (CCD). The rate of luminal alkalinization or acidification was measured as the rate of change of luminal fluid pH under stop-flow conditions using in vitro microperfused CCD segments. Outer CCD alkalinized the luminal fluid, consistent with net HCO3- secretion. Addition of ANG II, 10(-7) M, to the peritubular solution for 30 min significantly stimulated luminal alkalinization. The stimulatory effect of ANG II was not due to time-dependent effects and was blocked by peritubular addition of the ANG II type 1 (AT1) receptor antagonist, losartan, at 10(-6) M. Losartan, 10(-6) M, when added to the peritubular solution, did not alter the rate of luminal alkalinization independent of ANG II. In contrast, peritubular ANG II, 10(-7) M, did not alter inner CCD luminal acidification. Addition of ANG II to the peritubular solution at the lower concentration of 10(-10) M did not alter the rates of luminal alkalinization and acidification in the outer and inner CCD, respectively. Peritubular ANG II, 10(-7) M, but not vehicle, stimulated B cell apical HCO3- secretion occurring in response to peritubular Cl- removal. These studies demonstrate that ANG II acts through a basolateral AT1 receptor to stimulate outer CCD luminal alkalinization via, at least in part, B cell stimulation.

Reactive oxygen species have been implicated as mediators of tissue injury in glomerular inflammation. The expression of the antioxidant enzyme, manganese superoxide dismutase (MnSOD), was examined in primary cultures of rat glomerular epithelial cells (GEC) in response to inflammatory mediators. The results demonstrate that GEC respond to interleukin-1 (IL-1) and bacterial lipopolysaccharride (LPS) with an increase in MnSOD steady-state mRNA levels. The IL-1 alpha-mediated induction of MnSOD mRNA levels was both time- and dose-dependent. Maximal levels approximately 40-fold above controls, were observed at 12 hours with 2 ng/ml of IL-1 alpha. MnSOD protein levels were also markedly elevated by IL-1 alpha. The induction of MnSOD mRNA by IL-1 alpha required de novo transcription as well as some degree of protein synthesis. To elucidate the potential intracellular signal that mediates IL-1 alpha-dependent MnSOD expression, three classical signaling pathways were examined. We found no evidence that MnSOD induction by IL-1 alpha is mediated by either the cyclooxygenase or lipoxygenase pathway or via activation of protein kinase C. Based on the presence of IL-1 alpha in several forms of glomerular inflammation, the observed increase in MnSOD expression by this immunoregulatory cytokine must have an important role in the antioxidant defense of glomerular epithelial cells.

