Reversal of the diabetic bone signature with anabolic therapies in mice.

The mechanisms underlying the bone disease induced by diabetes are complex and not fully understood; and antiresorptive agents, the current standard of care, do not restore the weakened bone architecture. Herein, we reveal the diabetic bone signature in mice at the tissue, cell, and transcriptome levels and demonstrate that three FDA-approved bone-anabolic agents correct it. Diabetes decreased bone mineral density (BMD) and bone formation, damaged microarchitecture, increased porosity of cortical bone, and compromised bone strength. Teriparatide (PTH), abaloparatide (ABL), and romosozumab/anti-sclerostin antibody (Scl-Ab) all restored BMD and corrected the deteriorated bone architecture. Mechanistically, PTH and more potently ABL induced similar responses at the tissue and gene signature levels, increasing both formation and resorption with positive balance towards bone gain. In contrast, Scl-Ab increased formation but decreased resorption. All agents restored bone architecture, corrected cortical porosity, and improved mechanical properties of diabetic bone; and ABL and Scl-Ab increased toughness, a fracture resistance index. Remarkably, all agents increased bone strength over the healthy controls even in the presence of severe hyperglycemia. These findings demonstrate the therapeutic value of bone anabolic agents to treat diabetes-induced bone disease and suggest the need for revisiting the approaches for the treatment of bone fragility in diabetes.

LM22B-10 promotes corneal nerve regeneration through in vitro 3D co-culture model and in vivo corneal injury model.

Corneal nerve wounding often causes abnormalities in the cornea and even blindness in severe cases. In this study, we construct a dorsal root ganglion-corneal stromal cell (DRG-CSC, DS) co-culture 3D model to explore the mechanism of corneal nerve regeneration. Firstly, this model consists of DRG collagen grafts sandwiched by orthogonally stacked and orderly arranged CSC-laden plastic compressed collagen. Nerve bundles extend into the entire corneal stroma within 14 days, and they also have orthogonal patterns. This nerve prevents CSCs from apoptosis in the serum withdrawal medium. The conditioned medium (CM) for CSCs in collagen scaffolds contains NT-3, IL-6, and other factors. Among them, NT-3 notably promotes the activation of ERK-CREB in the DRG, leading to the growth of nerve bundles, and IL-6 induces the upregulation of anti-apoptotic genes. Then, LM22B-10, an activator of the NT-3 receptor TrkB/TrkC, can also activate ERK-CREB to enhance nerve growth. After administering LM22B-10 eye drops to regular and diabetic mice with corneal wounding, LM22B-10 significantly improves the healing speed of the corneal epithelium, corneal sensitivity, and corneal nerve density. Overall, the DS co-culture model provides a promising platform and tools for the exploration of corneal physiological and pathological mechanisms, as well as the verification of drug effects in vitro. Meanwhile, we confirm that LM22B-10, as a non-peptide small molecule, has future potential in nerve wound repair. STATEMENT OF SIGNIFICANCE: The cornea accounts for most of the refractive power of the eye. Corneal nerves play an important role in maintaining corneal homeostasis. Once the corneal nerves are damaged, the corneal epithelium and stroma develop lesions. However, the mechanism of the interaction between corneal nerves and corneal cells is still not fully understood. Here, we construct a corneal stroma-nerve co-culture in vitro model and reveal that NT-3 expressed by stromal cells promotes nerve growth by activating the ERK-CREB pathway in nerves. LM22B-10, an activator of NT-3 receptors, can also induce nerve growth in vitro. Moreover, it is used as eye drops to enhance corneal epithelial wound healing, corneal nerve sensitivity and density of nerve plexus in corneal nerve wounding model in vivo.

Mechanotransduction Regulates Reprogramming Enhancement in Adherent 3D Keratocyte Cultures.

