Biomedical subjects

Li-Wen Lai

Publications and source records attributed to Li-Wen Lai.

14 recordsLinked to original sources

PubMed2006

Functionally improved bone in calbindin-D28k knockout mice.

In vitro studies indicate that Calbindin-D28k, a calcium binding protein, is important in regulating the life span of osteoblasts as well as the mineralization of bone extracellular matrix. The recent creation of a Calbindin-D28k knockout mouse has provided the opportunity to study the physiological effects of the Calbindin-D28k protein on bone remodeling in vivo. In this experiment, histomorphometry, microCT, and bend testing were used to characterize bones in Calbindin-D28k KO (knockout) mice. The femora of Calbindin-D28k KO mice had significantly increased cortical bone volume (60.4% +/- 3.1) compared to wild-type (WT) mice (45.4% +/- 4.6). The increased bone volume was due to a decrease in marrow cavity area, and significantly decreased endosteal perimeters (3.397 mm +/- 0.278 in Calbindin-D28k KO mice, and 4.046 mm +/- 0.450 in WT mice). Similar changes were noted in the analysis of the tibias in both mice. The bone formation rates were similar in the femoral and tibial cortical bones of both mice. microCT analysis of the trabecular bone in the tibial plateau indicated that Calbindin-D28k KO mice had an increased bone volume (35.2% +/- 3.1) compared to WT mice (24.7% +/- 4.9) which was primarily due to increased trabecular number (8.99 mm(-1) +/- 0.94 in Calbindin-D28k KO mice compared to 6.75 mm(-1) +/- 0.85 in WT mice). Bone mineral content analysis of the tibias indicated that there is no difference in the calcium or phosphorus content between the Calbindin-D28k KO and WT mice. Cantilever bend testing of the femora demonstrated significantly lower strains in the bones of Calbindin-D28k KO mice (4135 micro strain/kg +/- 1266) compared to WT mice (6973 micro strain/kg +/- 998) indicating that the KO mice had stiffer bones. Three-point bending demonstrated increased failure loads in bones of Calbindin-D28k KO mice (31.6 N +/- 2.1) compared to WT mice (15.0 N +/- 1.7). In conclusion, Calbindin-D28k KO mice had increased bone volume and stiffness indicating that Calbindin-D28k plays an important role in bone remodeling.

Animals↗

PubMed2005

Novel delivery of oligonucleotides using a topical hydrogel tissue sealant in a murine partial nephrectomy model.

PURPOSE: Ischemia/reperfusion injury is a leading cause of renal damage and antisense gene therapy has been shown to ameliorate its effects. However, this approach has been limited by current delivery methods that require high concentrations of intravenous nucleic acids lacking specificity for targeting tissues. To overcome these limitations we developed a novel murine partial nephrectomy model to evaluate polyethylene-glycol (PEG) hydrogel tissue sealant as a topical oligonucleotide delivery system. MATERIALS AND METHODS: A total of 18 male C57BL/6 mice underwent left partial nephrectomy with vascular occlusion. Hydrogel primer and then sealant were applied to the cut surface and photopolymerized. Using this method 16 additional mice received hydrogel primer mixed with Cy5 labeled fluorescent oligonucleotide (10 to 100 microg). Kidneys were harvested at various time points and assessed for oligonucleotide penetration using fluorescence microscopy. RESULTS: A survival rate of 100% (34 subjects) was obtained using this mouse model of partial nephrectomy. PEG hydrogel provided adequate protection against renal hematoma and intraperitoneal blood. Fluorescent images revealed that 50 microg was the minimum dose resulting in complete progressive cellular penetration with time. In addition to direct diffusion from the application site, movement of oligonucleotide through the subcapsular space into the cortex was an observed mechanism of distribution. CONCLUSIONS: A murine partial nephrectomy model was successfully created using PEG hydrogel. In addition to achieving hemostasis, hydrogel served as a successful depot for delivering oligonucleotides throughout the kidney.

Animals↗

PubMed2005

Bilateral femoral head and distal tibial osteonecrosis in a patient with Fabry disease.

