Development of a concise and reliable method for quantifying the antibody loaded onto lipid nanoparticles modified with Herceptin.

Antibodies are essential components of the immune system with a wide range of molecular targets. They have been recognized as modalities for treating several diseases and more than 130 approved antibody-based therapeutics are available for clinical use. However, limitations remain associated with its efficacy, tissue permeability, and safety, especially in cancer treatment. Nanoparticles, particularly those responsive to external stimuli, have shown promise in improving the efficacy of antibody-based therapeutics and tissue-selective delivery. In this study, we developed a reliable and accurate method for quantifying the amount of antibody loaded onto lipid nanoparticles modified with Herceptin® (Trastuzumab), an antibody-based therapeutic used to treat HER2-positive cancers, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by silver staining. This method proved to be a suitable alternative to commonly used protein quantification techniques, which are limited by lipid interference present in the samples. Furthermore, the amount of Herceptin modified on the liposomes, measured by this method, was confirmed by Herceptin's antibody-dependent cell-mediated cytotoxicity activity. Our results demonstrate the potential of this method as a critical tool for developing tissue-selective antibody delivery systems, leading to improved efficacy and reduced side effects of antibody-based therapeutics.

Chain-specificity of laminin α1-5 LG45 modules in the recognition of carbohydrate-linked receptors and intramolecular binding.

Laminins are a family of heterotrimers composed of α-, ß-, and γ-chains in the basement membrane. Five α chains contain laminin globular (LG) domain consisting of five tandem modules (LG1-5 modules) at their C-terminus. Each LG45 modules is connected to a compact cloverleaf-shaped structure of LG1-3 through a flexible linker. Although the accumulated studies of the LG45 modules have suggested differences in each α chain regarding the binding of carbohydrate chain and intramolecular interaction, this remains unclear. In this study, to characterize their functions comparatively, we produced recombinant proteins of LG45 modules of human laminin α1-5 chains. Dystroglycan (DG) modified with matriglycan readily bound to the LG45 modules of α1 and α2 chains but not to the other α chains. In contrast, heparin bound to the LG45 modules of the α chains, except for α2. The binding of heparan sulfate/heparin-linked syndecans (SDCs) to LG45 modules was influenced by their core proteins. Furthermore, the α1 and α4LG45 modules bound to SDCs in a pH-dependent manner. A cell adhesion assay showed that HEK293 cells could readily adhere to the LG45 modules of α3-5 chains through a combination of SDCs and integrins. Moreover, α5LG45 modules bound to the E8 fragment, which includes the C-terminus of the laminin coiled-coil (LCC) domain and LG1-3 modules, but α2LG45 modules did not. The results suggested that although α5LG45 modules was fixed within the LG domain, α2LG45 modules was freely placed in the vicinity of LG1-3. Our findings provide information for investigation of the structural and functional diversity of basement membranes.

Structure-Activity Relationships of RGD-Containing Peptides in Integrin αvß5-Mediated Cell Adhesion.

The RGD motif is a cell adhesion sequence that binds to integrins, a receptor family for extracellular matrix proteins. We previously reported that the RGDX1X2 sequence, where X1X2 is VF or NY, is required for integrin αvß5-mediated cell adhesion. However, the importance and applications of the X1X2 combinations and their surrounding sequences of integrin αvß5-binding RGDX1X2-containing peptides have not been comprehensively elucidated. Therefore, we aimed to identify an RGD-containing peptide with enhanced integrin αvß5 binding activity. We synthesized various peptides based on the RGDVF and RGDNY peptides to optimize the N-terminal, C-terminal, and X1X2 combinations of the RGDX1X2 sequence. These peptides were immobilized on maleimide-functionalized bovine serum albumin-coated plates via a thiol-maleimide reaction, and cell adhesion was evaluated using HeLa cells and human dermal fibroblasts. Consequently, CPPP-RGDTF and CPPP-RGDTFI were identified as highly active peptides for integrin αvß5-mediated cell adhesion. CPPP-RGDTF and CPPP-RGDTFI are expected to serve as cell adhesion molecules for developing culture substrates and biomaterials. Furthermore, these findings provide important novel insights into the interaction between the RGD motifs and integrins.

Structural Requirement of hA5G18 Peptide (DDFVFYVGGYPS) from Laminin α5 Chain for Amyloid-like Fibril Formation and Cell Adhesion.