Suspended spheroid culture using ultralow attachment plates (ULAPs) is reported to effect corneal fibroblast reprogramming. Polydimethylsiloxane (PDMS), with hydrophobic and soft substrate properties, facilitates adherent spheroid formation that promotes cellular physical reprogramming into stem-like cells without using transcription factors. However, it is still unknown whether the biophysical properties of PDMS have the same effect on adult human corneal keratocyte reprogramming. Here, PDMS and essential 8 (E8) medium were utilized to culture keratocyte spheroids and fibroblast spheroids, and the reprogramming results were compared. We provide insights into the probable mechanisms of the PDMS effect on spheroids. qPCR analysis showed that the expression of some stem cell marker genes (OCT4, NANOG, SOX2, KLF4, CMYC, ABCG2 and PAX6) was significantly greater in keratocyte spheroids than in fibroblast spheroids. The endogenous level of stemness transcription factors (OCT4, NANOG, SOX2, KLF4 and CMYC) was higher in keratocytes than in fibroblasts. Immunofluorescence staining revealed Klf4, Nanog, Sox2, ABCG2 and Pax6 were positively stained in adherent 3D spheroids but weakly or negatively stained in adherent 2D cells. Furthermore, OCT4, NANOG, SOX2, KLF4, HNK1, ABCG2 and PAX6 gene expression was significantly higher in adherent 3D spheroids than in adherent 2D cells. Meanwhile, SOX2, ABCG2 and PAX6 were more upregulated in adherent 3D spheroids than in suspended 3D spheroids. The RNA-seq analysis suggested that regulation of the actin cytoskeleton, TGFß/BMP and HIF-1 signaling pathways induced changes in mechanotransduction, the mesenchymal-to-epithelial transition and hypoxia, which might be responsible for the effect of PDMS on facilitating reprogramming. In conclusion, compared to corneal fibroblasts, keratocytes were more susceptible to reprogramming due to higher levels of endogenous stemness transcription factors. Spheroid culture of keratocytes using PDMS had a positive impact on promoting the expression of some stem cell markers. PDMS, as a substrate to form spheroids, was better able to promote reprogramming than ULAPs. These results indicated that the physiological cells and culture conditions herein enhance reprogramming. Therefore, adherent spheroid culture of keratocytes using PDMS is a promising strategy to more safely promote reprogramming, suggesting its potential application for developing clinical implants in tissue engineering and regenerative medicine.

Characteristics of neural growth and cryopreservation of the dorsal root ganglion using three-dimensional collagen hydrogel culture versus conventional culture.

In vertebrates, most somatosensory pathways begin with the activation of dorsal root ganglion (DRG) neurons. The development of an appropriate DRG culture method is a prerequisite for establishing in vitro peripheral nerve disease models and for screening therapeutic drugs. In this study, we compared the changes in morphology, molecular biology, and transcriptomics of chicken embryo DRG cultured on tissue culture plates (T-DRG) versus three-dimensional collagen hydrogels (C-DRG). Our results showed that after 7 days of culture, the transcriptomics of T-DRG and C-DRG were quite different. The upregulated genes in C-DRG were mainly related to neurogenesis, axon guidance, and synaptic plasticity, whereas the downregulated genes in C-DRG were mainly related to cell proliferation and cell division. In addition, the genes related to cycles/pathways such as the synaptic vesicle cycle, cyclic adenosine monophosphate signaling pathway, and calcium signaling pathway were activated, while those related to cell-cycle pathways were downregulated. Furthermore, neurogenesis- and myelination-related genes were highly expressed in C-DRG, while epithelial-mesenchymal transition-, apoptosis-, and cell division-related genes were suppressed. Morphological results indicated that the numbers of branches, junctions, and end-point voxels per C-DRG were significantly greater than those per T-DRG. Furthermore, cells were scattered in T-DRG and more concentrated in C-DRG, with a higher ratio of 5-ethynyl-2'-deoxyuridine (EdU)-positive cells in T-DRG compared with C-DRG. C-DRG also had higher S100 calcium-binding protein B (S100B) and lower α-smooth muscle actin (α-SMA) expression than T-DRG, and contained fewer terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells after 48 hours of serum starvation. After cryopreservation, C-DRG maintained more intact morphological characteristics, and had higher viability and less TUNEL-positive cells than T-DRG. Furthermore, newly formed nerve bundles were able to grow along the existing Schwann cells in C-DRG. These results suggest that C-DRG may be a promising in vitro culture model, with better nerve growth and anti-apoptotic ability, quiescent Schwann cells, and higher viability. Results from this study provide a reference for the construction, storage, and transportation of tissue-engineered nerves. The study was approved by the Ethics Committee of Aier School of Ophthalmology, Central South University, China (approval No. 2020-IRB16), on March 15, 2020.

