Self-Rated Frailty and Mortality in Old Men: The Manitoba Follow-up Study.

BACKGROUND: While a multitude of definitions and operationalizations of frailty have been developed, rarely have these considered the perspective of the older adult themselves. This knowledge gap was addressed by examining older adults' self-rating of frailty. OBJECTIVES: To assess the validity of self-rated frailty and to determine whether self-rated frailty relates to mortality. DESIGN: The Manitoba Follow-up Study was initiated in 1948 as a prospective cohort study of 3,983 men. SETTING: Community dwelling older adult men. PARTICIPANTS: Survivors of the original cohort (231 men) were sent a quality of life survey in 2015. A response was received from 186 men, including 146 surveys completed by the participant himself and thus were eligible to include (completion rate of 78.4%). MEASUREMENTS: The quality of life survey is sent out annually to the study participants to ascertain information about mental, physical, and social functioning. In 2015, the Clinical Frailty Scale was adapted and added to the survey as a simple self-rating of frailty. RESULTS: The mean age of the 146 respondents in 2015 was 93.7 years (SD 2.7) Self-ratings of "moderate-severe" frailty, received from 132 men, were associated with worse measures of physical health and functional impairment, thus supporting the significance of self-rated frailty. Adjusted for age, the Hazard Ratio for mortality over the next 3 years was 3.3 (95% CI: 1.5, 7.1) for those who rated themselves as "mildly to severely frail" vs. "very fit or well, with no disease". CONCLUSION: The present study has illustrated that self-rated frailty is associated with other measures of health and that self-rated frailty predicts mortality over a three-year period. These findings support the utilization of older adult's self-ratings of frailty for new avenues of operationalizing frailty.

Social and societal implications of frailty, including impact on Canadian healthcare systems.

Frailty has many social and societal implications. Social circumstances are key both as contributors to frail older adults' health outcomes and as practical facilitators or barriers to intervention and supports. Frailty also has important societal implications for health systems and social care policy. In this discussion paper, we use a social ecology framework to consider the social and societal implications and impact of frailty at each level, from the individual, through relationships with family and friend caregivers, institutions, health systems, neighborhoods and communities, to society at large. We conclude by arguing that attention to these issues at a policy level is critical. We identify three target actions: 1) Social dimensions of frailty should be systematically considered when frailty is assessed. 2) Action is needed at the level of policies and programs to improve support for caregivers. 3) Policy review across all portfolios will benefit from a social frailty lens.

Covert stroke after non-cardiac surgery: a prospective cohort study.

BACKGROUND: Overt stroke after non-cardiac surgery has a substantial impact on the duration and quality of life. Covert stroke in the non-surgical setting is much more common than overt stroke and is associated with an increased risk of cognitive decline and dementia. Little is known about covert stroke after non-cardiac, non-carotid artery surgery. METHODS: We undertook a prospective, international cohort study to determine the incidence of covert stroke after non-cardiac, non-carotid artery surgery. Eligible patients were ≥65 yr of age and were admitted to hospital for at least three nights after non-cardiac, non-carotid artery surgery. Patients underwent a brain magnetic resonance study between postoperative days 3 and 10. The main outcome was the incidence of perioperative covert stroke. RESULTS: We enrolled a total of 100 patients from six centres in four countries. The incidence of perioperative covert stroke was 10.0% (10/100 patients, 95% confidence interval 5.5-17.4%). Five of the six centres that enrolled patients reported an incident covert stroke, and covert stroke was found in patients undergoing major general (3/27), major orthopaedic (3/41), major urological or gynaecological (3/22), and low-risk surgery (1/12). CONCLUSIONS: This international multicentre study suggests that 1 in 10 patients ≥65 yr of age experiences a perioperative covert stroke. A larger study is required to determine the impact of perioperative covert stroke on patient-important outcomes. CLINICAL TRIAL REGISTRATION: NCT01369537.

Validity of an early risk score for older adults.

