Synthetic tumor-derived human hypercalcemic factor exhibits parathyroid hormone-like vasorelaxation in renal arteries

Synthetic human tumor hypercalcemic factor (1-34, hHF) was compared with parathyroid hormone (human sequence, 1-34; hPTH) for vasorelaxant activity in isolated rabbit renal artery segments. The hHF exhibited a potent (IC50 = 1.3 x 10(-9) M) and profound (98%) relaxation which was significantly greater in magnitude than that obtained for hPTH (IC50 = 4.5 x 10(-9) M; maximal relaxation = 78%). The relaxations to both peptides were concentration-dependent and not associated with changes in cyclic AMP levels. These results demonstrate a parathyroid hormone-like response, independent of adenylate cyclase activation, in isolated renal arteries. Renal vasodilation may be important for the effects on renal function shared by these two peptides.     

Three Dimensional Culture of Human Renal Cell Carcinoma Organoids

Renal cell carcinomas arise from the nephron but are heterogeneous in disease biology, clinical behavior, prognosis, and response to systemic therapy. Development of patient-specific in vitro models that efficiently and faithfully reproduce the in vivo phenotype may provide a means to develop personalized therapies for this diverse carcinoma. Studies to maintain and model tumor phenotypes in vitro were conducted with emerging three-dimensional culture techniques and natural scaffolding materials. Human renal cell carcinomas were individually characterized by histology, immunohistochemistry, and quantitative PCR to establish the characteristics of each tumor. Isolated cells were cultured on renal extracellular matrix and compared to a novel polysaccharide scaffold to assess cell-scaffold interactions, development of organoids, and maintenance of gene expression signatures over time in culture. Renal cell carcinomas cultured on renal extracellular matrix repopulated tubules or vessel lumens in renal pyramids and medullary rays, but cells were not observed in glomeruli or outer cortical regions of the scaffold. In the polysaccharide scaffold, renal cell carcinomas formed aggregates that were loosely attached to the scaffold or free-floating within the matrix. Molecular analysis of cell-scaffold constructs including immunohistochemistry and quantitative PCR demonstrated that individual tumor phenotypes could be sustained for up to 21 days in culture on both scaffolds, and in comparison to outcomes in two-dimensional monolayer cultures. The use of three-dimensional scaffolds to engineer a personalized in vitro renal cell carcinoma model provides opportunities to advance understanding of this disease.     

Telocytes in the human kidney cortex

Renal interstitial cells play an important role in the physiology and pathology of the kidneys. As a novel type of interstitial cell, telocytes (TCs) have been described in various tissues and organs, including the heart, lung, skeletal muscle, urinary tract, etc. ( www.telocytes.com ). However, it is not known if TCs are present in the kidney interstitium. We demonstrated the presence of TCs in human kidney cortex interstitium using primary cell culture, transmission electron microscopy (TEM) and in situ immunohistochemistry (IHC). Renal TCs were positive for CD34, CD117 and vimentin. They were localized in the kidney cortex interstitial compartment, partially covering the tubules and vascular walls. Morphologically, renal TCs resemble TCs described in other organs, with very long telopodes (Tps) composed of thin segments (podomers) and dilated segments (podoms). However, their possible roles (beyond intercellular signalling) as well as their specific phenotype in the kidney remain to be established.     

Function of renal sex secretion and male hierarchy in the adder, Vipera berus, during reproduction

The function of the male renal sex segment secretions in the adder, Vipera berus, was studied. Renal sex segments in male adders were hypertrophied immediately before and during the mating period. Simultaneously, renal secretion was found in the entire ureter and coprodeum. Newly mated females have long, slightly convoluted, and hard posterior uteri, while females prior to mating, and nonreproductive ones, have weakly contracted or flaccid uteri. Uteri in newly mated females had a contracted sphincter muscle with reduced and compressed folded lumen. Uteri in an estrous female supplied with vital stained renal secretion became hard posteriorly. The stained secretion reached the entrance of the uteri, where the hard parts started. Estrous females artificially supplied with male sex secretion showed a significant decrease in acceptance of male courtship. Administration of renal sex secretion also interfered with reproduction.     