Effect of porcine corneal stromal extract on keratocytes from SMILE-derived lenticules.

Propagating large amounts of human corneal stromal cells (hCSCs) in vitro while maintaining the physiological quality of their phenotypes is necessary for their application in cell therapy. Here, a novel medium to propagate hCSCs obtained from small incision lenticule extraction (SMILE)-derived lenticules was investigated and the feasibility of intrastromal injection of these hCSCs was assessed. Primary hCSCs were cultured in porcine corneal stroma extract (pCSE) with RIFA medium including ROCK inhibitor Y27632, insulin-transferrin-selenium, fibroblast growth factor 2, L-ascorbate 2-phosphate and 0.5% FBS (RIFA medium + pCSE). Protein profiling of the pCSE was identified using nanoscale liquid chromatography coupled to tandem mass spectrometry (nano LC-MS/MS). After subculturing in RIFA medium + pCSE or 10% FBS normal medium (NM), hCSCs at P4 were transplanted into mouse corneal stroma. Compared with NM, ALDH3A1, keratocan and lumican were significantly more expressed in the RIFA medium + pCSE. ALDH3A1 was also more expressed in the RIFA medium + pCSE than in the RIFA medium. Fibronectin and α-SMA were less expressed in the RIFA medium + pCSE than in the NM. Using Metascape analysis, the pCSE with its anti-fibrosis, pro-proliferation and anti-apoptosis activities, was beneficial for hCSC cultivation. The intrastromally implanted hCSCs in the RIFA medium + pCSE had positive CD34 expression but negative CD45 expression 35 days after injection. We provide a valuable new medium that is advantageous for the proliferation of hCSCs with the properties of physiological keratocytes. Intrastromal injection of hCSCs in RIFA medium + pCSE has the potential for clinical cell therapy.

Epidemiology and clinical course of COVID-19 in Shanghai, China.

Background: Novel coronavirus pneumonia (COVID-19) is prevalent around the world. We aimed to describe epidemiological features and clinical course in Shanghai. Methods: We retrospectively analysed 325 cases admitted at Shanghai Public Health Clinical Center, between January 20 and February 29, 2020. Results: 47.4% (154/325) had visited Wuhan within 2 weeks of illness onset. 57.2% occurred in 67 clusters; 40% were situated within 53 family clusters. 83.7% developed fever during the disease course. Median times from onset to first medical care, hospitalization and negative detection of nucleic acid by nasopharyngeal swab were 1, 4 and 8 days. Patients with mild disease using glucocorticoid tended to have longer viral shedding in blood and feces. At admission, 69.8% presented with lymphopenia and 38.8% had elevated D-dimers. Pneumonia was identified in 97.5% (314/322) of cases by chest CT scan. Severe-critical patients were 8% with a median time from onset to critical disease of 10.5 days. Half required oxygen therapy and 7.1% high-flow nasal oxygen. The case fatality rate was 0.92% with median time from onset to death of 16 days. Conclusion: COVID-19 cases in Shanghai were imported. Rapid identification, and effective control measures helped to contain the outbreak and prevent community transmission.

Combination of four clinical indicators predicts the severe/critical symptom of patients infected COVID-19.