OBJECTIVES: To determine if a risk score developed in hospitalised older adults in the UK in 1962 is correlated with other measures of health and if this risk score predicts death or institutionalisation in community-living older adults. METHODS: A total of 1,735 older adults residing in the community in 1991 were followed over five years. We replicated the original risk index, a composite score of cognitive status, disability and continence. Other measures included age, gender, education, self-rated health (SRH), life satisfaction (LS) and frailty. Death and nursing home (NH) admission were determined five years later. RESULTS: The risk score was strongly associated with frailty, SRH and LS. The index predicted mortality and NH use: The adjusted odds ratio (95% confidence interval) from multinomial logistic regression models was 0.74 (0.69, 0.79) for death and 0.74 (0.67, 0.83) for NH. CONCLUSIONS: A risk score devised to measure inpatient rehabilitation also predicts outcomes in community-dwelling older adults. Cognition and function predict a variety of adverse outcomes in a variety of settings in different historic eras.

Agonists cause endocytosis of nicotinic acetylcholine receptors on cultured myotubes.

Regulated trafficking of neurotransmitter receptors in excitable cells may play an important role in synaptic plasticity. In addition, agonist-induced endocytosis of nicotinic acetylcholine receptors (nAChRs) in particular might be involved in nicotine tolerance and addiction. The existing evidence concerning regulated internalization of cell-surface nAChRs is indirect and equivocal, however. In the present study, radioligand binding and fluorescence microscopy were used to show that agonists cause substantial endocytosis of nAChRs on cultured myotubes. Exposure to carbachol or nicotine caused a decrease in the intensity of fluorescent labeling of clusters of cell-surface nAChRs that was blocked by low temperature. Overall, myotubes exposed to carbachol or nicotine bound 50-70% less [(125)I]-alpha-bungarotoxin on the cell surface than untreated cells. The effect of carbachol was significant within 5 min, increased progressively for at least 4 h, and had a sensitivity of 100 nM or less. Exposure to carbachol caused the appearance or dramatic expansion of an intracellular pool of nAChRs, which were localized to discrete, largely perinuclear structures. A pulse-chase labeling protocol allowed the selective labeling and localization of nAChRs that had been internalized from the cell surface. In untreated cells, very little internalization of nAChRs occurred over a period of 3 h, indicating that constitutive endocytosis of receptors over this period was minimal. Exposure to carbachol, however, caused a dramatic increase in the endocytosis of nAChRs. These results provide direct evidence that agonists, including the tobacco alkaloid nicotine, can cause substantial endocytosis of cell-surface nAChRs.

Glomerular endothelial cells and podocytes jointly synthesize laminin-1 and -11 chains.

BACKGROUND: The glomerular basement membrane (GBM) originates in development from fusion of subendothelial and subepithelial matrices. Subsequently, newly synthesized subepithelial matrix is added as glomerular capillary loops expand. During GBM assembly, the laminin-1 heterotrimer (alpha 1, beta 1, and gamma 1 chains), initially expressed in vascular clefts of comma- and S-shaped bodies, is eventually replaced by laminin-11 (alpha 5, beta 2, and gamma 1 chains), which persists into maturation. The cellular source(s) of these laminins is not known and prompted this study. METHODS: To determine which cells synthesize the various laminin chains, postfixation immunoelectron microscopy of developing mouse kidney was performed using monoclonal and polyclonal antibodies that specifically recognized laminin alpha 1, beta 1, alpha 5, or beta 2 chains. RESULTS: Intracellular labeling for laminin alpha 1, beta 1 (laminin-1), and alpha 5 and beta 2 (laminin-11) chains was observed in developing glomerular endothelial cells and podocytes of comma- and S-shaped nephric figures. Laminin-1 was also seen in unfused GBMs at this stage, whereas laminin-11 was only found intracellularly. In capillary loop stage GBMs, laminin alpha 1 chain was completely absent, whereas labeling for laminin alpha 5 was intense, indicating rapid substitution between alpha chains. In contrast, laminin beta 1 chain labeling remained strong both intracellularly and in GBMs of capillary loop stage glomeruli, and beta 2 was up-regulated as well. In maturing stage glomeruli, beta 1 labeling declined, and alpha 5 and beta 2 remained at high levels intracellularly in both endothelial cells and podocytes and in GBMs. CONCLUSIONS: Our results show that both endothelial cells and podocytes synthesize laminin-1 and -11 chains throughout glomerular development. The sustained and comparatively high level of laminin synthesis by endothelial cells was unexpected, suggesting that the endothelium may be an important source of GBM proteins in glomerular disease.