Natural Scaffolds for Renal Differentiation of Human Embryonic Stem Cells for Kidney Tissue Engineering

Despite the enthusiasm for bioengineering of functional renal tissues for transplantation, many obstacles remain before the potential of this technology can be realized in a clinical setting. Viable tissue engineering strategies for the kidney require identification of the necessary cell populations, efficient scaffolds, and the 3D culture conditions to develop and support the unique architecture and physiological function of this vital organ. Our studies have previously demonstrated that decellularized sections of rhesus monkey kidneys of all age groups provide a natural extracellular matrix (ECM) with sufficient structural properties with spatial and organizational influences on human embryonic stem cell (hESC) migration and differentiation. To further explore the use of decellularized natural kidney scaffolds for renal tissue engineering, pluripotent hESC were seeded in whole- or on sections of kidney ECM and cell migration and phenotype compared with the established differentiation assays for hESC. Results of qPCR and immunohistochemical analyses demonstrated upregulation of renal lineage markers when hESC were cultured in decellularized scaffolds without cytokine or growth factor stimulation, suggesting a role for the ECM in directing renal lineage differentiation. hESC were also differentiated with growth factors and compared when seeded on renal ECM or a new biologically inert polysaccharide scaffold for further maturation. Renal lineage markers were progressively upregulated over time on both scaffolds and hESC were shown to express signature genes of renal progenitor, proximal tubule, endothelial, and collecting duct populations. These findings suggest that natural scaffolds enhance expression of renal lineage markers particularly when compared to embryoid body culture. The results of these studies show the capabilities of a novel polysaccharide scaffold to aid in defining a protocol for renal progenitor differentiation from hESC, and advance the promise of tissue engineering as a source of functional kidney tissue.     

Transplantation of marrow-derived cardiac stem cells carried in designer self-assembling peptide nanofibers improves cardiac function after myocardial infarction

Progress in stem cell transplantation for the treatment of myocardial infarction is hampered by the poor retention and survival of the implanted cells. To enhance cell survival and differentiation and thereby improve the efficiency of stem cell therapy, we constructed a novel self-assembling peptide by attaching an RGDSP cell-adhesion motif to the self-assembling peptide RADA16. c-kit^pos/Nkx2.5^low/GATA4^low marrow-derived cardiac stem cells (MCSCs), which have a specific potential to differentiate into cardiomyocytes, were isolated from rat bone marrow. The cytoprotective effects of RGDSP scaffolds were assessed by exposure of MCSCs to anoxia in vitro. The efficacy of transplanting MCSCs in RGDSP scaffolds was evaluated in a female rat MI model. The designer self-assembling peptide self-assembled into RGDSP nanofiber scaffolds under physiological conditions. RGDSP scaffolds were beneficial for the growth of MCSCs and protected them from apoptosis and necrosis caused by anoxia. In a rat MI model, cardiac function was improved and collagen deposition was markedly reduced in the group receiving MCSCs in RGDSP scaffolds compared with groups receiving MCSCs alone, RGDSP scaffolds alone or MCSCs in RADA16 scaffolds. There were more surviving MCSCs in the group receiving MCSCs in RGDSP scaffolds than in the groups receiving MCSCs alone or MCSCs in RADA16 scaffolds. Most of the Y chromosome-positive cells expressed cardiac troponin T and connexin43 (Cx-43). These results suggest that RGDSP scaffolds provide a suitable microenvironment for the survival and differentiation of MCSCs. RGDSP scaffolds enhanced the efficacy of MCSC transplantation to repair myocardium and improve cardiac function.     

Porous bioactive glass scaffolds for local drug delivery in osteomyelitis: development and in vitro characterization

A new bioactive glass-based scaffold was developed for local delivery of drugs in case of osteomyelitis. Bioactive glass having a new composition was prepared and converted into porous scaffold. The bioactivity of the resulting scaffold was examined by in vitro acellular method. The scaffolds were loaded with two different drugs, an antibacterial or antifungal drug. The effects of the size of the scaffold, drug concentration, and dissolution medium on drug release were studied. The scaffolds were further coated with a degradable natural polymer, chitosan, to further control the drug release. Both the glass and scaffold were bioactive. The scaffolds released both the drugs for 6 weeks, in vitro. The results indicated that the bigger the size and the higher the drug concentration, the better was the release profile. The scaffolds appeared to be suitable for local delivery of the drugs in cases of osteomyelitis.     