Angiotensin II (ANG II) plays an important role in the regulation of solute transport in the kidney, and its effect on proximal tubule sodium and fluid transport has been studied extensively. Although there is evidence that ANG II receptors are present also in the distal nephron and collecting duct, little is known about the physiological role of ANG II in these segments of the renal tubule. Preliminary studies in our laboratory suggest that ANG II may have both structural and functional effects on intercalated cells in the cortical collecting duct (CCD). Therefore, the present study examines the effect of ANG II on H(+)-adenosinetriphosphatase (H(+)-ATPase) and H(+)-K(+)-ATPase activity in individual CCD segments microdissected from collagenase-treated rat kidneys. The H(+)-ATPase was measured as bafilomycin-sensitive ATPase activity, and H(+)-K(+)-ATPase was measured as Sch-28080-sensitive ATPase activity, by a fluorometric microassay. Preincubation of CCD segments with ANG II, 10(-10)-10(-5) M, caused a dose-dependent decrease in H(+)-ATPase activity with maximum inhibition at 10(-8) M of ANG II. The inhibitory effect of ANG II was abolished when tubules were incubated with ANG II in the presence of 10(-6) M losartan, indicating that the inhibition was mediated via specific AT1 receptors. The AT2-receptor antagonist, PD-123319, had no effect on the ANG II-mediated inhibition of H(+)-ATPase activity. Preincubation of CCD segments with 10(-10) or 10(-7) M ANG II had no effect on H(+)-K(+)-ATPase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Nitric oxide (NO) is a messenger molecule that is produced from L-arginine by NO synthase (NOS). Some NOS isoforms are present in cells constitutively, whereas others can be induced by cytokines. Recent evidence suggests that NO inhibits intracellular pH regulation by the vacuolar H(+)-adenosinetriphosphatase (ATPase) in macrophages, which contain an inducible form of NOS. The vacuolar H(+)-ATPase is involved in proton secretion in intercalated cells in the collecting duct. We have therefore examined the effect of NO on bafilomycin-sensitive H(+)-ATPase activity in individual cortical collecting ducts (CCD) microdissected from collagenase-treated kidneys of normal rats using a fluorometric microassay. Incubation of CCD with the NO donors, sodium nitroprusside (0.1 and 1 mM) or 3-morpholino-sydnonimine hydrochloride (SIN-1, 30 microM), caused a dose-dependent decrease in H(+)-ATPase activity. Incubation of CCD with lipopolysaccharide (LPS) and interferon-gamma, which induces NOS in macrophages, decreased H(+)-ATPase activity by 85%. This effect was prevented by simultaneous incubation with N omega-nitro-L-arginine, a competitive inhibitor of NOS, indicating that the decrease in H(+)-ATPase activity was caused by NO production. Incubation with 8-bromo-guanosine 3',5'-cyclic monophosphate (cGMP) also inhibited H(+)-ATPase activity, suggesting that NO may exert its effect in the CCD via activation of guanylyl cyclase and production of cGMP. Immunohistochemistry using antibodies to the macrophage-type NOS revealed strong labeling of intercalated cells in the CCD, confirming the presence of NOS in these cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Virtually all diseases affecting the native kidney recur in the kidney transplant with the exception of Alport syndrome, polycystic kidney disease, hypertension, chronic pyelonephritis, and chronic interstitial nephritis. Fortunately, in the majority of patients, recurrence of the original disease has minimal clinical impact, with only approximately 5% of all graft loss occurring as a result of recurrent disease. The primary renal diseases that commonly recur include membranoproliferative glomerulonephritis type II, IgA nephropathy, and focal and segmental glomerular sclerosis. The most common systemic disease that recurs is diabetic nephropathy. Living-related transplantation should be used with caution in patients with the hemolytic uremic syndrome, recurrent focal and segmental glomerular sclerosis, and membraneous glomerulonephritis. Fabry disease and primary hyperoxaluria type I are no longer absolute contraindications to kidney transplantation.

Intercalated cells are present in both the collecting duct, which is derived from the ureteric bud, and the connecting tubule (CNT), which is part of the nephron and thus is developed from the metanephric blastema. However, the embryologic origin of the intercalated cells has not been established. Two populations of intercalated cells, type A and type B, exist in the CNT and the cortical collecting duct (CCD). It is uncertain, however, whether these cells represent truly distinct cell types or whether one is derived from the other. In this study we have used specific antibodies to carbonic anhydrase II (CA II), H(+)-adenosinetriphosphatase (H(+)-ATPase), and band 3 protein to identify subpopulations of intercalated cells, to determine the site and time of their appearance, and to follow their differentiation in the developing rat kidney. Prenatal kidneys from 16-, 17-, 18-, and 20-day-old fetuses, and postnatal kidneys from 0-, 3-, 7-, 14-, and 21-day-old pups were preserved for immunohistochemical studies. Immunostaining for CA II and H(+)-ATPase appeared simultaneously in a subpopulation of cells in the CNT and the medullary collecting duct (MCD) of the 18-day-old fetus, suggesting that intercalated cells differentiate from separate foci, one in the nephron and one in the collecting duct. Cells with apical and cells with basolateral labeling for H(+)-ATPase appeared in the CNT and MCD at 18 days of gestation, indicating that type A and type B cells differentiate simultaneously during renal development. Band 3 immunostaining was very weak in the fetal kidney, but a striking increase in labeling was observed in the 3-day-old kidney, suggesting that there is an activation of acid-secreting cells shortly after birth. In the fetal kidney, immunostaining for CA II and H(+)-ATPase was observed in cells throughout the MCD and on the papillary surface. After birth, immunostaining gradually disappeared from both the papillary surface and the terminal inner MCD, and cells with basolateral labeling for H(+)-ATPase gradually disappeared from the outer MCD. The results of this study suggest that type A and type B intercalated cells represent distinct cell types that derive from undifferentiated cells at two separate foci, one in the nephron and one in the collecting duct. Our results also suggest that entire populations of intercalated cells are eliminated from the collecting duct during normal renal development.