Localization and functional characterization of Na+/H+ exchanger isoform NHE4 in rat thick ascending limbs.

The Na+/H+ exchanger NHE4 was cloned from a rat stomach cDNA library and shown to be expressed predominantly in the stomach and less dramatically in the kidney. The role and precise localization of NHE4 in the kidney are still unknown. A polyclonal antibody against a unique NHE4 decapeptide was used for immunohistochemistry in rat kidney. Simultaneous use of antibodies to Tamm-Horsfall glycoprotein and aquaporin-2 or -3 permitted identification of thick ascending limbs and collecting ducts, respectively. The results indicate that NHE4 is highly expressed in basolateral membranes of thick ascending limb and distal convoluted tubule, whereas collecting ducts from cortex to inner medulla and proximal tubules showed weaker basolateral NHE4 expression. Western blot analysis of NHE4 in membrane fractions prepared from the inner stripe of the outer medulla revealed the presence of a 95-kDa protein that was enriched in basolateral membrane vesicles isolated from medullary thick ascending limbs. The inhibition curve of H+-activated (22)Na uptake by 5-(N-ethyl-N-isopropyl)amiloride (EIPA) was consistent with the presence, beyond the EIPA high-affinity NHE1 isoform, of an EIPA low-affinity NHE with apparent half-maximal inhibition of 2.5 microM. Kinetic analyses showed that the extracellular Na+ dependence of NHE4 activity followed a simple hyperbolic relationship, with an apparent affinity constant of 12 mM. Intravesicular H+ activated NHE4 by a positive cooperative mechanism. NHE4 had an unusual low affinity for intravesicular H+ with a half-maximal activation value of pK 6.21. We conclude that NHE4, like NHE1, is expressed on the basolateral membrane of multiple nephron segments. Nevertheless, these two proteins exhibited dramatically different affinities for intracellular H+, suggesting that they may play distinct physiological roles in the kidney.

Polycystin-1 transforms the cAMP growth-responsive phenotype of M-1 cells.

BACKGROUND: Polycystic kidney disease (PKD) is characterized by the abnormal proliferation of tubular epithelial cells. It was recently shown that the growth of PKD cyst-lining cells is stimulated by cyclic adenosine monophosphate (cAMP), whereas the growth of normal human kidney cortex cells is inhibited. METHODS: We have examined the effects of overexpressing the C-terminal cytosolic tail of mouse polycystin-1, as a membrane-targeted fusion protein, on cAMP-responsive cell proliferation in stably transfected M-1 cortical collecting duct cells. Two cell lines that express high levels of the polycystin-1 fusion protein and two control cell lines that do not express the fusion protein were tested. RESULTS: Growth of parental M-1 cells and the control cell lines was inhibited by 8-Br-cAMP and by a variety of cAMP agonists. In contrast, growth of the polycystin-1-expressing clones was stimulated by cAMP. Consistent with this, the protein kinase A (PKA) inhibitor H-89 caused either a positive or a negative growth effect depending on the primary response to cAMP. PD98059 blocked the cAMP stimulation of cell proliferation, indicating that the pathway is MEK1 dependent. CONCLUSIONS: Expression of the polycystin-1 C-terminal tail disrupts normal cellular signaling and transforms the stably transfected M-1 cells to an abnormal PKD cell proliferation phenotype.

Glomerular laminin isoform transitions: errors in metanephric culture are corrected by grafting.