Fabry disease is a lysosomal storage disease caused by alpha-galactosidase A deficiency. The classic presentation of Fabry disease involves multiple organs, including kidneys, heart, skin, eyes, and nervous system. Osteonecrosis is rarely reported in patients with Fabry disease. In this article, we describe the case of a 37-year-old white man who had Fabry disease and no risk factors for osteonecrosis but who developed osteonecrosis in both femoral heads and in an unusual site, bilateral distal tibiae. Results of mutation analysis showed a nonsense mutation (R227X) in the alpha-galactosidase A gene. This case suggests that Fabry disease may be a risk factor for development of osteonecrosis. The enzyme replacement therapy currently available may be an effective method of preventing this complication.

Adult↗

PubMed2004

Effect of thiazide on renal gene expression of apical calcium channels and calbindins.

Thiazide diuretics are specific inhibitors of the Na-Cl cotransporter in the distal convoluted tubule (DCT). In addition to producing diuresis and natriuresis, they have a hypocalciuric effect. Recently, two apical calcium channels have been identified, transient receptor potential vanilloid 5 (TRPV5) and TRPV6; both are expressed in the DCT. We studied the effects of thiazides on mouse renal calcium handling and renal gene expression of TRPV5 and TRPV6, as well as calbindin-D(28k) and calbindin-D(9k), both of which are calcium transport facilitators located in the DCT. Upregulation of renal TRPV5 was found 4 h after intraperitoneal injection of chlorothiazide (CTZ) at both 25 and 50 mg/kg, but not at 100 mg/kg. Chronic treatment with CTZ at 25 mg/kg twice daily for 3 days, with or without salt supplementation of 0.8% NaCl and 0.1% KCl in the drinking water, caused hypocalciuria, but the gene expression patterns were different. Without salt supplementation, mice developed volume contraction and there were no changes in gene expression. When volume contraction was prevented by salt supplementation, there was a significant increase in gene expression of TRPV5, calbindin-D(28k), and calbindin-D(9k). Salt supplementation alone also induced significant upregulation of TRPV5, TRPV6, and both calbindins. The upregulation of TRPV5 by CTZ and salt supplementation and salt alone was further confirmed with immunofluorescent staining studies. Our studies suggest that thiazides induce hypocalciuria through different mechanisms depending on volume status. With volume contraction, increased calcium reabsorption in the proximal tubule plays the major role. Without volume contraction, hypocalciuria is probably achieved through increased calcium reabsorption in the DCT by the activation of a transcellular calcium transport system and upregulation of apical calcium channel TRPV5, calbindin-D(28k), and calbindin-D(9k).

Animals↗

PubMed2004

Effect of calbindin-D28K on cyclosporine toxicity in cultured renal proximal tubular cells.

Cyclosporine A (CsA) is known to have direct toxicity to renal tubular cells. Its toxicity may be mediated by intracellular calcium because CsA increases intracellular calcium concentration and enhances the activities of calcium-dependent calpains and caspases. Calbindin-D28k, a cytosolic calcium binding protein, has been used as an intracellular Ca2+ buffer to reduce calcium-mediated cytotoxicity in non-renal cells such as neuronal cells. We investigated the effects of gene transfer of calbindin-D28k cDNA on CsA cytotoxicity and intracellular calcium concentration ([Ca2+]i) in cultured murine proximal tubular (MCT) cells. A plasmid containing calbindin-D28k cDNA under the control of CMV promoter was transfected to MCT cells with liposomes. Cytotoxicity was assessed by LDH release and cell viability assay, and [Ca2+]i was measured ratiometrically with fura-2. Compared with MCT cells, cells transfected with calbindin-D28k cDNA showed a reduction in LDH release by 27, 30, 32, 33, and 19% (all P < 0.05), respectively, after 24 h exposure to 1, 2.5, 5, 10, and 25 microM CsA. Cell viability after CsA treatment was also significantly higher in CB cells. A mock transfection using plasmid without calbindin-D28k cDNA insert did not affect the LDH release or cell viability after CsA treatment. CsA treatment did not affect the protein and mRNA abundance of transfected calbindin-D28k cDNA. The expression of calbindin-D28k did not affect the baseline [Ca2+]i, but significantly suppressed CsA-induced elevation in [Ca2+]i. The expression of calbindin-D28k in renal tubular cells provides cytoprotective effects against CsA toxicity, probably through its buffering effects on [Ca2+]i.

Animals↗

PubMed2004

Bilateral symmetry of biomechanical properties in mouse femora.