In normal rabbit, immunolabeling of intercalated cells in the outer medullary collecting duct (OMCD) demonstrates band 3-like protein in the basolateral plasma membrane (15) and H(+)-adenosinetriphosphatase (H(+)-ATPase) in the apical plasma membrane and cytoplasmic vesicles (30). However, in type A intercalated cells in the cortical collecting duct (CCD), band 3-like protein is located primarily in multivesicular bodies and cytoplasmic vesicles (15), whereas H(+)-ATPase is present in cytoplasmic vesicles only in most intercalated cells (30). In this study, we observed the effect of chronic acid loading on immunolocalization of these transporters in the collecting duct. Adult New Zealand White rabbits received either normal tap water (controls) or 75 mM NH4Cl for 12 days plus eight daily gavages of 2-6 meq NH4Cl/kg body wt. At time of death, mean urine pH of acid-loaded animals was 5.96 (SD = 0.69), vs. 8.47 (SD = 0.07) in controls. Kidneys were fixed by in vivo perfusion and processed for light and electron microscopic immunoperoxidase localization of band 3-like protein and immunogold localization of H(+)-ATPase. In controls, band 3-like protein was largely confined to multivesicular bodies in the majority of positive-staining intercalated cells in the CCD and to the basolateral plasma membrane of intercalated cells in the OMCD. In acid-loaded rabbits, band 3 protein-positive intercalated cells in the inner CCD and the in the outer stripe of the OMCD (OMCDo) were strikingly stellate in form. Basolateral plasma membrane label was intensified, while the number of labeled multivesicular bodies was diminished. Morphometric analysis demonstrated an increase in the amount of basolateral plasma membrane in these intercalated cells. In control rabbits, H(+)-ATPase immunoreactivity in intercalated cells in the CCD was located predominantly over cytoplasmic vesicles. A minority of intercalated cells exhibited basolateral plasma membrane label, and only an occasional cell displayed apical plasma membrane label. In acid-loaded rabbits, H(+)-ATPase immunoreactivity was enhanced along the apical plasma membrane of intercalated cells in the inner CCD, and morphometric analysis demonstrated increased apical plasma membrane in band 3-positive intercalated cells in this segment. These results suggest that rabbits respond to acid loading via enhancement of both electrogenic proton secretion and Cl-/HCO3- exchange in intercalated cells in the inner CCD and the OMCDo.

The inner medullary collecting duct (IMCD) is the final portion of the mammalian renal tubule that is able to significantly regulate systemic acid-base balance. Although the H+ transporters of this segment are relatively well studied, little is known regarding the mechanisms of HCO3- transport. The mechanisms of HCO3- transport in primary cultures of rabbit IMCD were studied using the pH-sensitive dye, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, in CO2/HCO3(-)-containing solutions at 37 degrees C. Removal of Cl- from the extracellular solution caused reversible intracellular alkalinization, demonstrating the presence of Cl-/HCO3- exchange. Alkalinization with Cl- removal was independent of changes in membrane potential, did not require the presence of extracellular Na+, and was inhibited by the disulfonic stilbene, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS, 10(-4) M). Half-maximal intracellular pH (pHi) recovery with readdition of Cl- to the extracellular solution occurred at a Cl- concentration of 37.4 +/- 5.7 mM. When rabbit IMCD were cultured on permeable support membranes, Cl-/HCO3- exchange activity was found only on the basolateral membrane. However, there was no evidence of band 3 protein immunoreactivity. In contrast, no evidence for Na(+)-(HCO3-)n > 1 cotransport activity was found. Depolarization of IMCD cells by acute increases in extracellular K+ did not alter pHi, nor was Na(+)-dependent, 5-(N-ethyl-N-isopropyl)amiloride-insensitive pHi recovery from an acid load inhibited by DIDS (10(-4) M). Finally, recovery from intracellular alkalosis induced by incubation in 0 mM Cl-, 50 mM HCO3- extracellular solution required Cl- and was independent of Na+. These studies indicate that the major mechanism of HCO3- transport in primary cultures of the rabbit IMCD is via a band 3 protein-negative, Na(+)-independent, basolateral, Cl-/HCO3- exchanger.