肠道与泌尿系统的微生物对肾结石发病机制影响的研究进展

肾结石是成人泌尿系统的常见疾病。它会影响肾脏的生理机能,还会导致尿路感染从而对人体健康造成一定危害。肾结石周围存在一个多元化的微生物群落,而肠道微生物和泌尿系统微生物的变化可能引起肾结石的发生与发展。其中双歧杆菌(Bifidobacterium)、乳酸杆菌(Lactobacillus)和肠杆菌科(Enterobacteriaceae)与肾结石发生较为密切。本篇综述着重介绍了肠道微生物与泌尿系统微生物在肾结石形成过程中的关联性,同时也对肾-肠轴概念,肠道微生物从短链脂肪酸产生、草酸盐变化和炎症发生对肾结石的影响,以及肾结石的预防与治疗等方面做出了介绍。     

Oxidative stress‐induced renal telomere shortening as a mechanism of cyclosporine‐induced nephrotoxicity

Due to the association of oxidative stress and telomere shortening, it was aimed in the present study to investigate the possibility whether cyclosporine‐A exerts its nephrotoxic side effects via induction of oxidative stress‐induced renal telomere shortening and senescent phenotype in renal tissues of rats. Renal oxidative stress markers, 8‐hydroxydeoxyguanosine, malondialdehyde, and protein carbonyl groups were measured by standard methods. Telomere length and telomerase activity were also evaluated in kidney tissue samples. Results showed that cyclosporine‐A treatment significantly (P < 0.05) enhanced renal malondialdehyde, 8‐hydroxydeoxyguanosine, and protein carbonyl groups levels, decreased renal telomere length, and deteriorated renal function compared with the controls. Renal telomerase activity was not affected by cyclosporine‐A. Renal telomere length could be considered as an important parameter of both oxidative stress and kidney function. Telomere shortening and accelerated kidney aging may be caused by cyclosporine‐induced oxidative stress, indicating the potential mechanism of cyclosporine‐induced nephrotoxicity.     

Hemodynamic basis of renal arteriosclerosis in young greyhounds

Renal arteriosclerosis occurs with unusually high frequency in young race-trained greyhounds. Light and electron microscopic studies were used to examine the arterial walls of renal vessels in six greyhounds. Lesions characteristic of mechanical forces, namely pressure and shear stresses, were found consistently on the endothelial surfaces of damaged vessels. Such damage was found in both the main renal vessel and its branches. Although the patterns of endothelial damage showed quantitative differences among individuals, the qualitative features were remarkably similar in the group. Quantitatively, fibrous plaques were greatest in narrow and curved portions of renal vessels. The plaques were found on the outer luminal surface of the bend and the bifurcation segments, but were absent on the flow dividers. Hemodynamic forces appear to play an important role in the pathogenesis of renal fibrous plaques. Renal arteriosclerosis in greyhounds provides an excellent model for the study of pressure pulse velocity and shear stress damage under various physiological conditions.     

Relaxing effect of atrial natriuretic factor on endothelin-precontracted vascular strips

Endothelin (ET), a peptide recently isolated from the supernatant of cultured porcine aortic endothelial cells, is a potent vasoconstrictor. On the other hand, atrial natriuretic factor (ANF) is a powerful vasorelaxant found in cardiocytes. Its effect was investigated in ET-precontracted rabbit vascular strips. ANF-induced a dose-dependent relaxation of maximally-precontracted mesenteric, renal and aortic strips. Mesenteric artery strips were more sensitive to ANF than either renal or aortic strips. The relaxant effect of ANF on ET-precontracted arteries was more potent than that of other vasorelaxant agents, such as isoproterenol and sodium nitroprusside. Renal and aortic arteries were more sensitive to the vasoconstrictor effect of ET than mesenteric strips. From these results, we conclude that ANF may play a role as a physiological antagonist of ET. The different sensitivity of vascular segments to ET could be due to varying vascular ET receptor densities.     