BACKGROUND: Despite the death rate of COVID-19 is less than 3%, the fatality rate of severe/critical cases is high, according to World Health Organization (WHO). Thus, screening the severe/critical cases before symptom occurs effectively saves medical resources. METHODS AND MATERIALS: In this study, all 336 cases of patients infected COVID-19 in Shanghai to March 12th, were retrospectively enrolled, and divided in to training and test datasets. In addition, 220 clinical and laboratory observations/records were also collected. Clinical indicators were associated with severe/critical symptoms were identified and a model for severe/critical symptom prediction was developed. RESULTS: Totally, 36 clinical indicators significantly associated with severe/critical symptom were identified. The clinical indicators are mainly thyroxine, immune related cells and products. Support Vector Machine (SVM) and optimized combination of age, GSH, CD3 ratio and total protein has a good performance in discriminating the mild and severe/critical cases. The area under receiving operating curve (AUROC) reached 0.9996 and 0.9757 in the training and testing dataset, respectively. When the using cut-off value as 0.0667, the recall rate was 93.33 % and 100 % in the training and testing datasets, separately. Cox multivariate regression and survival analyses revealed that the model significantly discriminated the severe/critical cases and used the information of the selected clinical indicators. CONCLUSION: The model was robust and effective in predicting the severe/critical COVID cases.

The Construction of Retinal Pigment Epithelium Sheets with Enhanced Characteristics and Cilium Assembly Using iPS Conditioned Medium and Small Incision Lenticule Extraction Derived Lenticules.

In vitro generation of a functional retinal pigment epithelium (RPE) monolayer sheet is useful and promising for RPE cell therapy. Here, for the first time, we used induced pluripotent stem (iPS) supernatant as the conditioned medium (iPS-CM) and femtosecond laser intrastromal lenticule (FLI-lenticule) as a scaffold to construct an engineered RPE sheet. There are significant enhancements in RPE cell density, transepithelial electrical resistance (TER) and inhibitions of ultraviolet C (UVC)-irradiated apoptosis when RPE cells are cultured in iPS supernatant/Dulbecco's modified Eagle's medium (DMEM)-F12 of 1/2 (iPS-CM) compared with those in normal medium (NM, DMEM-F12). Using the assay of a panel of cytokines, combined with transcriptome and protein analyses, we discover that iPS-CM contains high levels of platelet-derived growth factor AA (PDGF-AA), insulin-like growth factor binding protein (IGFBP)-2, transforming growth factor (TGF)-α and IGFBP-6, which are responsible for the upregulation of gene and protein markers with RPE phenotypes and downregulation of gene and protein markers with epithelial-mesenchymal transition (EMT) phenotypes for RPE cells in iPS-CM when compared to those in NM. Moreover, compared to cultures on tissue culture plates (TCP), RPE cells on FLI-lenticule display more microvilli and cilium in accordance with the results in terms of RNA-Seq data, quantitative polymerase chain reaction (qPCR) expression, immunofluorescence staining, and western blot assays. Furthermore, acellular FLI-lenticule exhibits biocompatibility after rabbit subretinal implantation by 30 days through electroretinography and histological examination. Thus, we determined that engineered RPE sheets treated by iPS-CM in conjunction with FLI-lenticule scaffold aid in enhanced RPE characteristics and cilium assembly. Such a strategy to construct RPE sheets is a promising avenue for developing RPE cell therapy, disease models and drug screening tools. STATEMENT OF SIGNIFICANCE: In vitro generation of a functional RPE monolayer sheet is useful and promising for RPE cell therapy. Here, we constructed engineered RPE sheets treated by iPS-CM in conjunction with FLI-lenticule scaffolds to help in enhanced RPE characteristics and cilium assembly. Such a strategy to generate RPE sheets is a promising avenue for developing RPE cell therapy, disease models and drug screening tools.

Metal Hydride Nanoparticles with Ultrahigh Structural Stability and Hydrogen Storage Activity Derived from Microencapsulated Nanoconfinement.

