Previous studies have shown that high blood glucose-induced chronic microinflammation can cause inflammatory podocyte injury in patients with diabetic kidney disease(DKD). Therein, necroptosis is a new form of podocyte death that is closely associated with renal fibrosis(RF). To explore the effects and mechanisms in vivo of total flavones of Abelmoschus manihot(TFA), an extract from traditional Chinese herbal medicine Abelmoschus manihot for treating kidney diseases, on podocyte necroptosis and RF in DKD, and to further reveal its scientific connotation with multi-pathway and multi-target, the authors randomly divided all rats into four groups: a namely normal group, a model group, a TFA group and a rapamycin(RAP) group. After the modified DKD rat models were successfully established, four group rats were given double-distilled water, TFA suspension and RAP suspension, respectively by gavage every day. At the end of the 4th week of drug treatment, all rats were sacrificed, and the samples of their urine, blood and kidneys were collected. And then, the various indicators related to podocyte necroptosis and RF in the DKD model rats were observed, detected and analyzed, respectively. The results indicated that, general condition, body weight(BW), serum creatinine(Scr), urinary albumin(UAlb), and kidney hypertrophy index(KHI) in these modified DKD model rats were both improved by TFA and RAP. Indicators of RF, including glomerular histomorphological characteristics, fibronectin(FN) and collagen type Ⅰ(collagen Ⅰ) staining extent in glomeruli, as well as the protein expression levels of FN, collagen Ⅰ, transforming growth factor-β1(TGF-β1) and Smad2/3 in the kidneys were improved respectively by TFA and RAP. Podocyte damage, including foot process form and the protein expression levels of podocin and CD2AP in the kidneys was improved by TFA and RAP. In addition, tumor necrosis factor-α(TNF-α)-mediated podocyte necroptosis in the kidneys, including the morphological characteristics of podocyte necroptosis, the extent and levels of the protein expression of TNF-α and phosphorylated mixed lineage kinase domain like pseudokinase(p-MLKL) was improved respectively by TFA and RAP. Among them, RAP had the better effect on p-MLKL. More importantly, the activation of the receptor interacting serine/threonine protein kinase 1(RIPK1)/RIPK3/MLKL signaling axis in the kidneys, including the expression levels of its key signaling molecules, such as phosphorylated receptor interacting serine/threonine protein kinase 1(p-RIPK1), p-RIPK3, p-MLKL and cysteinyl aspartate specific proteinase-8(caspase-8) was improved respectively by TFA and RAP. Among them, the effect of TFA on p-RIPK1 was superior. On the whole, in this study, the authors demonstrated that TFA alleviates podocyte necroptosis and RF in DKD through inhibiting the activation of the TNF-α-mediated RIPK1/RIPK3/MLKL signaling axis in diabetic kidneys. The authors' findings provide new pharmacological evidence to reveal the scientific connotation of TFA in treating RF in DKD in more depth.