The hA5G18 peptide (DDFVFYVGGYPS) identified from the human laminin α5 chain G domain promotes cell attachment and spreading when directly coated on a plastic plate, but does not show activity when it is conjugated on a chitosan matrix. Here, we focused on the structural requirement of hA5G18 for activity. hA5G18 was stained with Congo red and formed amyloid-like fibrils. A deletion analysis of hA5G18 revealed that FVFYV was a minimum active sequence for the formation of amyloid-like fibrils, but FVFYV did not promote cell attachment. Next, we designed functional fibrils using FVFYV as a template for amyloid-like fibrils. When we conjugated an integrin binding sequence Arg-Gly-Asp (RGD) to the FVFYV peptide with Gly-Gly (GG) as a spacer, FVFYVGGRGD promoted cell attachment in a plate coat assay, but a negative control sequence RGE conjugated peptide, FVFYVGGRGE, also showed activity. However, when the peptides were conjugated to Sepharose beads, the FVFYVGGRGD beads showed cell attachment activity, but the FVFYVGGRGE beads did not. These results suggest that RGD and RGE similarly contribute to cell attachment activity in amyloid-like fibrils, but only RGD contributes the activity on the Sepharose beads. Further, we conjugated a basic amino acid (Arg, Lys, and His) to the FVFYV peptide. Arg or Lys-conjugated FVFYV peptides, FVFYVGGR and FVFYVGGK, showed cell attachment activity when they were coated on a plate, but a His-conjugated FVFYV peptide FVFYVGGH did not show activity. None of the basic amino acid-conjugated peptides showed cell attachment in a Sepharose bead assay. The cell attachment and spreading on FVFYVGGR and FVFYVGGK were inhibited by an anti-integrin ß1 antibody. These results suggest that the Arg and Lys residues play critical roles in the interaction with integrins in amyloid-like fibrils. FVFYV is useful to use as a template for amyloid-like fibrils and to develop multi-functional biomaterials.

Effect of Amino Acid Substitution on Cell Adhesion Properties of Octa-arginine.

Octa-arginine (R8) is a cell-permeable peptide with excellent cell adhesion properties. Surface-immobilized R8 mediates cell attachment via cell surface receptors, such as heparan sulfate proteoglycans and integrin ß1, and promotes cell spreading and proliferation. However, it is not clear how these properties are affected by specific peptide composition and if they could be improved. Here, we synthesized XR8 peptides, in which half of the original R8 arginine residues were replaced with another amino acid (X). We then aimed to investigate the effect of the substitution on cell adhesion and proliferation on XR8-conjugated agarose matrices. The XR8-matrix showed slightly better cell attachment when X was a hydrophobic or aromatic amino acid. However, hydrophobic XR8-matrices tended to promote cell proliferation to a less extent. Eventually, YR8-matrix most efficiently promoted cell adhesion, spreading, and proliferation among the XR8-matrices tested. Collectively, these observations indicate that the properties of residue X play a major role in the biological activity of XR8-matrices and shed light on the interaction between small peptides and the cell membrane. Further, YR8 is a promising cell-adhesive peptide for the development of cell culture substrates and biomaterials.

A heterozygous LAMA5 variant may contribute to slowly progressive, vinculin-enhanced familial FSGS and pulmonary defects.

The LAMA5 gene encodes laminin α5, an indispensable component of glomerular basement membrane and other types of basement membrane. A homozygous pathological variant in LAMA5 is known to cause a systemic developmental syndrome including glomerulopathy. However, the roles of heterozygous LAMA5 gene variants in human renal and systemic diseases have remained unclear. We performed whole-exome sequencing analyses of a family with slowly progressive nephropathy associated with hereditary focal segmental glomerulosclerosis, and we identified what we believe to be a novel probable pathogenic variant of LAMA5, NP_005551.3:p.Val3687Met. In vitro analyses revealed cell type-dependent changes in secretion of variant laminin α5 laminin globular 4-5 (LG4-5) domain. Heterozygous and homozygous knockin mice with a corresponding variant of human LAMA5, p.Val3687Met, developed focal segmental glomerulosclerosis-like pathology with reduced laminin α5 and increased glomerular vinculin levels, which suggested that impaired cell adhesion may underlie this glomerulopathy. We also identified pulmonary defects such as bronchial deformity and alveolar dilation. Reexaminations of the family revealed phenotypes compatible with reduced laminin α5 and increased vinculin levels in affected tissues. Thus, the heterozygous p.Val3687Met variant may cause a new syndromic nephropathy with focal segmental glomerulosclerosis through possibly defective secretion of laminin α5. Enhanced vinculin may be a useful disease marker.