Metal hydrides (MHs) have recently been designed for hydrogen sensors, switchable mirrors, rechargeable batteries, and other energy-storage and conversion-related applications. The demands of MHs, particular fast hydrogen absorption/desorption kinetics, have brought their sizes to nanoscale. However, the nanostructured MHs generally suffer from surface passivation and low aggregation-resisting structural stability upon absorption/desorption. This study reports a novel strategy named microencapsulated nanoconfinement to realize local synthesis of nano-MHs, which possess ultrahigh structural stability and superior desorption kinetics. Monodispersed Mg2 NiH4 single crystal nanoparticles (NPs) are in situ encapsulated on the surface of graphene sheets (GS) through facile gas-solid reactions. This well-defined MgO coating layer with a thickness of ≈3 nm efficiently separates the NPs from each other to prevent aggregation during hydrogen absorption/desorption cycles, leading to excellent thermal and mechanical stability. More interestingly, the MgO layer shows superior gas-selective permeability to prevent further oxidation of Mg2 NiH4 meanwhile accessible for hydrogen absorption/desorption. As a result, an extremely low activation energy (31.2 kJ mol-1 ) for the dehydrogenation reaction is achieved. This study provides alternative insights into designing nanosized MHs with both excellent hydrogen storage activity and thermal/mechanical stability exempting surface modification by agents.

Increased expression of p21WAF1/CIP1 in kidney proximal tubules mediates fibrosis.

Tissue fibrosis is a major cause of death in developed countries. It commonly occurs after either acute or chronic injury and affects diverse organs, including the heart, liver, lung, and kidney. Using the renal ablation model of chronic kidney disease, we previously found that the development of progressive renal fibrosis was dependent on p21(WAF1/Cip1) expression; the genetic knockout of the p21 gene greatly alleviated this disease. In the present study, we expanded on this observation and report that fibrosis induced by two different acute injuries to the kidney is also dependent on p21. In addition, when p21 expression was restricted only to the proximal tubule, fibrosis after injury was induced in the whole organ. One molecular fibrogenic switch we describe is transforming growth factor-ß induction, which occurred in vivo and in cultured kidney cells exposed to adenovirus expressing p21. Our data suggests that fibrosis is p21 dependent and that preventing p21 induction after stress could be a novel therapeutic target.

Proximal tubule PPARα attenuates renal fibrosis and inflammation caused by unilateral ureteral obstruction.

We examined the effects of increased expression of proximal tubule peroxisome proliferator-activated receptor (PPAR)α in a mouse model of renal fibrosis. After 5 days of unilateral ureteral obstruction (UUO), PPARα expression was significantly reduced in kidney tissue of wild-type mice but this downregulation was attenuated in proximal tubules of PPARα transgenic (Tg) mice. When compared with wild-type mice subjected to UUO, PPARα Tg mice had reduced mRNA and protein expression of proximal tubule transforming growth factor (TGF)-ß1, with reduced production of extracellular matrix proteins including collagen 1, fibronectin, α-smooth muscle actin, and reduced tubulointerstitial fibrosis. UUO-mediated increased expression of microRNA 21 in kidney tissue was also reduced in PPARα Tg mice. Overexpression of PPARα in cultured proximal tubular cells by adenoviral transduction reduced aristolochic acid-mediated increased production of TGF-ß, demonstrating PPARα signaling reduces epithelial TGF-ß production. Flow cytometry studies of dissociated whole kidneys demonstrated reduced macrophage infiltration to kidney tissue in PPARα Tg mice after UUO. Increased expression of proinflammatory cytokines including IL-1ß, IL-6, and TNF-α in wild-type mice was also significantly reduced in kidney tissue of PPARα Tg mice. In contrast, the expression of anti-inflammatory cytokines IL-10 and arginase-1 was significantly increased in kidney tissue of PPARα Tg mice when compared with wild-type mice subjected to UUO. Our studies demonstrate several mechanisms by which preserved expression of proximal tubule PPARα reduces tubulointerstitial fibrosis and inflammation associated with obstructive uropathy.