Glomerular basement membrane (GBM) assembly and maturation are marked by the replacement of laminin-1 (containing alpha 1-, beta 1-, and gamma 1-chains) with laminin-11 (consisting of alpha 5-, beta 2-, and gamma 1-chains). Similarly, the alpha 1- and alpha 2-chains of type IV collagen are replaced by collagen alpha 3-, alpha 4-, and alpha 5(IV)-chains. The cellular origins of these molecules and mechanisms for isoform removal and substitution are unknown. To explore glomerular laminin isoform transitions in vitro, we assessed metanephric organ cultures. Standard culture conditions do not support endothelial cell differentiation, and glomerular structures that form in vitro are avascular. Nevertheless, extensive podocyte development occurs in these cultures, including the formation of foot processes and assembly of a GBM-like matrix. Here, we show that the podocyte-specific markers, glomerular epithelial protein 1 and nephrin, which are normally expressed in capillary loop stage glomeruli in vivo, are also expressed by glomerular figures that form in organ culture. However, the GBM-like segments that form in vitro do not undergo normal laminin isoform switching. Instead, both laminin alpha 1- and alpha 5-chains are present, as is the beta 1-chain, but not beta 2. When avascular organ-cultured kidneys are grafted into anterior eye chambers, however, kidney-derived angioblasts establish extensive vasculature by 6 days, and glomeruli are lined by endothelial cells. We evaluated embryonic day 12 (E12) vascular endothelial growth factor receptor (Flk1)-lacZ kidneys that had first been grown in organ culture for 6--7 days and then grafted into wild-type mice. Correct laminin isoform substitution occurred and correlated with the appearance of endothelial cells expressing Flk1. Our findings indicate that endothelial cells, and/or factors present in the circulation, mediate normal GBM laminin isoform transitions in vivo.

Molecular cloning, expression, and distribution of glomerular epithelial protein 1 in developing mouse kidney.

BACKGROUND: Glomerular epithelial protein 1 (GLEPP1) is a receptor-like membrane protein tyrosine phosphatase (RPTP) with a large ectodomain consisting of multiple fibronectin type III repeats, a single transmembrane segment, and a single cytoplasmic phosphatase active site sequence. In adult human and rabbit kidneys, GLEPP1 is found exclusively on apical membranes of podocytes and especially on surfaces of foot processes. Although neither ligand nor function for this protein is known, other RPTPs with similar topologies have been implicated in mediating adherence behavior of cells. METHODS: To evaluate potential roles of GLEPP1 further, we cloned the full-length mouse GLEPP1 cDNA and examined its expression patterns in developing kidney by Northern blot analysis, in situ hybridization, and immunofluorescence microscopy. RESULTS: Nucleotide sequencing showed that mouse GLEPP1 was approximately 80% identical to rabbit and human GLEPP1 and approximately 91% identical at the amino acid level. The membrane-spanning and phosphatase domains of mouse GLEPP1 shared> 99% homology with PTPphi, a murine macrophage cytoplasmic phosphatase. Northern analysis identified a single GLEPP1 transcript of approximately 5.5 kb in fetal kidney that became approximately threefold more abundant in adults. In situ hybridization of newborn mouse kidney revealed GLEPP1 mRNA in visceral epithelial cells (developing podocytes) of comma- and S-shaped nephric figures, and expression increased in capillary loop and maturing stage glomeruli. Beginning on embryonic day 14, GLEPP1 protein was first observed on cuboidal podocytes of capillary loop stage glomeruli, but nascent podocytes of earlier comma- and S-shaped nephric figures were negative. At later stages of glomerular maturation, where foot process elongation and interdigitation occurs, GLEPP1 immunolabeling intensified on podocytes and then persisted at high levels in fully developed glomeruli. CONCLUSION: Our findings are consistent with a role for GLEPP1 in mediating and maintaining podocyte differentiation specifically.

Macula densa Na(+)/H(+) exchange activities mediated by apical NHE2 and basolateral NHE4 isoforms.