Laminin α5_CD239_Spectrin is a candidate association that compensates the linkage between the basement membrane and cytoskeleton in skeletal muscle fibers.

The linkage between the basement membrane (BM) and cytoskeleton is crucial for muscle fiber stability and signal transduction. Mutations in the linkage molecules can cause various types of muscular dystrophies. The different severities and times of onset suggest that compensatory linkages occur at the sarcolemma. Cluster of differentiation 239 (CD239) binds to the α5 subunit of laminin-511 extracellularly and is connected to spectrin intracellularly, resulting in a linkage between the BM and cytoskeleton. In this study, we explored the linkage of laminin α5_CD239_spectrin in skeletal muscles. Although laminin α5, CD239, and spectrin were present in embryonic skeletal muscles, they disappeared in adult skeletal muscle tissues, except for the soleus and diaphragm. Laminin α5_CD239_spectrin was localized in the skeletal muscle tissues of Duchenne muscular dystrophy and congenital muscular dystrophy mouse models. The experimental regeneration of skeletal muscle increased the CD239-mediated linkage, indicating that it responds to regeneration, but not to genetic influence. Furthermore, in silico analysis showed that laminin α5_CD239_spectrin was upregulated by steroid therapy for muscular dystrophy. Therefore, CD239-mediated linkage may serve as a therapeutic target to prevent the progression of muscular dystrophy.

RGDX1 X2 motif regulates integrin αvß5 binding for pluripotent stem cell adhesion.

The arginine-glycine-aspartic acid (RGD) motif is a cell adhesion sequence that binds to integrins. Some RGD-containing peptides promote adhesion of both embryonic stem cells and induced pluripotent stem cells (iPSCs); however, not all such RGD-containing peptides are active. In this study, we elucidated the role of RGD-neighboring sequences on iPSC adhesion using diverse synthetic peptides and recombinant proteins. Our results indicate that iPSC adhesion requires RGDX1 X2  sequences, such as RGDVF and RGDNY, and that the X1 X2 residues are essential for the adhesion via integrin αvß5 but not αvß3. iPSCs express integrin αvß5 but not αvß3; therefore, iPSC adhesion requires the RGDX1 X2 -containing sequences. The importance of the X1 X2 residues was confirmed with both HeLa and A549 cells, which express integrin αvß5 but not αvß3. Analysis of RGD-neighboring sequences provides important insights into ligand-binding specificity of integrins. Identification of integrin αvß5-binding motifs is potentially useful in drug development, drug delivery, cell culture, and tissue engineering.

Octa-arginine and Octa-lysine Promote Cell Adhesion through Heparan Sulfate Proteoglycans and Integrins.

Octa-arginine (R8) has been extensively studied as a cell-penetrating peptide. R8 binds to diverse transmembrane heparan sulfate proteoglycans (HSPGs), including syndecans, and is internalized by cells. R8 is also reported to bind to integrin ß1. In this study, we evaluated the biological activities of R8 and octa-lysine (K8), a peptide similar to R8, with a focus on cell adhesion. R8 and K8 were immobilized on aldehyde-agarose matrices via covalent conjugation, and the effect of these peptides on cell attachment, spreading, and proliferation was examined using human dermal fibroblasts. The results indicated that R8- and K8-matrices mediate cell adhesion mainly via HSPGs. Moreover, R8- and K8-matrices interacted with integrin ß1 and promote cell spreading and proliferation. These results are useful for further understanding of the R8-membrane interactions and the cellular uptake mechanisms. In addition, the R8- and K8-matrices may potentially be used as a multi-functional biomaterial to promote cell adhesion, spreading, and proliferation.

Development of an Antibody Delivery Method for Cancer Treatment by Combining Ultrasound with Therapeutic Antibody-Modified Nanobubbles Using Fc-Binding Polypeptide.