In contrast to results obtained in the rat kidney, studies of H+ATPase localization in the rabbit kidney have failed to demonstrate basolateral plasma membrane H+ATPase immunoreactivity in intercalated cells in the cortical collecting duct (CCD). Previous studies have relied on light microscopic immunofluorescence techniques, which have limited resolution. Therefore, the immunogold procedure was used to localize H+ATPase in rabbit collecting ducts at the ultrastructural level. Rabbit kidneys were preserved in vivo with periodate-lysine-paraformaldehyde or glutaraldehyde solutions, and samples of cortex were embedded in Lowicryl K4M. Thin sections were labeled for H+ATPase by the immunogold procedure with a rabbit polyclonal antibody against the 70-kd subunit of bovine brain H+ATPase. Three patterns of localization of H+ATPase were observed. The majority of intercalated cells in the CCD exhibited label over cytoplasmic vesicles only. In these cells, no label was associated with either the apical or basolateral plasma membranes. In a second group of cells, label for H+ATPase was observed along the basolateral plasma membrane and over cytoplasmic vesicles throughout the cell. Rarely, intercalated cells with H+ATPase label along the apical plasma membrane and over the apical cytoplasmic vesicles were observed in the CCD. In the initial collecting tubule and connecting segment, intercalated cells with either pronounced apical or basolateral plasma membrane label prevailed, whereas few cells exhibited label restricted to the cytoplasmic vesicles. In summary, in the rabbit CCD, three patterns of H+ATPase distribution exist in intercalated cells, two of which conform to published models of type A and type B intercalated cells.

Nitric oxide (NO) is a messenger molecule that functions as a vasodilator and accounts for the biologic activity of endothelium-derived relaxing factor (EDRF). The enzyme responsible for NO production, NO synthase (NOS), exists in different isoforms. Some are expressed constitutively in various tissues, whereas others require induction by endotoxin and cytokines. There is evidence that NO plays an important role in the regulation of basal renal vascular resistance and in the tubuloglomerular feedback response. Specific antibodies to a constitutive NOS isolated from rat brain (B-NOS) and an inducible NOS isolated from rat aortic smooth muscle (VSM-NOS) were used to establish the cellular and subcellular distribution of NOS in the kidney. Kidneys from normal rats were preserved and processed for light and electron microscopic immunohistochemical studies using both a preembedding and a postembedding horseradish peroxidase technique. By light microscopy, strong immunostaining for B-NOS was observed in the juxtaglomerular apparatus, where it was located in cells of the macula densa. In contrast, immunostaining for VSM-NOS was specifically located in the terminal afferent arteriole and occasionally also in the initial efferent arteriole in the juxtaglomerular apparatus. In addition, faint immunostaining was present in the entire distal tubule. There was no labeling of arcuate or interlobular arteries. The injection of lipopolysaccharide was associated with increased immunostaining for VSM-NOS in the afferent arteriole but had no effect on B-NOS staining. By electron microscopy, B-NOS immunostaining was present throughout the cytoplasm of the macula densa cells, where it appeared to be associated mainly with small vesicles. Immunostaining for VSM-NOS in the afferent arteriole exhibited a patchy distribution in some cells, whereas other cells were stained throughout the cytoplasm. These results indicate that two distinct isoforms of NOS are present in the juxtaglomerular apparatus and support the hypothesis that NOS is involved in the regulation of tubuloglomerular feedback and glomerular capillary pressure.

