Active Tobacco Use in Patients with Claudication Does Not Affect Outcomes after Endovascular Interventions.

BACKGROUND: With the advent of endovascular procedures, the indications for intervention in claudicants have become less strict. Many interventionalists, however, will not intervene in patients with lifestyle-limiting claudication unless they have discontinued tobacco use. Many patients are unable to comply with this goal, and there is little published evidence to suggest that continued tobacco use results in poorer outcomes. We sought to determine if it is justified to deny this group of patients endovascular, potentially lifestyle-improving, procedures based on their outcomes. METHODS: A retrospective chart review was performed between 2007 and 2011 at a midsize community teaching hospital. Patients included had documented lifestyle-limiting claudication, underwent endovascular therapy, and had no previous vascular intervention. Patients were divided into 2 groups: active smokers (AS) and nonsmokers (NS) including former and never smokers. The primary outcome was the need for reintervention and the secondary outcomes were the need for surgical revascularization, limb loss, myocardial infarction (MI), stroke, and death. RESULTS: One hundred thirty-eight patients met inclusion criteria with 89 being male (64.5%). Forty-seven (34%) were active smokers versus 91 (66%) who were nonsmokers. Mean age at initial intervention for all 138 subjects was 66.34 years (standard deviation 10.7) and was not statistically different between the AS and NS groups. Mean follow-up was 3.6 years and was not significantly different between the two groups. Between the two groups (AS vs NS), there was no statistically significant difference between the rate of reintervention, surgical bypass, and limb loss. We also did not observe any significant difference in the rate of MI, stroke, or death during our follow-up period. CONCLUSIONS: Although tobacco use has been shown to negatively impact bypass patency, our data show that it does not appear to increase the need for reintervention, conversion to open surgical revascularization, limb loss, or other morbidities in patients undergoing endovascular interventions for claudication. We continue to strongly recommend all our patients who smoke to discontinue tobacco use. Our results, however, do not support the notion that those patients who are unable to quit should be denied the potential benefit of an endovascular intervention. The most important limitation of our study is the small numbers of patients available for review. Larger studies will be necessary to confirm our findings.

α-Intercalated cells defend the urinary system from bacterial infection.

α-Intercalated cells (A-ICs) within the collecting duct of the kidney are critical for acid-base homeostasis. Here, we have shown that A-ICs also serve as both sentinels and effectors in the defense against urinary infections. In a murine urinary tract infection model, A-ICs bound uropathogenic E. coli and responded by acidifying the urine and secreting the bacteriostatic protein lipocalin 2 (LCN2; also known as NGAL). A-IC-dependent LCN2 secretion required TLR4, as mice expressing an LPS-insensitive form of TLR4 expressed reduced levels of LCN2. The presence of LCN2 in urine was both necessary and sufficient to control the urinary tract infection through iron sequestration, even in the harsh condition of urine acidification. In mice lacking A-ICs, both urinary LCN2 and urinary acidification were reduced, and consequently bacterial clearance was limited. Together these results indicate that A-ICs, which are known to regulate acid-base metabolism, are also critical for urinary defense against pathogenic bacteria. They respond to both cystitis and pyelonephritis by delivering bacteriostatic chemical agents to the lower urinary system.

Aberrant expression of laminin-332 promotes cell proliferation and cyst growth in ARPKD.

Basement membrane abnormalities have often been observed in kidney cysts of polycystic kidney disease (PKD) patients and animal models. There is an abnormal deposition of extracellular matrix molecules, including laminin-α3,ß3,γ2 (laminin-332), in human autosomal dominant PKD (ADPKD). Knockdown of PKD1 paralogs in zebrafish leads to dysregulated synthesis of the extracellular matrix, suggesting that altered basement membrane assembly may be a primary defect in ADPKD. In this study, we demonstrate that laminin-332 is aberrantly expressed in cysts and precystic tubules of human autosomal recessive PKD (ARPKD) kidneys as well as in the kidneys of PCK rats, an orthologous ARPKD model. There was aberrant expression of laminin-γ2 as early as postnatal day 2 and elevated laminin-332 protein in postnatal day 30, coinciding with the formation and early growth of renal cysts in PCK rat kidneys. We also show that a kidney cell line derived from Oak Ridge polycystic kidney mice, another model of ARPKD, exhibited abnormal lumen-deficient and multilumen structures in Matrigel culture. These cells had increased proliferation rates and altered expression levels of laminin-332 compared with their rescued counterparts. A function-blocking polyclonal antibody to laminin-332 significantly inhibited their abnormal proliferation rates and rescued their aberrant phenotype in Matrigel culture. Furthermore, abnormal laminin-332 expression in cysts originating from collecting ducts and proximal tubules as well as in precystic tubules was observed in a human end-stage ADPKD kidney. Our results suggest that abnormal expression of laminin-332 contributes to the aberrant proliferation of cyst epithelial cells and cyst growth in genetic forms of PKD.