A key challenge in treating solid tumors is that the tumor microenvironment often inhibits the penetration of therapeutic antibodies into the tumor, leading to reduced therapeutic efficiency. It has been reported that the combination of ultrasound-responsive micro/nanobubble and therapeutic ultrasound (TUS) enhances the tissue permeability and increases the efficiency of delivery of macromolecular drugs to target tissues. In this study, to facilitate efficient therapeutic antibody delivery to tumors using this combination system, we developed therapeutic antibody-modified nanobubble (NBs) using an Fc-binding polypeptide that can quickly load antibodies to nanocarriers; since the polypeptide was derived from Protein G. TUS exposure to this Herceptin®-modified NBs (Her-NBs) was followed by evaluation of the antibody's own ADCC activity, resulting the retained activity. Moreover, the utility of combining therapeutic antibody-modified NBs and TUS exposure as an antibody delivery system for cancer therapy was assessed in vivo. The Her-NBs + TUS group had a higher inhibitory effect than the Herceptin and Her-NBs groups. Overall, these results suggest that the combination of therapeutic antibody-modified NBs and TUS exposure can enable efficient antibody drug delivery to tumors, while retaining the original antibody activity. Hence, this system has the potential to maximize the therapeutic effects in antibody therapy for solid cancers.

Laminin ß2 variants associated with isolated nephropathy that impact matrix regulation.

Mutations in LAMB2, encoding laminin ß2, cause Pierson syndrome and occasionally milder nephropathy without extrarenal abnormalities. The most deleterious missense mutations that have been identified affect primarily the N-terminus of laminin ß2. On the other hand, those associated with isolated nephropathy are distributed across the entire molecule, and variants in the ß2 LEa-LF-LEb domains are exclusively found in cases with isolated nephropathy. Here we report the clinical features of mild isolated nephropathy associated with 3 LAMB2 variants in the LEa-LF-LEb domains (p.R469Q, p.G699R, and p.R1078C) and their biochemical characterization. Although Pierson syndrome missense mutations often inhibit laminin ß2 secretion, the 3 recombinant variants were secreted as efficiently as WT. However, the ß2 variants lost pH dependency for heparin binding, resulting in aberrant binding under physiologic conditions. This suggests that the binding of laminin ß2 to negatively charged molecules is involved in glomerular basement membrane (GBM) permselectivity. Moreover, the excessive binding of the ß2 variants to other laminins appears to lead to their increased deposition in the GBM. Laminin ß2 also serves as a potentially novel cell-adhesive ligand for integrin α4ß1. Our findings define biochemical functions of laminin ß2 variants influencing glomerular filtration that may underlie the pathogenesis of isolated nephropathy caused by LAMB2 abnormalities.

Development of A2G80 peptide-gene complex for targeted delivery to muscle cells.

Therapeutic strategies based on antisense oligonucleotides and therapeutic genes are being extensively investigated for the treatment of hereditary muscle diseases and hold great promise. However, the cellular uptake of these polyanions to the muscle cells is inefficient. Therefore, it is necessary to develop more effective methods of gene delivery into the muscle tissue. The A2G80 peptide (VQLRNGFPYFSY) from the laminin α2 chain has high affinity for α-dystroglycan (α-DG) which is expressed on the membrane of muscle cells. In this study, we designed a peptide-modified A2G80 with oligoarginine and oligohistidine (A2G80-R9-H8), and prepared peptide/plasmid DNA (pDNA) complex, to develop an efficient gene delivery system for the muscle tissue. The peptide/pDNA complex showed α-DG-dependent cellular uptake of the A2G80 sequence and significantly improved gene transfection efficiency mediated by the oligohistidine sequence in C2C12 myoblast cells. Further, the peptide/pDNA complex promoted efficient and sustained gene expression in the Duchenne muscular dystrophy mouse models. The A2G80-R9-H8 peptide has the potential for use as a specific carrier for targeting muscle in gene therapy in muscular dystrophy.

Alpha-dystroglycan binding peptide A2G80-modified stealth liposomes as a muscle-targeting carrier for Duchenne muscular dystrophy.