We have used in situ hybridization histochemistry with isoform-specific, digoxigenin-labeled cRNA probes to characterize systematically the cellular distribution of mRNAs encoding alpha- and beta-subunit isoforms of the Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase) in the normal rat kidney. Transcripts encoding the alpha 1-, alpha 2-, alpha 3-, beta 1-, and beta 2-subunits were detected in virtually all of the nephron segments, with prominent hybridization signal in the S3 segment of the proximal tubule, the cortical and medullary thick ascending limb of Henle's loop, the distal convoluted tubule, the cortical collecting duct along its entire length, and the renal pelvic epithelium. Several differences in the cell-specific pattern of expression of the various isoforms were observed. Among the alpha-isoforms, the alpha 3-subunit appeared to be preferentially expressed in the glomerular podocytes and mesangial cells, papillary interstitial cells, and renal pelvic epithelium. The beta-isoforms also differed in their distribution pattern, with the beta 1-subunit expressed to a greater degree in the glomerulus and renal pelvic epithelium and the beta 2-subunit preferentially expressed in the papillary interstitial cells and papillary surface epithelium. The detection and expression pattern of alpha- and beta-subunit mRNAs in structures throughout the kidney is compatible with the possibility of six structurally unique Na(+)-K(+)-ATPase isozymes and suggests a potentially greater role for isozymes comprised of the alpha 2-, alpha 3-, and beta 2-subunits in renal sodium and potassium transport.

A disease syndrome similar to the hemolytic uremic syndrome of people is described in three dogs with acute renal failure. In each dog, hemorrhagic gastroenteritis preceded the onset of anuric acute renal failure. Evidence of microangiopathic hemolytic anemia (schizocytes, thrombocytopenia, and increased concentrations of fibrin split products) was present in the three dogs. Serum chemistry results showed increased concentrations of blood urea nitrogen, creatinine, and phosphorus. Ultrasound examination performed in one dog revealed increased echogenicity of the renal cortices. Treatment for anuric acute renal failure using a continuous dopamine and furosemide infusion established urine production in one of three dogs. Microscopic examination of tissue from the two dogs that underwent necropsy showed occlusion of the renal vasculature by fibrin thrombi consistent with microangiopathic arteriolar thrombosis. The pathophysiology and current knowledge of human hemolytic uremic syndrome is compared with hemolytic uremic syndrome in these dogs.

Northern blotting studies have demonstrated mRNA for the serotonin 5-HT1A receptor in human neonatal kidney (B. K. Kobilka, T. Frielle, S. Collins, T. Yang-Feng, T. S. Kobilka, U. Francke, R. J. Lefkowitz, and M. G. Caron. Nature Lond. 329: 75-79, 1987). To confirm expression of receptor protein in kidney, we raised antibodies to two peptides derived from the third intracellular loop of the human 5-HT1A receptor. Specific immunoglobulin G (IgG) was purified sequentially on protein A-Sepharose and peptide-Affigel 10 columns. Each IgG was able to: 1) quantitatively immunoprecipitate [3H]8-OH-2-(di-n-propylamino)1,2,3,4-tetrahydronaphthalene ([3H]8-OH-DPAT)-labeled human and rat receptors; 2) immunoblot a new protein in cells transfected with human 5-HT1A receptor DNA; and 3) immunoautoradiographically label areas of rat brain (frontal cortex, hippocampus, and lateral septum) in a highly characteristic pattern similar to that labeled by 125I-Bolton-Hunter-8-methoxy-2-(N-propyl-N-propylamino)Tetralin, a specific 5-HT1A receptor autoradiography ligand. By use of a light microscopic immunoperoxidase labeling technique, incubation of each IgG antibody with sections of rat and human kidney demonstrated an identical pattern of immunoreactivity. Specific labeling of basolateral plasma membranes was detected throughout medullary and cortical thick ascending limbs (TAL), in distal convoluted tubules (DCT), in connecting tubule cells of the connecting tubule, and in principal cells of the initial collecting tubule. There was no labeling in the inner medulla, glomeruli, or blood vessels. The labeling was blocked by preincubation with the corresponding peptide, but not with noncorresponding peptide or carrier protein. There was no labeling with preimmune IgG. Electron microscopic immunoperoxidase labeling confirmed the specific localization of the IgG antibody along the basolateral plasma membrane in all positively staining cells in rat kidney. Radioligand binding studies with the specific 5-HT1A receptor ligand [3H]8-OH-DPAT confirmed the presence of 5-HT1A receptor binding sites in bulk-isolated rat medullary TAL. These studies provide the first evidence that the 5-HT1A receptor is expressed on the basolateral surface of TAL and DCT cells of human and rat kidney. The specific localization to these cells suggests a possible role for the 5-HT1A receptor in the regulation of salt and water transport in mammalian kidney.