Bone healing and remodeling are commonly examined in animal models by comparing one femur (experimental) to the contralateral femur (control) with the assumption that they are identical with respect to their biomechanical properties. While past studies have characterized the symmetry in geometrical properties in many types of animal bones, few studies have compared the symmetry in the biomechanical properties. The purpose of this study was to determine whether there is symmetry in the mechanical properties of mouse femora. Strain gauges were attached to the posterior surface of the femora of C57BL/6 mice, parallel to the long axis of the bone. The femora were mechanically tested in cantilever bending while strain values were recorded. Moments of inertia, cortical areas, and moduli of elasticity were determined from strains and cross-sectional properties. Mouse femora demonstrated an average strain difference of 0.4% in tension and 1.4% in compression. Elastic moduli differed by 6.6% and 0.9% in tension and compression, respectively, and failure strength differed by an average of 2.0%. Statistical analysis showed there were no significant differences in strain, modulus, or failure load values for the mice, indicating mechanical and geometrical symmetry of mouse femora in cantilever bending.

Anatomy, Cross-Sectional↗

PubMed2004

Gene therapy for renal disorders.

During the last 20 years there have been major improvements in renal replacement therapy, including dialysis and kidney transplantation; however, the treatment options for renal diseases are still limited. Gene therapy is a potential modality for many renal diseases for which we are as yet unable to offer specific treatment. This article reviews the recent data on gene therapy in animal models applicable to human renal diseases and evaluates its efficacy, safety and clinical relevance. Several approaches appear to be promising, including adeno-associated viral vectors for long-term gene expression, electroporation for muscular gene delivery, ultrasound/microbubble-mediated gene targeting, macrophage-based gene therapy and small interfering RNAs.

Dependovirus↗

PubMed2004

A novel alpha-galactosidase a mutant (M42L) identified in a renal variant of Fabry disease.

A 65-year-old man presented to our institution for workup of proteinuria. His serum creatinine level was 1.7 mg/dL (130 micromol/L), and he had proteinuria with protein of almost 5 g/24 h. Fabry disease was diagnosed by means of kidney biopsy and low serum and leukocyte levels of alpha-galactosidase A. Review of his history, family history, physical examinations, and diagnostic studies did not show other findings typical of this disease. His renal function continued to decline, and he eventually underwent a living unrelated renal transplantation 5 years later. Three years after transplantation, his creatinine level is 1.7 mg/dL (130 micromol/L), and corrected iothalamate clearance is 53 mL/min/1.73 m2 . Genetic studies showed that he has a novel missense mutation (M42L) in exon 1. Methionine at codon 42 is highly conserved in eukaryotic alpha-galactosidase A orthologues. This genotype predicts a minor misfolding of alpha-galactosidase A because of a small difference in hydrophobicity between methionine and leucine. His mutation resulted in a very low, but detectable, serum level of alpha-galactosidase A (0.002 U/L; normal range, 0.016 to 0.2 U/L). Cases of Fabry disease that present with predominantly renal manifestations are rare and require a high index of suspicion for diagnosis. Because treatment for Fabry disease recently has become available, it is important for clinicians to be aware of this disease and pursue the diagnosis in cases of otherwise unexplained renal dysfunction.

Aged↗

PubMed2003

Pathogenesis of renal ischemia/reperfusion injury: lessons from knockout mice.

Ischemia/reperfusion-induced acute renal failure is a common clinical problem associated with a high morbidity and mortality. Upon hypoxic injury, the depletion of ATP causes mitochondrial dysfunction, and accumulation of intracellular sodium, calcium and reactive oxygen species. Subsequently, multiple enzyme systems including proteases, nitric oxide synthases, phospholipases and endonuclease are activated and responsible for cytoskeleton disruption, membrane damage, and DNA degradation, and eventually cell death. Ischemia/reperfusion injury also activates complement, cytokines, and chemokines, which are cytotoxic themselves, but also attract leukocytes into the ischemic area to cause further damage. The vascular endothelial cell injury and dysfunction prolong ischemia and induce vascular congestion, edema, and further infiltration of inflammatory cells. Many players in renal ischemia/reperfusion injury and their mechanisms have been investigated using genetically manipulated mouse models. In this review, we focus on the information gathered from these studies. Deficiency of the Na/Ca exchanger, inducible nitric oxide synthase, Caspase-1, A3 adenosine receptor, C3, C5, C6, Factor B, or midkine protects the kidney against I/R injury. Conversely, deficiency of the interleukin-1 receptor, osteopontin, C4, or recombination activation gene-1 is not protective, while the absence of adrenomedullin or endothelin receptor B delays the recovery of ischemia/reperfusion injury. The knowledge obtained from these studies provides new direction for designing potential therapeutic agents for treating ischemia/reperfusion injury.