Safe and efficient gene therapy for the treatment of Duchenne muscular dystrophy (DMD), a genetic disorder, is required. For this, the muscle-targeting delivery system of genes and nucleic acids is ideal. In this study, we focused on the A2G80 peptide, which has an affinity for α-dystroglycan expressed on muscle cell membranes, as a muscle targeted nanocarrier for DMD and developed A2G80-modified liposomes. We also prepared A2G80-modified liposomes coated with long- and short-chain PEG, called A2G80-LSP-Lip, to improve the blood circulation of liposomes using microfluidics. The liposomes had a particle size of approximately 80 nm. A2G80-LSP-Lip showed an affinity for the muscle tissue section of mice by overlay assay. When the liposomes were administered to DMD model mice (mdx mice) via the tail vein, A2G80-LSP-Lip accumulated efficiently in muscle tissue compared to control liposomes. These results suggest that A2G80-LSP-Lip can function as a muscle-targeting liposome for DMD via systemic administration, and may be a useful tool for DMD treatment.

Conformational dependence of integrin-binding peptides derived from homologous loop regions in the laminin α chains.

Laminin α chains (α1-α5 chains) are expressed in a tissue- and developmental stage-specific manner and have diverse chain-specific biological functions. Especially, laminin globular (LG) modules (LG1-LG5) located at the C-terminus of the α chains play a critical role in the biological activities of laminins. Each LG module is composed of a 14-stranded ß-sheet (A-N) sandwich structure. We previously screened cell attachment activity of the loop regions between the E and F strands in the LG modules using 17 homologous peptides (EF peptides) and found that four active EF peptides bind to integrin α2ß1. One of the four peptides, G4EF1 demonstrated improved cell attachment activity when cyclized. Here, we focused on the remaining three integrin α2ß1-binding EF peptides (G5EF1, G3EF3, and G5EF5) and analyzed the relationship between their peptide conformation and cell attachment activity. First, we determined their active core sequences and found that G5EF1z (IGLEIVDGKVLFHVNN), G3EF3z (LLVTLEDGHIALST), and G5EF5z (KVLTEQVL) are the core sequences. Cyclic peptides of the core sequences (cycloG5EF1z, cycloG3EF3z, and cycloG5EF5z) enhanced integrin-mediated cell adhesion activity compared with their linear peptides. The results indicated that cell adhesion activity of the integrin α2ß1-binding EF peptides is conformation dependent and that the loop structure is critical for their activity. This suggests that conformation of the loop regions plays an important role for the activities of the LG modules.

Development of Three-Dimensional Cell Culture Scaffolds Using Laminin Peptide-Conjugated Agarose Microgels.

Three-dimensional (3D) cell scaffolds are essential for tissue engineering. So far, various polymer hydrogels have been utilized to design 3D scaffolds as a biomaterial. In this study, we focused on a biocompatible polysaccharide, agarose, as a potential biomaterial candidate. We have previously established a laminin-derived cell adhesive peptide library, and these peptides are useful for incorporating cell adhesiveness into inert materials. We synthesized aldehyde-functionalized agarose (CHO-agarose) by (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) oxidation of agarose and developed peptide-agarose scaffolds using two laminin peptides, CGG-A99 (CGGAGTFALRGDNPQG, binds to αvß3 integrin) and CGG-AG73 (CGGRKRLQVQLSIRT, binds to syndecan). The peptides were effectively loaded onto the CHO-agarose gels via thiazolidine formation without coupling reagents. Two-dimensional (2D) cell culture assay using human dermal fibroblasts (HDFs) showed that the peptide-agarose gels have potent cell adhesion activity and promoted cell proliferation. Furthermore, we developed a simple preparation method of 3D cell culture scaffolds using peptide-agarose microgels. HDFs cultured in a 3D environment of the A99-agarose microgel scaffold exhibited cell spreading morphology and proliferated significantly. These results suggest that 3D cell culture systems using peptide-agarose microgel scaffolds are promising as a biomaterial for tissue engineering.

Evaluation of extracellular matrix mimetic laminin bioactive peptide and elastin-like polypeptide.