Humans ; Rats ; Animals ; Diabetic Nephropathies/drug therapy* ; Abelmoschus ; Flavones/pharmacology* ; Podocytes ; Tumor Necrosis Factor-alpha/metabolism* ; Necroptosis ; Receptor-Interacting Protein Serine-Threonine Kinases/metabolism* ; Fibrosis ; Threonine/pharmacology* ; Collagen/metabolism* ; Serine/pharmacology* ; Diabetes Mellitus/drug therapy*

OBJECTIVES: To explore the mechanism of transforming growth factor-β1 (TGF-β1) induce renal fibrosis. METHODS: Renal fibroblast NRK-49F cells treated with and without TGF-β1 were subjected to RNA-seq analysis. DESeq2 was used for analysis. Differentially expressed genes were screened with the criteria of false discovery rate<0.05 and l o g 2 F C >1. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed for differentially expressed genes. Genes encoding transcription factors were further screened for differential expression genes. Then, the expression of these genes during renal fibrosis was verified using unilateral ureteral obstruction (UUO)-induced mouse renal fibrosis model and a public gene expression dataset (GSE104954). RESULTS: After TGF-β1 treatment for 6, 12 and 24 h, 552, 1209 and 1028 differentially expressed genes were identified, respectively. GO analysis indicated that these genes were significantly enriched in development, cell death, and cell migration. KEGG pathway analysis showed that in the early stage of TGF-β1 induction (TGF-β1 treatment for 6 h), the changes in Hippo, TGF-β and Wnt signaling pathways were observed, while in the late stage of TGF-β1 induction (TGF-β1 treatment for 24 h), the changes of extracellular matrix-receptor interaction, focal adhesion and adherens junction were mainly enriched. Among the 291 up-regulated differentially expressed genes treated with TGF-β1 for 6 h, 13 genes (Snai1, Irf8, Bhlhe40, Junb, Arid5a, Vdr, Lef1, Ahr, Foxo1, Myc, Tcf7, Foxc2, Glis1) encoded transcription factors. Validation in a cell model showed that TGF-β1 induced expression of 9 transcription factors (encoded by Snai1, Irf8, Bhlhe40, Junb, Arid5a, Vdr, Lef1, Myc, Tcf7), while the expression levels of the other 4 genes did not significantly change after TGF-β1 treatment. Validation results in UUO-induced mouse renal fibrosis model showed that Snai1, Irf8, Bhlhe40, Junb, Arid5a, Myc and Tcf7 were up-regulated after UUO, Vdr was down-regulated and there was no significant change in Lef1. Validation based on the GSE104954 dataset showed that IRF8 was significantly overexpressed in the renal tubulointerstitium of patients with diabetic nephropathy or IgA nephropathy, MYC was highly expressed in diabetic nephropathy, and the expressions of the other 7 genes were not significantly different compared with the control group. CONCLUSIONS TGF-β1 induces differentially expressed genes in renal fibroblasts, among which Irf8 and Myc were identified as potential targets of chronic kidney disease and renal fibrosis.
Mice ; Animals ; Humans ; Transforming Growth Factor beta1/metabolism* ; Diabetic Nephropathies/pathology* ; Transcriptome ; Signal Transduction ; Kidney ; Ureteral Obstruction/pathology* ; Fibrosis ; Interferon Regulatory Factors ; Transforming Growth Factor beta/metabolism* ; DNA-Binding Proteins/metabolism* ; Transcription Factors/metabolism*

Lignans derived from Eucommia ulmoides Oliver (Eucommia lignans) inhibit the progression of inflammatory diseases, while their effect on the progression of diabetic nephropathy (DN) remained unclear. This work was designed to assess the function of Eucommia lignans in DN. The major constituents of Eucommia lignans were analyzed by UPLC-Q-TOF-MS/MS. The binding between Eucommia lignans and aldose reductase (AR) was predicted by molecular docking. Eucommia lignans (200, 100, and 50 mg·kg^-1) were used in model animals to evaluate their renal function changes. Rat glomerular mesangial cells (HBZY-1) were transfected with sh-AR, sh-AMPK, and oe-AR in the presence of high glucose (HG) or HG combined with Eucommia lignans to evaluate whether Eucommia lignans affected HG-induced cell injury and mitochondrial dysfunction through the AR/Nrf2/HO-1/AMPK axis. Eucommia lignans significantly attenuated the progression of DN in vivo. Eucommia lignans notably reversed HG-induced upregulation of inflammatory cytokines and mitochondrial injury, while downregulating the levels of Cyto c, caspase 9, AR, and NOX4 in HBZY-1 cells. In contrast, HG-induced downregulation of Nrf2, HO-1 and p-AMPKα levels were abolished by Eucommia lignans. Meanwhile, knockdown of AR exerted similar therapeutic effect of Eucommia lignans on DN progression, and AR overexpression reversed the effect of Eucommia lignans. Eucommia lignans alleviated renal injury through the AR/Nrf2/HO-1/AMPK axis. Thus, these findings might provide evidence for the use of Eucommia lignans in treating DN.