Immunomagnetic separation,primary culture,and characterization of cortical thick ascending limb plus distal convoluted tubule cells from mouse kidney

Summary Renal cortical thick ascending limbs of Henle’s loop (CAL) and distal convoluted tubules (DCT) represent sites at which much of the final regulation of urinary ionic composition, particularly that of calcium, is accomplished in both humans and in rodents. We sought in the present work to develop an efficient means for isolating parathyroid hormone (PTH)-sensitive cells from these nephron segments and to grow them in primary culture. [CAL+DCT] cells were isolated from mouse kidney using an antiserum against the Tamm-Horsfall glycoprotein which, in the renal cortex, is produced exclusively by these cells. A second antibody conjugated to coated ferrous particles permitted magnetic separation of [CAL+DCT] cells from Tamm-Horsfall negative renal cortical cells. Approximately 3 × 10⁶ cells per kidney with a trypan blue exclusion greater than 94% were isolated by these procedures. Experiments were performed to characterize the cells after 7 to 10 days in primary culture. PTH and isoproterenol, but neither calcitonin nor vasopressin, stimulated cyclic AMP (cAMP) formation in [CAL+DCT] cells, consistent with the pattern of hormone-activated cAMP synthesis found in freshly isolated CAL and DCT segments. Alkaline phosphatase, an enzyme present dominantly in proximal tubule brush border membranes, was virtually absent from [CAL+DCT] cells but was present in Tamm-Horsfall negative cells. Similarly, Na-glucose cotransport was absent in [CAL+DCT] cells but present in Tamm-Horsfall negative renal cortical cells. Finally, transport-related oxygen consumption in [CAL+DCT] cells was blocked by bumetanide and by chlorothiazide, diuretics that inhibit sodium transport in CAL and DCT nephron segments. These results demonstrate that PTH-sensitive [CAL+DCT] cells can be isolated in relatively high yield and viability and grown in cell culture. Primary cultures of these cells exhibit a phenotype appropriate to their site of origin in the nephron. Experimental work reported here was supported by grants from the National Institutes of Health, Bethesda, MD GM34399, American Heart Association (grant-in-aid 88-0721), and the Hitchcock Foundation. J. H. Pizzonia was supported by a Ford Foundation Fellowship and this work constitutes partial fulfillment of the requirements for a doctoral degree at Dartmouth College. B. J. Bacskai was supported by a Pharmaceutical Manufacturers Association Foundation Advanced Predoctoral Fellowship. P. A. Friedman was an Established Investigator of the American Heart Association during the tenure of these studies.     

Isolation of endophytic fungi from leaves of Pasania edulis and their within-leaf distributions

 Endophytic fungi were isolated from leaves of Pasania edulis, one of the most important trees of the warm temperate forests in southern Kyushu, by the surface sterilization method using H₂O₂ as a sterilizing agent. From a tree in the Experimental Nursery of Kagoshima University, located at the city of Kagoshima, Phyllosticta sp. and Colletotrichum spp. were frequently isolated. From a stand in a laurel forest in Mt. Takakuma, an ascomycetous fungus (Ascomycete sp. 1) and Phomopsis sp. were frequently isolated. Phyllosticta sp. was isolated more frequently from petiole segments and leaf segments with midrib and Phomopsis sp. from petiole segments and leaf-base segments with midrib than other segments. Colletotrichum spp. were isolated less frequently from petioles and Ascomycete sp. 1 from petiole segments and leaf-base segments with midrib than other segments. As possible causes of such biases in within-leaf distributions of the endophytes, differences in infection modes and negative interactions of major endophytes within leaves are suggested. Received: December 13, 2001 / Accepted: June 7, 2002 Acknowledgments The authors thank the staff members of the Experimental Forests of Kagoshima University for enabling the present study. Correspondence to:K. Hata     

A novel tripolymer coating demonstrating the synergistic effect of chitosan, collagen type 1 and hyaluronic acid on osteogenic differentiation of human bone marrow derived mesenchymal stem cells

The biomimetic approach mimicking in vivo micro environment is the key for developing functional tissue engineered constructs. In this study, we used a tripolymer combination consisting of a natural polymer, chitosan and two extracellular matrix components; collagen type 1 and hyaluronic acid to coat tissue culture plate to evaluate their effect on osteogenic differentiation of human bone marrow derived mesenchymal stem cells (hMSCs). The polymers were blended at different mixing ratios and the tissue culture plates were coated either by polyblend method or by surface modification method. hMSCs isolated from adult bone marrow were directed to osteoblast differentiation on the coated plates. Our results showed that the tripolymer coating of the tissue culture plate enhanced mineralization as evidenced by calcium quantification exhibiting significantly higher amount of calcium compared to the untreated or individual polymer coated plates. We found that the tripolymer coated plates having a 1:1 mixing ratio of chitosan and collagen type 1, surface modified with hyaluronic acid is an ideal combination to achieve the synergistic effect of these polymers on in vitro osteogenic differentiation of hMSCs. These results thus, establish a novel biomimetic approach of surface modification to enhance osteoblast differentiation and mineralization. Our findings hold great promise in implementing a biomimetic surface coating to improve osteoconductivity of implants and scaffolds for various orthopaedic and bone tissue engineering applications.     