The extracellular matrix (ECM) is comprised of a large network of proteins that are essential for tissue development and repair. A bioactive RGD-containing peptide from laminin α1 chain, A99 (AGTFALRGDNPQG), promotes strong cell attachment and has demonstrated utility in cell culture and tissue engineering. Various materials can be utilized as a scaffold for bioactive peptides; however, it may be advantageous to design materials that use bioconjugation strategies that do not affect bioactivity, generate homogenous products, and can be produced at scale. This report is the first to compare the methods for preparing chemically conjugated and recombinant A99 to elastin-like polypeptides (ELPs) as the scaffold and characterize the biological and cell attachment activity using human dermal fibroblasts (HDFs). ELPs are biocompatible protein-polymers that are also thermo-responsive. Below a lower critical solution temperature (LCST), they are highly soluble. Above the LCST, ELPs phase separate into a polymer-rich liquid, known as a coacervate. Both chemically conjugated and recombinant fusion between A99 and an ELP (A99-ELP-R) show dose-dependent cell attachment. In addition, coating above the LCST provides better cell spreading compared to coating at 4°C. ELPs provide an excellent structural framework for deposition of bioactive peptides of the ECM, and their intrinsic biophysical properties make laminin peptide-ELPs promising biomaterials for cell culture and tissue engineering.

Tissue substructure-specific deposition of the ß3-containing laminin-332 in the biliary epithelium of human and mouse livers.

Laminin is a family of basement membrane proteins, whose selective and spatiotemporal expression profiles are linked to their various functions in development, maintenance, and functional regulation of different tissues. In the liver, α1-and α5-containing laminin isoforms have been documented to be critically involved in the developmental process of the epithelial tissue of the bile duct. However, possible roles of other laminin isoforms in bile duct formation and function remain elusive. Here, we evaluated public single-cell RNA sequencing databases on human liver cells to reveal expression landscape of laminin genes, and found that genes for laminin-332 subunits were conjointly expressed in the EPCAM+ biliary epithelial cell population. Expression of the ß3 and γ2 subunit genes was restricted to biliary epithelial cells in the liver and, remarkably, showed apparent heterogeneity among them. We confirmed the heterogeneous nature of the laminin-ß3 expression in murine livers, which was firmly related to morphological substructures in the biliary epithelium. Finally, we generated the liver epithelial tissue-specific laminin- ß3 knockout mice and found that this laminin subunit was dispensable under physiological conditions. Together, our present findings have identified the ß3 subunit and the related laminin-332 isoform as useful markers and potentially important regulatory molecules for future understanding of pathophysiology in the hepatobiliary system.

Identification of active sequences in human laminin α5 G domain.

Human laminin-511 (α5ß1γ1) and its truncated protein, laminin-511 E8 fragment, bind to integrin α6ß1 and have been widely used for embryonic stem cell and induced pluripotent stem cell culture under feeder-free conditions. In this study, we focused on human laminin α5 chain G domain, which is thought to be critical for the biological functions of laminin-511, and screened its biologically active sequences using a synthetic peptide library. We synthesized 115 peptides (hA5G1-hA5G115) covering the entire laminin α5 chain G domain and evaluated cell attachment activity using both the peptide-coated plate and peptide-chitosan matrix (peptide-ChtM) assays. Seventeen peptides demonstrated cell attachment activity in the assays. Both hA5G18 and hA5G26-coated plates and hA5G74-ChtMs promoted integrin ß1-mediated cell attachment. These findings are useful for the study of molecular mechanisms of laminin-511, and the active peptides have a potential for use as a molecular probe for cell adhesion receptors.

Disrupted tubular parathyroid hormone/parathyroid hormone receptor signaling and damaged tubular cell viability possibly trigger postsurgical kidney injury in patients with advanced hyperparathyroidism.