Animals ; Rats ; AMP-Activated Protein Kinases/genetics* ; Diabetes Mellitus ; Diabetic Nephropathies/prevention & control* ; Eucommiaceae/metabolism* ; Lignans/therapeutic use* ; Molecular Docking Simulation ; NF-E2-Related Factor 2/metabolism* ; Tandem Mass Spectrometry

Diabetic kidney disease is an important microvascular complication of diabetes and the leading cause of end-stage renal disease. Its pathological characteristics mainly include epithelial mesenchymal transition(EMT) in glomerulus, podocyte apoptosis and autophagy, and damage of glomerular filtration barrier. Transforming growth factor-β(TGF-β)/Smad signaling pathway is specifically regulated by a variety of mechanisms, and is a classic pathway involved in physiological activities such as apoptosis, proliferation and differentiation. At present, many studies have found that TGF-β/Smad signaling pathway plays a key role in the pathogenesis of diabetic kidney disease. Traditional Chinese medicine has significant advantages in the treatment of diabetic kidney disease for its multi-component, multi-target and multi-pathway characteristics, and some traditional Chinese medicine extracts, traditional Chinese medicines and traditional Chinese medicine compound prescription improve the renal injury of diabetic kidney disease by regulating TGF-β/Smad signaling pathway. This study clarified the mechanism of TGF-β/Smad signaling pathway in diabetic kidney disease by expounding the relationship between the key targets of the pathway and diabetic kidney disease, and summarized the research progress of traditional Chinese medicine in the treatment of diabetic kidney disease by interfering with TGF-β/Smad signaling pathway in recent years, to provide reference for drug research and clinical treatment of diabetic kidney disease in the future.
Humans ; Diabetic Nephropathies/genetics* ; Medicine, Chinese Traditional ; Kidney/pathology* ; Transforming Growth Factor beta/metabolism* ; Signal Transduction ; Epithelial-Mesenchymal Transition ; Smad Proteins/metabolism* ; Transforming Growth Factor beta1/metabolism* ; Diabetes Mellitus/genetics*

This study investigated the effect of multi-glycosides of Tripterygium wilfordii(GTW) on renal injury in diabetic kidney disease(DKD) rats through Nod-like receptor protein 3(NLRP3)/cysteine-aspartic acid protease-1(caspase-1)/gsdermin D(GSDMD) pyroptosis pathway and the mechanism. To be specific, a total of 40 male SD rats were randomized into the normal group(n=8) and modeling group(n=34). In the modeling group, a high-sugar and high-fat diet and one-time intraperitoneal injection of streptozotocin(STZ) were used to induce DKD in rats. After successful modeling, they were randomly classified into model group, valsartan(Diovan) group, and GTW group. Normal group and model group were given normal saline, and the valsartan group and GTW group received(ig) valsartan and GTW, respectively, for 6 weeks. Blood urea nitrogen(BUN), serum creatinine(Scr), alanine ami-notransferase(ALT), albumin(ALB), and 24 hours urinary total protein(24 h-UTP) were determined by biochemical tests. The pathological changes of renal tissue were observed based on hematoxylin and eosin(HE) staining. Serum levels of interleukin-1β(IL-1β) and interleukin-18(IL-18) were detected by enzyme-linked immunosorbent assay(ELISA). Western blot was used to detect the expression of pyroptosis pathway-related proteins in renal tissue, and RT-PCR to determine the expression of pyroptosis pathway-related genes in renal tissue. Compared with the normal group, the model group showed high levels of BUN, Scr, ALT, and 24 h-UTP and serum levels of IL-1β and IL-18(P<0.01), low level of ALB(P<0.01), severe pathological damage to kidney, and high protein and mRNA levels of NLRP3, caspase-1, and GSDMD in renal tissue(P<0.01). Compared with the model group, valsartan group and GTW group had low levels of BUN, Scr, ALT, and 24 h-UTP and serum levels of IL-1β and IL-18(P<0.01), high level of ALB(P<0.01), alleviation of the pathological damage to the kidney, and low protein and mRNA levels of NLRP3, caspase-1, and GSDMD in renal tissue(P<0.01 or P<0.05). GTW may inhibit pyroptosis by decreasing the expression of NLRP3/caspase-1/GSDMD in renal tissue, thereby relieving the inflammatory response of DKD rats and the pathological injury of kidney.