Computational design of protein antigens that interact with the CDR H3 loop of HIV broadly neutralizing antibody 2F5

Rational design of proteins with novel binding specificities and increased affinity is one of the major goals of computational protein design. Epitope‐scaffolds are a new class of antigens engineered by transplanting viral epitopes of predefined structure to protein scaffolds, or by building protein scaffolds around such epitopes. Epitope‐scaffolds are of interest as vaccine components to attempt to elicit neutralizing antibodies targeting the specified epitope. In this study we developed a new computational protocol, MultiGraft Interface, that transplants epitopes but also designs additional scaffold features outside the epitope to enhance antibody‐binding specificity and potentially influence the specificity of elicited antibodies. We employed MultiGraft Interface to engineer novel epitope‐scaffolds that display the known epitope of human immunodeficiency virus 1 (HIV‐1) neutralizing antibody 2F5 and that also interact with the functionally important CDR H3 antibody loop. MultiGraft Interface generated an epitope‐scaffold that bound 2F5 with subnanomolar affinity (K_D = 400 pM) and that interacted with the antibody CDR H3 loop through computationally designed contacts. Substantial structural modifications were necessary to engineer this antigen, with the 2F5 epitope replacing a helix in the native scaffold and with 15% of the native scaffold sequence being modified in the design stage. This epitope‐scaffold represents a successful example of rational protein backbone engineering and protein–protein interface design and could prove useful in the field of HIV vaccine design. MultiGraft Interface can be generally applied to engineer novel binding partners with altered specificity and optimized affinity. Proteins 2014; 82:2770–2782. © 2014 Wiley Periodicals, Inc.     

Chitin-based scaffolds are an integral part of the skeleton of the marine demosponge Ianthella basta

The skeletons of demosponges, such as Ianthella basta, are known to be a composite material containing organic constituents. Here, we show that a filigree chitin-based scaffold is an integral component of the I. basta skeleton. These chitin-based scaffolds can be isolated from the sponge skeletons using an isolation and purification technique based on treatment with alkaline solutions. Solid-state ¹³C NMR, Raman, and FT-IR spectroscopies, as well as chitinase digestion, reveal that the isolated material indeed consists of chitin. The morphology of the scaffolds has been determined by light and electron microscopy. It consists of cross-linked chitin fibers approximately 40–100 nm in diameter forming a micro-structured network. The overall shape of this network closely resembles the shape of the integer sponge skeleton. Solid-state ¹³C NMR spectroscopy was used to characterize the sponge skeleton on a molecular level. The ¹³C NMR signals of the chitin-based scaffolds are relatively broad, indicating a high amount of disordered chitin, possibly in the form of surface-exposed molecules. X-ray diffraction confirms that the scaffolds isolated from I. basta consist of partially disordered and loosely packed chitin with large surfaces. The spectroscopic signature of these chitin-based scaffolds is closer to that of α-chitin than β-chitin.     

Renal failure induces telomere shortening in the rat heart

BackgroundRenal failure aggravates pathological cardiac remodelling induced by myocardial infarction (MI). Cardiac remodelling is associated with telomere shortening, a marker for biological ageing. We investigated whether mild and severe renal failure shorten cardiac telomeres and excessively shorten telomeres after MI. MethodsRats were subjected to sham, unilateral (UNX) or 5/6th nephrectomy (5/6NX) to induce none, mild or severe renal failure. MI was induced by left coronary artery ligation. Renal function parameters and blood pressure were measured. DNA was isolated from non-infarcted cardiac tissue. Telomere length was assessed by quantitative polymerase chain reaction (PCR). ResultsProteinuria was unchanged in UNX and MI compared with control, but strongly increased in 5/6NX, UNX+MI and 5/6NX+MI. Serum creatinine levels were increased fourfold in 5/6NX and tenfold in 5/6NX+MI. 5/6NX and groups with both renal failure and MI showed an approximate 20% reduction of telomere length, similar to the MI group. No excess telomere shortening was observed in hearts from rats with renal ablation after MI. ConclusionSevere renal failure, but not mild renal failure, leads to shortening of cardiac telomeres to a similar extent as found after MI. Renal failure did not induce excessive telomere shortening after MI. (Neth Heart J 2009;17:190–4.)     