Adenosine↗

PubMed2003

Renal gene transfer: nonviral approaches.

Gene therapy has the potential to become an important modality for treating both hereditary and acquired renal diseases. Since renal diseases may involve different cell types in the kidney, it is critical to achieve efficient gene transfer specifically to each cell type. We reviewed the literature on nonviral gene transfer techniques, which are designed to target the kidney specifically. A variety of approaches have been developed to target glomeruli, tubules, renal vasculature, and interstitium with different degree of success. Besides using delivery systems based on liposomes, polycations, and viral fusion proteins, investigators have adopted newer approaches including electroporation and hydrodynamic-based gene transfer, and demonstrated that they are efficient and safe in animal models. Potential clinical applications and safety concerns of gene therapy for renal diseases are discussed.

DNA↗

PubMed2002

Cytoprotective effects of calbindin-D(28k) against antimycin-A induced hypoxic injury in proximal tubular cells.

Intracellular calcium plays an important role on the pathogenesis of hypoxia-induced cellular injury. Calbindin-D(28k), a cytosolic vitamin D-dependent calcium binding protein, can serve as a buffer to limit a surge in intracellular Ca2+ concentration ([Ca2+]i) induced by various stimulations. To evaluate the possible cytoprotective effect of calbindin-D(28k) against hypoxic injury in proximal tubular cells, a plasmid containing calbindin-D(28k) cDNA under the control of CMV immediate-early gene promoter was transfected into the murine proximal tubular epithelial (MCT) cells. The expression of calbindin-D(28k) in the transfected cells was verified with Northern blot analysis, Western blot analysis, and immunofluorescent staining. The non-transfected and transfected MCT cells were subjected to chemical hypoxia induced by antimycin A (10 microM) and glucose deprivation for 30-120 min. The transfection of calbindin-D(28k) reduced lactate dehydrogenase (LDH) release by 41%, 41%, 24%, and 24%, respectively, at 30, 60, 90 and 120 min after hypoxia when compared to the non-transfected cells (all p < 0.05). Cell viability after hypoxic injury was also significantly higher in transfected cells than non-transfected cells. Transfection with the plasmid without calbindin-D(28k) cDNA did not affect LDH release or cell viability after chemical hypoxic injury. [Ca+2]i was measured ratiometrically with fura-2 after exposure to chemical hypoxia. The rate of initial rise in [Ca2+]i and final [Ca+2]i at 30-120 min were significantly lowered in transfected cells. In conclusion, this study demonstrated that transfection of calbindin-D(28k) gene into MCT cells provide protective effects against chemical hypoxic injury probably through its buffering effects on [Ca+2]i.

Animals↗

PubMed2002

Cyclosporine A-induced hypercalciuria in calbindin-D28k knockout and wild-type mice.