Functional and immunohistochemical studies were performed to localize and identify Na(+)/H(+) exchanger (NHE) isoforms in macula densa cells. By using the isolated perfused thick ascending limb with attached glomerulus preparation dissected from rabbit kidney, intracellular pH (pH(i)) was measured with fluorescence microscopy by using 2',7'-bis-(2-carboxyethyl)-5-(and -6) carboxyfluorescein. NHE activity was assayed by measuring the initial rate of Na(+)-dependent pH(i) recovery from an acid load imposed by prior lumen and bath Na(+) removal. Removal of Na(+) from the bath resulted in a significant, DIDS-insensitive, ethylisopropyl amiloride (EIPA)-inhibitable decrease in pH(i). This basolateral transporter showed very low affinity for EIPA and Hoechst 694 (IC(50) = 9.0 and 247 microM, respectively, consistent with NHE4). The recently reported apical NHE was more sensitive to inhibition by these drugs (IC(50) = 0.86 and 7.6 microM, respectively, consistent with NHE2). Increasing osmolality, a known activator of NHE4, greatly stimulated basolateral NHE. Immunohistochemical studies using antibodies against NHE1-4 peptides demonstrated expression of NHE2 along the apical and NHE4 along the basolateral, membrane, whereas NHE1 and NHE3 were not detected. These results suggest that macula densa cells functionally and immunologically express NHE2 at the apical membrane and NHE4 at the basolateral membrane. These two isoforms likely participate in Na(+) transport, pH(i), and cell volume regulation and may be involved in tubuloglomerular feedback signaling by these cells.

Key methodological features of randomized controlled trials of Alzheimer's disease therapy. Minimal clinically important difference, sample size and trial duration.

BACKGROUND: The results of clinical trials are routinely presented in terms of statistical significance, which may or may not indicate clinical significance. Analysis of the minimal clinically important difference (MCID) of cognitive scales has received little attention to date. OBJECTIVES: By reviewing the key methodological features (sample size, duration, statistical and clinical significance) of clinical trials examining the efficacy of tacrine in the treatment of Alzheimer's disease (AD), we assessed their ability to detect clinically important changes in cognition. DESIGN: The value for the MCID of the Mini-Mental State Examination (MMSE) was determined by surveying specialists in neurology and geriatric medicine. This value was then used to interpret the clinical significance of the results of published randomized controlled trials (RCTs) assessing the efficacy of tacrine in the treatment of AD and to retrospectively determine their optimal sample size and trial duration. RESULTS: The mean survey MCID for the MMSE was 3.72 (95% confidence interval 3.50-3.95) points. Only 2 of 12 tacrine RCTs using the MMSE found a statistically significant difference in MMSE scores for patients taking tacrine compared with those taking placebo. These improvements were not clinically significant when compared with the survey MMSE MCID. For parallel trials of tacrine in AD, the smallest sample size and minimum trial duration required to demonstrate a clinically significant difference were calculated to be 53 subjects and 1 year, respectively. Five of the 7 parallel trials met the required sample size; however, none of them met the criteria for trial duration. CONCLUSIONS: When using the MMSE as an outcome measure, no tacrine trial reported results that were clinically significant as perceived by clinicians working with dementia patients. Application of a range of plausible MCIDs to the parallel design RCTs also demonstrated that 2 of 7 of these trials did not have sufficient sample size, and none had sufficient duration of treatment to reliably detect clinically meaningful changes in cognition. Future clinical trials in this area will need to incorporate the evolving knowledge of MCIDs in order to increase their chance of detecting clinically relevant results. CopyrightCopyright 1999S.KargerAG,Basel

Nephrin localizes to the slit pore of the glomerular epithelial cell.

BACKGROUND: Recognition that mutation of the protein nephrin, encoded by the NPHS1 gene, singly results in the cellular alterations that result in foot process effacement, and nephrotic range proteinuria emphasizes the pivotal role that this protein plays in regulating glomerular filter integrity. This article reports the development of reagents necessary to study the biology of nephrin in mouse, and describes the initial characterization of the nephrin protein. METHODS: A cDNA including the full-length mouse nephrin open reading frame was cloned and sequenced. Immuno-affinity purified polyclonal antiserum directed against the cytoplasmic domain of mouse nephrin was developed. RESULTS: Nephrin identified in mouse glomerular extract was found to be a glycoprotein with an apparent molecular mass of 185 kDa. As detected by indirect immunofluorescence microscopy and immunogold electron microscopy, nephrin was located only in visceral glomerular epithelial cells, where it was targeted to intercellular junctions of mature podocyte foot processes. In developing glomeruli of newborn mouse, antinephrin immunolocalized to the earliest slit pore regions between differentiating podocytes, sites where slit diaphragms first become visible. CONCLUSION: As a putative cell adhesion molecule of the immunoglobulin superfamily, nephrin likely participates in cell-cell interactions between podocyte foot processes and may represent a component of the slit diaphragm.