Reduced kidney lipoprotein lipase and renal tubule triglyceride accumulation in cisplatin-mediated acute kidney injury.

Peroxisome proliferator-activated receptor-α (PPARα) activation attenuates cisplatin (CP)-mediated acute kidney injury by increasing fatty acid oxidation, but mechanisms leading to reduced renal triglyceride (TG) accumulation could also contribute. Here, we investigated the effects of PPARα and CP on expression and enzyme activity of kidney lipoprotein lipase (LPL) as well as on expression of angiopoietin protein-like 4 (Angptl4), glycosylphosphatidylinositol-anchored-HDL-binding protein (GPIHBP1), and lipase maturation factor 1 (Lmf1), which are recognized as important proteins that modulate LPL activity. CP caused a 40% reduction in epididymal white adipose tissue (WAT) mass, with a reduction of LPL expression and activity. CP also reduced kidney LPL expression and activity. Angptl4 mRNA levels were increased by ninefold in liver and kidney tissue and by twofold in adipose tissue of CP-treated mice. Western blots of two-dimensional gel electrophoresis identified increased expression of a neutral pI Angptl4 protein in kidney tissue of CP-treated mice. Immunolocalization studies showed reduced staining of LPL and increased staining of Angptl4 primarily in proximal tubules of CP-treated mice. CP also increased TG accumulation in kidney tissue, which was ameliorated by PPARα ligand. In summary, a PPARα ligand ameliorates CP-mediated nephrotoxicity by increasing LPL activity via increased expression of GPHBP1 and Lmf1 and by reducing expression of Angptl4 protein in the proximal tubule.

MicroRNA-21 promotes fibrosis of the kidney by silencing metabolic pathways.

Scarring of the kidney is a major public health concern, directly promoting loss of kidney function. To understand the role of microRNA (miRNA) in the progression of kidney scarring in response to injury, we investigated changes in miRNA expression in two kidney fibrosis models and identified 24 commonly up-regulated miRNAs. Among them, miR-21 was highly elevated in both animal models and in human transplanted kidneys with nephropathy. Deletion of miR-21 in mice resulted in no overt abnormality. However, miR-21(-/-) mice suffered far less interstitial fibrosis in response to kidney injury, a phenotype duplicated in wild-type mice treated with anti-miR-21 oligonucleotides. Global derepression of miR-21 target mRNAs was readily detectable in miR-21(-/-) kidneys after injury. Analysis of gene expression profiles up-regulated in the absence of miR-21 identified groups of genes involved in metabolic pathways, including the lipid metabolism pathway regulated by peroxisome proliferator-activated receptor-α (Pparα), a direct miR-21 target. Overexpression of Pparα prevented ureteral obstruction-induced injury and fibrosis. Pparα deficiency abrogated the antifibrotic effect of anti-miR-21 oligonucleotides. miR-21 also regulated the redox metabolic pathway. The mitochondrial inhibitor of reactive oxygen species generation Mpv17l was repressed by miR-21, correlating closely with enhanced oxidative kidney damage. These studies demonstrate that miR-21 contributes to fibrogenesis and epithelial injury in the kidney in two mouse models and is a candidate target for antifibrotic therapies.

Transgenic expression of proximal tubule peroxisome proliferator-activated receptor-alpha in mice confers protection during acute kidney injury.