BACKGROUND: It is known that cyclosporine A (CsA) treatment induces high bone-turnover osteopenia and hypercalciuria. It has been proposed that down-regulation of renal calbindin-D28k by CsA results in renal calcium wasting. We investigated the role of the kidney and bone in CsA-induced hypercalciuria in calbindin-D28k knockout (KO) and wild-type (WT) mice. METHODS: Two sets of experiments were performed. In experiment 1, KO and WT mice were treated with CsA 20 mg/kg/day intraperitoneally (IP) for 7 days. In experiment 2, to eliminate the CsA effect on bone resorption, pamidronate (APD) 2.5 mg/kg IP was given every 4 days with the first dose given 4 days prior to the 7-day course of CsA. Serum levels of creatinine, calcium, and osteocalcin, as well as renal calcium excretion were measured to assess CsA's effects on calcium homeostasis. Effects of CsA on the expression of calbindin-D28k, and two calcium channels in the apical membrane of the distal tubule, epithelial calcium channel (ECaC) and alpha1G-subunit of a voltage-dependent Ca channel (alpha1G), in the kidney were examined by semiquantitative reverse transcription polymerase chain reaction (RT-PCR). RESULTS: KO mice had a threefold increase in renal calcium excretion when compared with WT mice at the baseline. This difference disappeared when calcium load was reduced by overnight fasting. After the CsA treatment, both WT and KO mice had a significant increase of renal calcium excretion (urine Ca/Cr ratio in WT, 0.11 +/- 0.01 to 1.29 +/- 0.17; in KO, 0.39 +/- 0.04 to 1.18 +/- 0.13; both P < 0.01). CsA treatment decreased renal calbindin-D28k mRNA by 61%, but did not affect the expression of ECaC and alpha1G. Baseline serum osteocalcin level of KO mice was significantly lower than that of WT mice. After CsA treatment, both groups had a 50% increase in the serum osteocalcin level, indicating increased bone turnover. When mice were treated with both CsA and APD, the increase in serum osteocalcin level was prevented, and renal calcium excretion was significantly lower than that in mice treated with CsA alone. However, there was still a significant increase in the urine Ca/Cr ratio in WT and KO mice compared with pretreatment levels (urine Ca/Cr in WT, 0.11 +/- 0.01 to 0.76 +/- 0.05, P < 0.01; in KO, 0.39 +/- 0.05 to 0.79 +/- 0.06; P < 0.01). CONCLUSION: Calbindin-D28k KO mice have diet-dependent hypercalciuria and a lower bone turnover rate. CsA treatment suppresses the expression of calbindin-D28k in mice, but has no effects on ECaC and alpha1G gene expression at the mRNA level. The pathogenesis of CsA-induced hypercalciuria involves both down-regulation of calbindin-D28k with subsequent impaired renal calcium reabsorption and CsA-induced high turnover bone disease. Additionally, our results suggest that mechanism(s) independent of calbindin-D28k within the kidney also may contribute to the CsA-induced calcium leak.

Animals↗

PubMed2002

Chimeric RNA/DNA oligonucleotide-based gene therapy.

BACKGROUND: Chimeric RNA/DNA oligonucleotides, emerging as a potential strategy for gene therapy, have been shown to induce site-specific correction of point mutations in several genetic disease models. METHODS: Six recent studies of chimeric RNA/DNA oligonucleotide-based gene therapy in genetic disease models are reviewed. Chimeric RNA/DNA oligonucleotides, complementary to 25 to 30 residues of genomic DNA flanking the mutation site with the exception of a mismatch in the center, were delivered via different routes and delivery vehicles to target different tissues and organs. Corrections of the mutation at genotypic and phenotypic levels were assessed using various methods, including allele-specific polymerase chain reaction assay, restriction enzyme digestion, colony-lifting assays, sequencing, Northern and Western blot analyses, enzyme activity assay, immunohistochemical staining, and functional studies. RESULTS: The gene correction frequency varied, ranging from less than 1% to more than 40%. This represented several magnitudes higher conversion rate compared with homologous recombination frequency, which is in the range of 10(-5) to 10(-6). The resulting phenotype changes lasted longer than one year in some studies. CONCLUSION: Chimeric RNA/DNA oligonucleotide-based gene therapy has the potential to develop into powerful therapeutic modality for genetic diseases. It can offer permanent expression and normal regulation of corrected genes in appropriate cells or tissues. Further efforts to elucidate the mechanisms of chimeric RNA/DNA oligonucleotide-based gene therapy are warranted in order to increase the efficacy and safety of this method.

Animals↗

PubMed2002

Gene therapy for renal disorders: what are the benefits for the elderly?

Chronic renal failure is one of the major health problems for the elderly. Currently, about 50% of all patients receiving chronic dialysis for end-stage renal disease (ESRD) are aged 65 years or older. Their first-year mortality rate is as high as 30%. The leading causes of ESRD in the elderly are diabetic nephropathy, hypertension and large vessel diseases, and glomerulonephritis. The elderly are also prone to developing acute renal failure induced by ischaemic injury or nephrotoxic drugs. Gene transfer in experimental animals have been tested in all of these conditions, as well as in animal kidney transplantation models, with various degrees of success. However, there are many obstacles to be overcome before gene therapy can be tested clinically for renal disorders. In particular, the major challenges include determining how to prolong and control transgene expression or antisense inhibition and how to minimise the adverse effects of viral or nonviral vectors. Once these problems are solved, gene therapy will have a role in treating age-related renal impairment.

Aged↗