Assessing the quality of newspaper medical advice columns for elderly readers.

BACKGROUND: Medical advice columns in newspapers can provide a valuable service by educating the general public about important health-related issues. However, these columns may be harmful if the advice or information given in them is incomplete, inappropriate or misleading. The objective of this study was to assess the safety and appropriateness of advice given to elderly readers of newspapers in medical advice columns. METHODS: Medical advice columns published in Canadian newspapers in 1995 were identified from a CD-ROM database. The articles that were selected were published in English and contained medical advice pertinent to elderly people about topics that could be found in a textbook of geriatric medicine. Fifty articles, randomly selected from the 109 articles that met these criteria, were independently assessed by 5 geriatricians. A scoring system was used to rate the ability to determine to which population the article applied, how well fact was distinguished from opinion, the degree to which critical issues were addressed, the safety and the appropriateness of the advice. When the kappa statistic for inter-rater agreement was 0.74 or less, a 2-stage Delphi process was used in an attempt to reach consensus. RESULTS: Agreement (kappa > 0.74) was eventually achieved for 232 (92.8%) of the 250 ratings. In 4 (8%) of the articles there was a high probability that the advice given could be applied to the wrong patient population; in 7 (14%) there was a high probability that opinion might be interpreted as fact; and in 11 (22%) the major critical issues were not identified. Of greatest concern, however, the advice in 25 (50%) of the articles was judged to be inappropriate, and in 14 (28%) advice may have been dangerous and potentially life-threatening. INTERPRETATION: Although medical advice columns have the potential to improve the health of elderly readers, a significant percentage of these articles contain inappropriate or even potentially dangerous advice.

Use of a thromboembolic risk score to improve thromboprophylaxis in surgical patients.

Two hundred admissions to a general surgical ward were audited prospectively before and after the introduction of a thromboembolic risk score. This was based on the Thromboembolic Risk Factors (THRIFT) Consensus Group guidelines for thrombo-prophylaxis. The results showed an overall improvement in compliance from 65% to 79%. High risk patients formed 24% of the patients studied. In this group, compliance improved significantly from 14% to 58%. The overall prevalence of important thromboembolic risk factors was calculated. Of the patients, 26.5% had a Body Mass Index (BMI) of > 25, and 10% gave a past or family history of thromboembolism. Of female patients, 24% were taking oestrogens. We conclude that quantitative assessment of all patients for thromboembolic risk can improve the implementation of thromboprophylaxis, particularly in high risk patients.

Subcortical vascular dementia: survey of treatment patterns and research considerations.

BACKGROUND: Since few studies have examined the effectiveness of therapies for subcortical vascular dementia, treatment guidelines are not available. Current patterns in the treatment of such dementias have not been studied. OBJECTIVE: To determine the practice patterns of Canadian specialists for the treatment of subcortical vascular dementia, and to survey their opinions regarding issues which are important in the design of a randomized controlled trial (RCT) in this field. DESIGN: National survey of all specialists certified in Neurology or Geriatric Medicine. RESULTS: Of responding physicians (78%) prescribed antithrombotic therapy for patients with vascular dementia. Most begin treatment with aspirin 325 mg daily (64%). The next three most common initial treatments were; no pharmacotherapy (12%), aspirin 650 mg daily (11%), and aspirin 1300 mg daily (11%). If the dementia continued to progress despite initial therapy, the treatment options were more varied. Most specialists (69%) believed that an RCT to assess the efficacy of aspirin in vascular dementia is warranted. The majority (69%) also felt that serial neuroimaging would be required for participants in such a trial, with magnetic resonance imaging being cited most frequently (41%). The majority of specialists considered three years as the minimum duration for such a trial. CONCLUSIONS: Specialist physician practice patterns vary significantly for the treatment of patients with subcortical vascular dementia. Most physicians believe that an RCT testing the efficacy of aspirin in this condition is required. However, before such a trial can be conducted, many methodological difficulties need to be addressed.