Our previous studies suggest that peroxisome proliferator-activated receptor-alpha (PPARalpha) plays a critical role in regulating fatty acid beta-oxidation in kidney tissue and this directly correlated with preservation of kidney morphology and function during acute kidney injury. To further study this, we generated transgenic mice expressing PPARalpha in the proximal tubule under the control of the promoter of KAP2 (kidney androgen-regulated protein 2). Segment-specific upregulation of PPARalpha expression by testosterone treatment of female transgenic mice improved kidney function during cisplatin or ischemia-reperfusion-induced acute kidney injury. Ischemia-reperfusion injury or treatment with cisplatin in wild-type mice caused inhibition of fatty-acid oxidation, reduction of mitochondrial genes of oxidative phosphorylation, mitochondrial DNA, fatty-acid metabolism, and the tricarboxylic acid cycle. Similar injury in testosterone-treated transgenic mice resulted in amelioration of these effects. Similarly, there were increases in the levels of 4-hydroxy-2-hexenal-derived lipid peroxidation products in wild-type mice, which were also reduced in the transgenic mice. Similarly, necrosis of the S3 segment was reduced in the two injury models in transgenic mice compared to wild type. Our results suggest proximal tubule PPARalpha activity serves as a metabolic sensor. Its increased expression without the use of an exogenous PPARalpha ligand in the transgenic mice is sufficient to protect kidney function and morphology, and to prevent abnormalities in lipid metabolism associated with acute kidney injury.

Leydig cells express the myelin proteolipid protein gene and incorporate a new alternatively spliced exon.

Although the myelin proteolipid protein gene (Plp1) is highly expressed in the central nervous system encoding the most abundant myelin protein in oligodendrocytes, it is also expressed in other tissues, including testis. Transgenic studies with mice that harbor Plp1-lacZ fusion genes suggest that Leydig cells are the source of Plp1 gene expression in testis. However, virtually nothing is known about Plp1 gene regulation in Leydig cells, which is the focus of this study. The first intron contains both positive and negative regulatory elements that are important in regulating Plp1 gene expression in oligodendrocytes. To test whether these elements are functional in Leydig cells, a battery of Plp1-lacZ fusion genes with partial deletion of Plp1 intron 1 sequence was transfected into the mouse Leydig cell line, TM3. Results presented here suggest that an enhancer, which is very potent in oligodendrocytes, is only nominally active in TM3 cells. The intron also contains several negative regulatory elements that are operative in TM3 cells. Moreover a new exon (exon 1.2) was identified within the first 'intron' resulting in novel splice variants in TM3 cells. Western blot analysis suggests that these splice variants, along with those containing another alternatively spliced exon (exon 1.1) derived from intron 1 sequence, give rise to multiple Plp1 gene products in the mouse testis.

Anti-inflammatory effect of fibrate protects from cisplatin-induced ARF.

Recently, we demonstrated that peroxisome proliferator-activated receptor-alpha (PPARalpha) ligand ameliorates cisplatin-induced acute renal failure (ARF) by preventing inhibition of substrate oxidation, and also by preventing apoptosis and necrosis of the proximal tubule (Li S, Bhatt R, Megyesi J, Gokden N, Shah SV, and Portilla D. Am J Physiol Renal Physiol 287: F990-F998, 2004). In the following studies, we examined the protective effect of PPARalpha ligand on cisplatin-induced inflammatory responses during ARF. Mice subjected to a single intraperitoneal injection of cisplatin developed ARF at day 3. Cisplatin increased mRNA and protein expression of TNF-alpha, RANTES, and also upregulated endothelial adhesion molecules ICAM-1/VCAM-1 and chemokine receptors CCR1/CCR5. Cisplatin also led to neutrophil infiltration in the corticomedullary region. Pretreatment of wild-type mice with WY-14,643, a fibrate class of PPARalpha ligands, before cisplatin significantly suppressed cisplatin-induced upregulation of cytokine/chemokine expression, prevented neutrophil accumulation, and ameliorated renal dysfunction. In contrast, treatment with PPARalpha ligand before cisplatin did not prevent cytokine/chemokine production, neutrophil accumulation, and did not protect kidney function in PPARalpha null mice. In addition, we observed that cisplatin-induced NF-kappaB binding activity in nuclear extracts from wild-type mice was markedly reduced by treatment with PPARalpha ligand. These results demonstrate that PPARalpha exerts an anti-inflammatory effect in kidney tissue by a mechanism that includes inhibition of NF-kappaB DNA binding activity, and this effect results in inhibition of neutrophil infiltration, cytokine/chemokine release, and amelioration of cisplatin-induced ARF.