Galectin-3 mediates oligomerization of secreted hensin using its carbohydrate-recognition domain.

A multidomain, multifunctional 230-kDa extracellular matrix (ECM) protein, hensin, regulates the adaptation of rabbit kidney to metabolic acidosis by remodeling collecting duct intercalated cells. Conditional deletion of hensin in intercalated cells of the mouse kidney leads to distal renal tubular acidosis and to a significant reduction in the number of cells expressing the basolateral chloride-bicarbonate exchanger kAE1, a characteristic marker of α-intercalated cells. Although hensin is secreted as a monomer, its polymerization and ECM assembly are essential for its role in the adaptation of the kidney to metabolic acidosis. Galectin-3, a unique lectin with specific affinity for ß-galactoside glycoconjugates, directly interacts with hensin. Acidotic rabbits had a significant increase in the number of cells expressing galectin-3 in the collecting duct and exhibited colocalization of galectin-3 with hensin in the ECM of microdissected tubules. In this study, we confirmed the increased expression of galectin-3 in acidotic rabbit kidneys by real-time RT-PCR. Galectin-3 interacted with hensin in vitro via its carbohydrate-binding COOH-terminal domain, and the interaction was competitively inhibited by lactose, removal of the COOH-terminal domain of galectin-3, and deglycosylation of hensin. Galectin-9, a lectin with two carbohydrate-recognition domains, is also present in the rabbit kidney; galectin-9 partially oligomerized hensin in vitro. Our results demonstrate that galectin-3 plays a critical role in hensin ECM assembly by oligomerizing secreted monomeric hensin. Both the NH2-terminal and COOH-terminal domains are required for this function. We suggest that in the case of galectin-3-null mice galectin-9 may partially substitute for the function of galectin-3.

Secreted cyclophilin A, a peptidylprolyl cis-trans isomerase, mediates matrix assembly of hensin, a protein implicated in epithelial differentiation.

Hensin is a rabbit ortholog of DMBT1, a multifunctional, multidomain protein implicated in the regulation of epithelial differentiation, innate immunity, and tumorigenesis. Hensin in the extracellular matrix (ECM) induced morphological changes characteristic of terminal differentiation in a clonal cell line (clone C) of rabbit kidney intercalated cells. Although hensin is secreted in monomeric and various oligomeric forms, only the polymerized ECM form is able to induce these phenotypic changes. Here we report that hensin secretion and matrix assembly were inhibited by the peptidylprolyl cis-trans isomerase (PPIase) inhibitors cyclosporin A (CsA) and a derivative of cyclosporin A with modifications in the d-Ser side chain (Cs9) but not by the calcineurin pathway inhibitor FK506. PPIase inhibition led to failure of hensin polymerization in the medium and ECM, plus the loss of apical cytoskeleton, apical microvilli, and the columnar epithelial shape of clone C cells. Cyclophilin A was produced and secreted into the media to a much greater extent than cyclophilins B and C. Our results also identified the direct CsA-sensitive interaction of cyclophilin A with hensin, suggesting that cyclophilin A is the PPIase that mediates the polymerization and matrix assembly of hensin. These results are significant because this is the first time a direct role of peptidylprolyl cis-trans isomerase activity has been implicated in the process of epithelial differentiation.

Role of integrins in the assembly and function of hensin in intercalated cells.

Epithelial differentiation proceeds in at least two steps: Conversion of a nonepithelial cell into an epithelial sheet followed by terminal differentiation into the mature epithelial phenotype. It was recently discovered that the extracellular matrix (ECM) protein hensin is able to convert a renal intercalated cell line from a flat, squamous shape into a cuboidal or columnar epithelium. Global knockout of hensin in mice results in embryonic lethality at the time that the first columnar cells appear. Here, antibodies that either activate or block integrin beta1 were used to demonstrate that activation of integrin alpha v beta 1 causes deposition of hensin in the ECM. Once hensin polymerizes and deposits into the ECM, it binds to integrin alpha 6 and mediates the conversion of epithelial cells to a cuboidal phenotype capable of apical endocytosis; therefore, multiple integrins play a role in the terminal differentiation of the intercalated cell: alpha v beta 1 generates polymerized hensin, and another set of integrins (containing alpha 6) mediates signals between hensin and the interior of the cells.