Plasma membrane cholesterol is a key molecule in shear stress-dependent activation of extracellular signal-regulated kinase.

Shear stress, the dragging force generated by fluid flow, differentially activates extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) in bovine aortic endothelial cells (BAEC) (Jo, H., Sipos, K., Go, Y. M., Law, R., Rong, J., and McDonald, J. M. (1997) J. Biol. Chem. 272, 1395-1401). Here, we examine whether cholesterol-enriched compartments in the plasma membrane are responsible for such differential regulation. Pretreatment of BAEC with a cholesterol-binding antibiotic, filipin, did not inhibit shear-dependent activation of JNK. In contrast, filipin and other membrane-permeable cholesterol-binding agents (digitonin and nystatin), but not the lipid-binding agent xylazine, inhibited shear-dependent activation of ERK. The effect of cholesterol-binding drugs did not appear to be due to membrane permeabilization, since treatment of BAEC with a detergent, Triton X-100 which also permeabilizes membranes, did not inhibit shear-dependent activation of ERK. Furthermore, shear-dependent activation of ERK, but not JNK, was inhibited by cyclodextrin, a membrane-impermeable cholesterol-binding agent, which removes cell-surface cholesterol. Moreover, the effects of cyclodextrin were prevented by adding cholesterol during the incubation. These results indicate that cholesterol or cholesterol-sensitive compartments in the plasma membrane play a selective and essential role in activation of ERK, but not JNK, by shear stress. Although exposure to shear stress (1 h) increased the number of caveolae by 3-fold, treatment with filipin had no effect in either control or shear-exposed cells suggesting that caveolae density per se is not a crucial determinant in shear-dependent ERK activation. In summary, the current study suggests that cholesterol-sensitive microdomains in the plasma membrane, such as caveolae-like domains, play a critical role in differential activation of ERK and JNK by shear stress.

Origins and formation of microvasculature in the developing kidney.

Regulation of microvessel assembly in the developing kidney is not known and may occur through vasculogenic, angiogenic, or both processes. To examine this question, we grafted rat and mice embryonic (E) day 12 (E12) kidneys, which have only a rudimentary vasculature, into anterior eye chambers of mouse and rat hosts. Species-specific, monoclonal anti-basement membrane antibodies showed that glomerular basement membranes, mesangial matrices, and microvessel basement membranes were always derived from the graft. When wild-type E12 mouse kidneys were grafted into anterior chambers of ROSA26 mice, in which the beta-galactosidase transgene is expressed ubiquitously, glomerular and microvascular endothelial cells stemmed from the graft, even after maintenance of kidneys in organ culture for 6 days before grafting. Immunolabeling with antibodies against the vascular endothelial growth factor (VEGF) receptor, Flk1, the EphB1 receptor, and its ligand, ephrin-B1, labeled discrete mesenchymal cells in embryonic and newborn kidney cortex, as well as developing microvessel and glomerular endothelium. In adult kidneys, Flk1 labeled glomeruli weakly, other vascular structures were unlabeled. When wild-type E12 kidneys were grafted under renal capsules of adult ROSA26 hosts, endothelium developing within the graft again came from the implanted kidney. In contrast, when E12 kidneys were grafted into renal cortices of newborns, glomeruli within grafts now contained host-derived endothelium. Similarly, when ROSA26 E12 kidneys were implanted into newborn wild-type hosts, chimeric vessels containing graft- and host-derived endothelium were seen in nearby host tissue. Our results indicate that cells capable of forming the entire microvascular tree of grafted metanephroi are already present in the E12 kidney. We hypothesize that Flk1/VEGF and EphB1/ephrin-B1 mediate renal endothelial mitosis-motility and cell guidance-aggregation behavior, respectively.