PPAR-alpha ligand ameliorates acute renal failure by reducing cisplatin-induced increased expression of renal endonuclease G.

Cisplatin injury to the kidney is characterized, in part, by inhibition of substrate oxidation, inflammation, and tubular cell death in the form of apoptosis and necrosis. Recently, we demonstrated that cisplatin-induced inhibition of substrate oxidation can be reversed by the administration of peroxisome proliferator-activated receptor-alpha (PPAR-alpha) ligands, resulting in amelioration of renal function. We therefore hypothesize that by improving fatty acid oxidation in vivo might protect renal function by reducing both apoptosis and necrosis in cisplatin-treated mice. Mice subjected to a single intraperitoneal injection of cisplatin developed acute renal failure (ARF) at days 3 and 4. At day 4 after cisplatin injection mRNA, protein levels and enzyme activity of proapoptotic renal endonuclease G (Endo G) were increased compared with saline-treated mice. In situ hybridization and immunohistochemical studies localized the increased expression of Endo G mRNA to the cytosolic compartment and Endo G protein to the nuclear compartment of proximal tubules in cisplatin-treated mice. Pretreatment of PPAR-alpha wild-type mice with PPAR-alpha ligand WY-14643 reduced significantly cisplatin-induced increased protein expression and enzyme activity of Endo G and prevented the nuclear translocation of mitochondrial Endo G. Morphological examination of tubular injury in the PPAR-alpha wild-type mice that received PPAR-alpha ligand and cisplatin did show significant amelioration of acute tubular necrosis, as well as a significant reduction in the number of apoptotic cells in the proximal tubule when compared with the cisplatin-treated group. In contrast, in PPAR-alpha-null mice treated with the ligand and cisplatin, Endo G protein expression was not reduced and this was accompanied by lack of protection of kidney function. We conclude that PPAR-alpha ligand protects against cisplatin-induced renal injury via a PPAR-alpha-dependent mechanism by reducing the expression and enzyme activity of proximal tubule Endo G, which results in amelioration of both proximal tubule cell apoptosis and necrosis.

PPAR alpha ligand protects during cisplatin-induced acute renal failure by preventing inhibition of renal FAO and PDC activity.

Previous studies demonstrated that during cisplatin-induced acute renal failure, there is a significant reduction in proximal tubule fatty acid oxidation. We now report on the effects of peroxisome proliferator-activated receptor-alpha (PPAR alpha) ligand Wy-14643 (WY) on the abnormalities of medium chain fatty acid oxidation and pyruvate dehydrogenase complex (PDC) activity in kidney tissue of cisplatin-treated mice. Cisplatin causes a significant reduction in mRNA levels and enzyme activity of mitochondrial medium chain acyl-CoA dehydrogenase (MCAD). PPAR alpha ligand WY ameliorated cisplatin-induced acute renal failure and prevented cisplatin-induced reduction of mRNA levels and enzyme activity of MCAD. In contrast, in cisplatin-treated PPAR alpha null mice, WY did not protect kidney function and did not reverse cisplatin-induced decreased expression of MCAD. Cisplatin inhibited renal PDC activity before the development of acute tubular necrosis, and PDC inhibition was reversed by pretreatment with PPAR alpha agonist WY. Cisplatin also induced increased mRNA and protein levels of pyruvate dehydrogenase kinase-4 (PDK4), and PPAR alpha ligand WY prevented cisplatin-induced increased expression of PDK4 protein levels in wild-type mice. We conclude that PPAR alpha agonists have therapeutic potential for cisplatin-induced acute renal failure. Use of PPAR alpha ligands prevents acute tubular necrosis by ameliorating cisplatin-induced inhibition of two distinct metabolic processes, MCAD-mediated fatty acid oxidation and PDC activity.