Differentiation of columnar epithelia: the hensin pathway.

Epithelia, the most common variety of cells in complex organisms exist in many shapes. They are sheets of polarized cells that separate two compartments and selectively transport materials from one to the other. After acquiring these general characteristics, they differentiate to become specialized types such as squamous columnar or transitional epithelia. High density seeding converts a kidney-derived cell line from flat ;generic' epithelial cells to columnar cells. The cells acquire all the characteristics of differentiated columnar cells, including microvilli, and the capacity for apical endocytosis. The high seeding density induces the deposition of a new protein termed hensin and polymerization of hensin is the crucial event that dictates changes in epithelial phenotype. Hensin is widely expressed in most epithelia. Its deletion in mice leads to embryonic lethality at the time of generation of the first columnar epithelium, the visceral endoderm. Moreover many human cancers have deletions in the hensin gene, which indicates that it is a tumor suppressor.

Galectin-3 expression is induced in renal beta-intercalated cells during metabolic acidosis.

The adaptation of the cortical collecting duct (CCD) to metabolic acidosis requires the polymerization and deposition in the extracellular matrix of the novel protein hensin. HCO3(-)-secreting beta-intercalated cells remove apical Cl-:HCO3(-) exchangers and may reverse functional polarity to secrete protons. Using intercalated cells in culture, we found that galectin-3 facilitated hensin polymerization, thereby causing their differentiation into the H+-secreting cell phenotype. We examined the expression of galectin-3 in the rabbit kidney and its relationship to hensin during metabolic acidosis. In control kidneys, galectin-3 was expressed in the cortical and medullary collecting ducts. In the outer cortex 26 +/- 3% of CCD cells expressed galectin-3 compared with 64 +/- 3% of the cells of the inner cortex. In the CCD, galectin-3 was rarely expressed in beta-intercalated cells, being primarily present in alpha-intercalated and principal cells. During metabolic acidosis, the intensity of cellular staining for galectin-3 increased and more cells began to express it; the percentage of CCD cells expressing galectin-3 increased from 26 +/- 3 to 66 +/- 3% in the outer cortex and from 64 +/- 3 to 78 +/- 4% in the inner cortex. This was particularly evident in beta-intercalated cells where expression was found in only 8 +/- 2% in control animals but in 75 +/- 2% during metabolic acidosis in the outer cortex and similarly for the inner cortex (26 +/- 6 to 90 +/- 7%). Importantly, both galectin-3 and hensin were found in the extracellular matrix of microdissected CCDs; and during metabolic acidosis, many more cells exhibited this extracellular colocalization. Thus galectin-3 may play several important roles in the CCD, including mediating the adaptation of beta-intercalated cells during metabolic acidosis.

Cyclosporin A produces distal renal tubular acidosis by blocking peptidyl prolyl cis-trans isomerase activity of cyclophilin.

Cyclosporin A (CsA), a widely used immunosuppressant, causes distal renal tubular acidosis (dRTA). It exerts its immunosuppressive effect by a calcineurin-inhibitory complex with its cytosolic receptor, cyclophilin A. However, CsA also inhibits the peptidyl prolyl cis-trans isomerase (PPIase) activity of cyclophilin A. We studied HCO(3)(-) transport and changes in beta-intercalated cell pH on luminal Cl(-) removal in isolated, perfused rabbit cortical collecting tubules (CCDs) before and after exposure to media pH 6.8 for 3 h. Acid incubation causes adaptive changes in beta-intercalated cells by extracellular deposition of hensin (J Clin Invest 109: 89, 2002). Here, CsA prevented this adaptation. The unidirectional HCO(3)(-) secretory flux, estimated as the difference between net flux and that after Cl(-) removal from the lumen, was -6.7 +/- 0.2 pmol.min(-1).mm(-1) and decreased to -1.3 +/- 0.2 after acid incubation. CsA in the bath prevented the adaptive decreases in HCO(3)(-) secretion and apical Cl(-):HCO(3)(-) exchange. To determine the mechanism, we incubated CCDs with FK-506, which inhibits calcineurin activity independently of the host cell cyclophilin. FK-506 did not prevent the acid-induced adaptive decrease in unidirectional HCO(3)(-) secretion. However, [AD-Ser](8) CsA, a CsA derivative, which does not inhibit calcineurin but inhibits PPIase activity of cyclophilin A, completely blocked the effect of acid incubation on apical Cl(-):HCO(3)(-) exchange. Acid incubation resulted in prominent "clumpy" staining of extracellular hensin and diminished apical surface of beta-intercalated cells [smaller peanut agglutinin (PNA) caps]. CsA and [AD-Ser](8) CsA prevented most hensin staining and the reduction of apical surface; PNA caps were more prominent. We suggest that hensin polymerization around adapting beta-intercalated cells requires the PPIase activity of cyclophilins. Thus CsA is able to prevent this adaptation by inhibition of a peptidyl prolyl cis-trans isomerase activity. Such inhibition may cause dRTA during acid loading.