Intercalated cells (ICs) in the collecting duct and the connecting tubule (CNT) are involved in H+ secretion and HCO3- reabsorption. H+ secretion is mediated by an H(+)-adenosinetriphosphatase in the apical plasma membrane, whereas a band 3-like Cl(-)-HCO3- exchanger in the basolateral membrane is responsible for HCO3- reabsorption. Recent studies have reported that a band 3-like protein is also present in mitochondria in rabbit ICs. The purpose of this study was to establish the subcellular location of the band 3-like Cl(-)-HCO3- exchanger in rabbit ICs by electron microscopic immunocytochemistry using a monoclonal antibody, IVF12, against erythrocyte band 3 protein. Rabbit kidneys were preserved by in vivo perfusion with a paraformaldehyde-lysine-periodate solution and processed for immunocytochemistry using a horseradish peroxidase preembedding technique. Band 3 immunostaining was observed on the basolateral plasma membrane of ICs in the outer medullary collecting duct and type A cells in the cortical collecting duct (CCD) and CNT. In addition, distinct staining for band 3 was present in numerous small vesicles and in multivesicular bodies in type A ICs in the CCD and CNT. However, there was no evidence of band 3 immunostaining of mitochondria or of the apical plasma membrane in any cells of the collecting duct. These observations suggest that basolateral Cl(-)-HCO3- exchangers in type A ICs in the rabbit kidney are stored in intracellular vesicles and possibly degraded in the vascular-lysosomal system when these cells are in a resting state. The previously reported band 3 immunolabeling of mitochondria could not be confirmed.

We examined the effect of Cl- depletion metabolic alkalosis (CDA) on H(+)-ATPase and band 3 protein localization in intercalated cells (IC) of the rat cortical collecting duct (CCD) and the outer medullary collecting duct (OMCD). After 30 min of peritoneal dialysis against 0.15 M NaHCO3 to produce CDA, or Ringer bicarbonate to serve as controls (CON), both groups were infused intravenously with an 80 mM Cl- solution for 90 min. For CDA vs. CON, physiological parameters were as follows: plasma total CO2, 38.0 +/- 1.1 vs. 27.8 +/- 0.6 meq/l (P less than 0.001); urinary total CO2 excretion, 141 +/- 89 vs. 20 +/- 3 neq.min-1.100 g body wt-1; and urinary Cl- excretion, 20 +/- 10 vs. 486 +/- 144 neq.min-1.100 g body wt-1 (P less than 0.001). H(+)-ATPase was localized in thin sections using a rabbit polyclonal antibody against the 70-kDa subunit of bovine brain H(+)-ATPase. Band 3 protein was localized using a polyclonal antibody against the 43-kDa subunit of the cytoplasmic domain of human erythrocyte band 3 protein. In CON rats, H(+)-ATPase localized along the apical plasma membrane and over the apical cytoplasmic vesicles of type A ICs in the CCD and ICs of the OMCD. H(+)-ATPase was observed along the basolateral plasma membrane and over cytoplasmic vesicles throughout type B ICs. In CDA rats, H(+)-ATPase was only observed over apical cytoplasmic vesicles in type A ICs and in the majority of OMCD ICs. In type B ICs, H(+)-ATPase staining was intensified along the basal plasma membrane in CDA. Band 3 protein was consistently localized in the basolateral plasma membrane of all type A cells in the CCD and ICs of the OMCD in both CON and CDA. In summary, stimulation of HCO3- secretion in rats caused withdrawal of H(+)-ATPase from the apical plasma membrane and storage in apical cytoplasmic vesicles of ICs of the OMCD and type A ICs of the CCD. H(+)-ATPase appeared to be inserted into the basal plasma membrane of type B ICs. These findings suggest that, during correction of CDA, proton secretion by type A and OMCD ICs is suppressed and proton transport across the basolateral plasma membrane of type B ICs is stimulated.