Thermo-responsive non-woven scaffolds for "smart" 3D cell culture

The thermo‐responsive polymer poly(N‐isopropylacrylamide) has received widespread attention for its in vitro application in the non‐invasive, non‐destructive release of adherent cells on two dimensional surfaces. In this study, 3D non‐woven scaffolds fabricated from poly(propylene) (PP), poly(ethylene terephthalate) (PET), and nylon that had been grafted with PNIPAAm were tested for their ability to support the proliferation and subsequent thermal release of HC04 and HepG2 hepatocytes. Hepatocyte viability and proliferation were estimated using the Alamar Blue assay and Hoechst 33258 total DNA quantification. The assays revealed that the pure and grafted non‐woven scaffolds maintained the hepatocytes within the matrix and promoted 3D proliferation comparable to that of the commercially available Algimatrix? alginate scaffold. Albumin production and selected cytochrome P450 genes expression was found to be superior in cells growing on pure and grafted non‐woven PP scaffolds as compared to cells grown as a 2D monolayer. Two scaffolds, namely, PP‐g‐PNIPAAm‐A and PP‐g‐PNIPAAm‐B were identified as having far superior thermal release capabilities; releasing the majority of the cells from the matrices within 2 h. This is the first report for the development of 3D non‐woven, thermo‐responsive scaffolds able to release cells from the matrix without the use of any enzymatic assistance or scaffold degradation. Biotechnol. Bioeng. 2012; 109:2147–2158. © 2012 Wiley Periodicals, Inc.     

Which method of estimating renal function is the best predictor of mortality after coronary artery bypass grafting?

Objectives

Definitions of renal function in patients undergoing coronary artery bypass graft surgery (CABG) vary in the literature. We sought to investigate which method of estimating renal function is the best predictor of mortality after CABG.

Methods

We analysed the preoperative and postoperative renal function data from all patients undergoing isolated CABG from January 1998 through December 2007. Preoperative and postoperative renal function was estimated using serum creatinine (SeCr) levels, creatinine clearance (CrCl) determined by the Cockcroft-Gault formula and the glomerular filtration rate (e-GFR) estimated by the Modification of Diet in Renal Disease (MDRD) formula. Receiver operator characteristic (ROC) curves and area under the ROC curves were calculated.

Results

In 9987 patients, CrCl had the best discriminatory power to predict early as well as late mortality, followed by e-GFR and finally SeCr. The odds ratios for preoperative parameters for early mortality were closer to 1 than those of the postoperative parameters.

Conclusions

Renal function determined by the Cockcroft-Gault formula is the best predictor of early and late mortality after CABG. The relationship between renal function and mortality is non-linear. Renal function as a variable in risk scoring systems such as the EuroSCORE needs to be reconsidered.     

Tight junction claudins and the kidney in sickness and in health

The epithelial cell tight junction has several functions including the control of paracellular transport between epithelial cells. Renal paracellular transport has been long recognized to exhibit unique characteristics within different segments of the nephron, functions as an important component of normal renal physiology and has been speculated to contribute to renal related pathology if functioning abnormally. The discovery of a large family of tight junction associated 4-transmembrane spanning domain proteins named claudins has advanced our understanding on how the paracellular permeability properties of tight junctions are determined. In the kidney, claudins are expressed in a nephron-specific pattern and are major determinants of the paracellular permeability of tight junctions in different nephron segments. The combination of nephron segment claudin expression patterns, inherited renal diseases, and renal epithelial cell culture models is providing important clues about how tight junction claudin molecules function in different segments of the nephron under normal and pathological conditions. This review discusses early observations of renal tubule paracellular transport and more recent information on the discovery of the claudin family of tight junction associated membrane proteins and how they relate to normal renal function as well as diseases of the human kidney.