Terminal differentiation of epithelia from trophectoderm to the intercalated cell: the role of hensin.

The intercalated cells of the collecting tubules of mammalian kidneys were discovered by Haggege and Richet to change their morphology in response to a variety of physiologic stimuli related to changes in acid base status. Recent studies showed that the conversion of beta to alpha intercalated cell under the influence of acidification of the medium is due to the deposition of hensin in the extracellular matrix of these cells and activation of a novel inductive signal transduction pathway. The conversion of beta to alpha cells is shown to be a process of terminal differentiation. Hensin is secreted as a monomer, and activation of the cell induces two activities that convert it to a dimer by folding and into a fiber by bundling of the folded dimers by galectin 3. Only the fiber is functional. Hensin is expressed in most epithelial cells, and its staining pattern suggests that it might be involved in the terminal differentiation of most epithelia. There is loss of heterozygosity of hensin in a large number of epithelial and neural tumors, making it likely that it is a tumor suppressor gene.

Genomic analysis of alachlor-induced oncogenesis in rat olfactory mucosa.

Alachlor induces olfactory mucosal tumors in rats in a highly ordered temporal process. We used GeneChip analysis to test the hypothesis that histological progression and oncogenic transformation are accompanied by gene expression changes that might yield clues as to the molecular pathogenesis of tumor formation. Acute alachlor exposure caused upregulation of matrix metalloproteinases (MMP)-2 and -9, tissue inhibitor of metalloproteinase-1, carboxypeptidase Z, and other genes related to extracellular matrix homeostasis. Heme oxygenase was upregulated acutely and maintained elevated expression. Expression of ebnerin, related to the putative human tumor suppressor gene DMBT1, progressively increased in alachlor-treated olfactory mucosa. Progression from adenomas to adenocarcinoma was correlated with upregulation of genes in the wnt signaling pathway. Activated wnt signaling was confirmed by immunohistochemical localization of beta-catenin to nuclei of adenocarcinomas, but not earlier lesions. These observations suggest that initiation and progression of alachlor-induced olfactory mucosal tumors is associated with alterations in extracellular matrix components, induction of oxidative stress, upregulation of ebnerin, and final transformation to a malignant state by wnt pathway activation.

Acid incubation reverses the polarity of intercalated cell transporters, an effect mediated by hensin.

Metabolic acidosis causes a reversal of polarity of HCO(3)(-) flux in the cortical collecting duct (CCD). In CCDs incubated in vitro in acid media, beta-intercalated (HCO(3)(-)-secreting) cells are remodeled to functionally resemble alpha-intercalated (H(+)-secreting) cells. A similar remodeling of beta-intercalated cells, in which the polarity of H(+) pumps and Cl(-)/HCO(3)(-) exchangers is reversed, occurs in cell culture and requires the deposition of polymerized hensin in the ECM. CCDs maintained 3 h at low pH ex vivo display a reversal of HCO(3)(-) flux that is quantitatively similar to an effect previously observed in acid-treated rabbits in vivo. We followed intracellular pH in the same beta-intercalated cells before and after acid incubation and found that apical Cl/HCO(3) exchange was abolished following acid incubation. Some cells also developed basolateral Cl(-)/HCO(3)(-) exchange, indicating a reversal of intercalated cell polarity. This adaptation required intact microtubules and microfilaments, as well as new protein synthesis, and was associated with decreased size of the apical surface of beta-intercalated cells. Addition of anti-hensin antibodies prevented the acid-induced changes in apical and basolateral Cl(-)/HCO(3)(-) exchange observed in the same cells and the corresponding suppression of HCO(3)(-) secretion. Acid loading also promoted hensin deposition in the ECM underneath adapting beta-intercalated cells. Hence, the adaptive conversion of beta-intercalated cells to alpha-intercalated cells during acid incubation depends upon ECM-associated hensin.