BACKGROUND: Parathyroidectomy (PTX) that alleviates clinical manifestations of advanced hyperparathyroidism, including hypercalcemia and hypophosphatemia, is considered the best protection from calcium overload in the kidney. However, little is known about the relationship between postsurgical robust parathyroid hormone (PTH) reduction and perisurgical renal tubular cell viability. Post-PTX kidney function is still a crucial issue for primary hyperparathyroidism (PHPT) and tertiary hyperparathyroidism after kidney transplantation (THPT). METHODS: As a clinical study, we examined data from 52 consecutive patients (45 with PHPT, 7 with THPT) who underwent PTX in our center between 2015 and 2017 to identify post-PTX kidney injury. Their clinical data, including urinary liver-type fatty acid-binding protein (L-FABP), a tubular biomarker for acute kidney injury (AKI), were obtained from patient charts. An absolute change in serum creatinine level of 0.3 mg/dL (26.5 µmol/L) on Day 2 after PTX defines AKI. Post-PTX calcium supplement dose adjustment was performed to strictly maintain serum calcium at the lower half of the normal range. To mimic post-PTX-related kidney status, a unique parathyroidectomized rat model was produced as follows: 13-week-old rats underwent thyroparathyroidectomy (TPTX) and/or 5/6 subtotal nephrectomy (NX). Indicated TPTX rats were given continuous infusion of a physiological level of 1-34 PTH using a subcutaneously implanted osmotic minipump. Immunofluorescence analyses were performed by polyclonal antibodies against PTH receptor (PTHR) and a possible key modulator of kidney injury, Klotho. RESULTS: Patients' estimated glomerular filtration rate (eGFR) did not have any clinically relevant change (62.5 ± 22.0 versus 59.4 ± 21.9 mL/min/1.73 m2, NS), whereas serum calcium (2.7 ± 0.18 versus 2.2 ± 0.16 mmol/L, P < 0.0001) and phosphorus levels (0.87 ± 0.19 versus 1.1 ± 0.23 mmol/L, P < 0.0001) were normalized and PTH decreased robustly (181 ± 99.1 versus 23.7 ± 16.8 pg/mL, P < 0.0001) after successful PTX. However, six patients who met postsurgical AKI criteria had lower eGFR and greater L-FABP than those without AKI. Receiver operating characteristics (ROC) analysis revealed eGFR <35 mL/min/1.73 m2 had 83% accuracy. Strikingly, L-FABP >9.8 µg/g creatinine had 100% accuracy in predicting post-PTX-related AKI. Rat kidney PTHR expression was lower in TPTX. PTH infusion (+PTH) restored tubular PTHR expression in rats that underwent TPTX. Rats with TPTX, +PTH and 5/6 NX had decreased PTHR expression compared with those without 5/6 NX. 5/6 NX partially cancelled tubular PTHR upregulation driven by +PTH. Tubular Klotho was modestly expressed in normal rat kidneys, whereas enhanced patchy tubular expression was identified in 5/6 NX rat kidneys. This Klotho and expression and localization pattern was absolutely canceled in TPTX, suggesting that PTH indirectly modulated the Klotho expression pattern. TPTX +PTH recovered tubular Klotho expression and even triggered diffusely abundant Klotho expression. 5/6 NX decreased viable tubular cells and eventually downregulated tubular Klotho expression and localization. CONCLUSIONS: Preexisting tubular damage is a potential risk factor for AKI after PTX although, overall patients with hyperparathyroidism are expected to keep favorable kidney function after PTX. Patients with elevated tubular cell biomarker levels may suffer post-PTX kidney impairment even though calcium supplement is meticulously adjusted after PTX. Our unique experimental rat model suggests that blunted tubular PTH/PTHR signaling may damage tubular cell viability and deteriorate kidney function through a Klotho-linked pathway.

Characterization of dystroglycan binding in adhesion of human induced pluripotent stem cells to laminin-511 E8 fragment.

Human induced pluripotent stem cells (hiPSCs) grow indefinitely in culture and have the potential to regenerate various tissues. In the development of cell culture systems, a fragment of laminin-511 (LM511-E8) was found to improve the proliferation of stem cells. The adhesion of undifferentiated cells to LM511-E8 is mainly mediated through integrin α6ß1. However, the involvement of non-integrin receptors remains unknown in stem cell culture using LM511-E8. Here, we show that dystroglycan (DG) is strongly expressed in hiPSCs. The fully glycosylated DG is functionally active for laminin binding, and although it has been suggested that LM511-E8 lacks DG binding sites, the fragment does weakly bind to DG. We further identified the DG binding sequence in LM511-E8, using synthetic peptides, of which, hE8A5-20 (human laminin α5 2688-2699: KTLPQLLAKLSI) derived from the laminin coiled-coil domain, exhibited DG binding affinity and cell adhesion activity. Deletion and mutation studies show that LLAKLSI is the active core sequence of hE8A5-20, and that, K2696 is a critical amino acid for DG binding. We further demonstrated that hiPSCs adhere to hE8A5-20-conjugated chitosan matrices. The amino acid sequence of DG binding peptides would be useful to design substrata for culture system of undifferentiated and differentiated stem cells.