Two populations of intercalated cells, type A and type B, are present in the rat cortical collecting duct (CCD). Type A cells are involved in proton secretion and contain an apical H(+)-adenosinetriphosphatase (ATPase) and a basolateral Cl(-)-HCO3- exchanger. Type B cells are believed to be involved in HCO3- secretion, which is mediated by a Cl(-)-HCO3- exchange process and is Cl- dependent. The aim of this study was to examine the morphological and immunocytochemical response of type B intercalated cells in the rat to increased delivery of Cl- to the CCD. This was accomplished by chronic infusion of a loop diuretic, bumetanide (30 mg.kg body wt-1.day-1), via an osmotic minipump, and simultaneous administration of 0.9% sodium chloride in the drinking water for 6 days. The kidneys were preserved by in vivo perfusion with a periodate-lysine-paraformaldehyde fixative and processed for horseradish peroxidase and protein A gold immunocytochemistry, using rabbit polyclonal antibodies against carbonic anhydrase II, proton ATPase, and band 3 protein. Chronic infusion of bumetanide in combination with a high salt intake was associated with significant changes in the intercalated cells. Type B cells were increased in size and exhibited numerous apical microvilli, increased basolateral membrane area, and marked cytoplasmic and basolateral labeling for H(+)-ATPase. In contrast, type A cells were small and had sparse apical microprojections. H(+)-ATPase immunolabeling was observed primarily over apical tubulovesicles, and there was decreased basolateral immunolabeling for band 3 protein and occasional labeling for band 3 in lysosome-like structures. These observations support the hypothesis that increased delivery of Cl- to the CCD is associated with stimulation of type B intercalated cells to secrete HCO3-. The observations in type A cells are consistent with the cells being in a resting or inactivated state.

The phosphoinositide system plays a critical role in mesangial cell contraction. myo-Inositol depletion occurs in glomeruli from diabetic animals and may result in mesangial cell dysfunction. The hypothesis that mesangial cell exposure to high concentrations of glucose could lead to abnormalities in phosphoinositide metabolism and receptor-mediated inositol phosphate release was tested. When compared with controls (5 mM glucose), inositol phosphate release in mesangial cells exposed to 28 mM glucose was decreased by 27% after maximal stimulation with angiotensin II, by 41% after arginine vasopressin, and by 63% after the thromboxane A2 analog, U46619. Increasing the concentration of glucose to 50 mM caused a further reduction (from 27 to 54%) in maximal angiotensin II stimulation of inositol phosphate release. High glucose decreased incorporation of myo-inositol into phospholipids but did not change phosphoinositide mass. High glucose also resulted in increased de novo synthesis of diacylglycerol which was associated with membrane translocation of protein kinase C. myo-inositol supplementation prevented the reduction in phosphoinositide hydrolysis whereas sorbinil did not. It was concluded that high concentrations of glucose cause abnormalities in myo-inositol metabolism in mesangial cells which lead to reduced receptor-mediated phosphoinositide hydrolysis. These abnormalities appear to be related to desensitization of receptor-mediated phosphoinositide responses due to negative feedback by protein kinase C which becomes activated as a result of enhanced de novo diacylglycerol formation from glucose. These changes are unrelated to the polyol pathway and can be prevented by myo-inositol supplementation.

Pregnancy is an unusual occurrence in women with ESRD. We report a successful pregnancy in a patient with ESRD who was receiving hemodialysis; she gave birth to a healthy female infant at 34 weeks' gestational age. We discuss possible physiologic causes of gonadal dysfunction in patients with ESRD.