Rats ; Male ; Animals ; Diabetic Nephropathies/genetics* ; Interleukin-18/metabolism* ; Glycosides/pharmacology* ; Tripterygium ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Rats, Sprague-Dawley ; Caspase 1/metabolism* ; Pyroptosis ; Uridine Triphosphate/pharmacology* ; Kidney ; Valsartan/pharmacology* ; RNA, Messenger/metabolism* ; Diabetes Mellitus

This study aimed to explore the effects and mechanisms of total flavones of Abelmoschus manihot(TFA), the extracts from traditional Chinese medicine indicated for kidney diseases, on insulin resistance(IR) and podocyte epithelial-mesenchymal transition(EMT) in diabetic kidney disease(DKD), and further to reveal the scientific connotation. Thirty-two rats were randomly divided into a normal group, a model group, a TFA group, and a rosiglitazone(ROS) group. The modified DKD model was induced in rats by methods including high-fat diet feeding, unilateral nephrectomy, and streptozotocin(STZ) intraperitoneal injection. After modeling, the rats in the four groups were given double-distilled water, TFA suspension, and ROS suspension correspondingly by gavage every day. At the end of the 8th week of drug administration, all rats were sacrificed, and the samples of urine, blood, and kidney tissues were collected. The parameters and indicators related to IR and podocyte EMT in the DKD model rats were examined and observed, including the general condition, body weight(BW) and kidney weight(KW), the biochemical parameters and IR indicators, the protein expression levels of the key signaling molecules and structural molecules of slit diaphragm in the renal insulin receptor substrate(IRS) 1/phosphatidylinositol 3-kinase(PI3K)/serine-threonine kinase(Akt) pathway, foot process form and glomerular basement membrane(GBM) thickness, the expression of the marked molecules and structural molecules of slit diaphragm in podocyte EMT, and glomerular histomorphological characteristics. The results showed that for the DKD model rats, both TFA and ROS could improve the general condition, some biochemical parameters, renal appearance, and KW. The ameliorative effects of TFA and ROS were equivalent on BW, urinary albumin(UAlb)/urinary creatinine(UCr), serum creatinine(Scr), triglyceride(TG), and KW. Secondly, they could both improve IR indicators, and ROS was superior to TFA in improving fast insulin(FIN) and homeostasis model assessment of insulin resistance(HOMA-IR). Thirdly, they could both improve the protein expression levels of the key signaling molecules in the IRS1/PI3K/Akt pathway and glomerulosclerosis in varying degrees, and their ameliorative effects were similar. Finally, both could improve podocyte injury and EMT, and TFA was superior to ROS. In conclusion, this study suggested that podocyte EMT and glomerulosclerosis could be induced by IR and the decreased activation of the IRS1/PI3K/Akt pathway in the kidney in DKD. Similar to ROS, the effects of TFA in inhibiting podocyte EMT in DKD were related to inducing the activation of the IRS1/PI3K/Akt pathway and improving IR, which could be one of the scientific connotations of TFA against DKD. This study provides preliminary pharmacological evidence for the development and application of TFA in the field of diabetic complications.
Rats ; Animals ; Diabetic Nephropathies/drug therapy* ; Proto-Oncogene Proteins c-akt/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; Abelmoschus/chemistry* ; Podocytes ; Rats, Sprague-Dawley ; Epithelial-Mesenchymal Transition ; Flavones/pharmacology* ; Insulin Resistance ; Reactive Oxygen Species ; Diabetes Mellitus

Renal tubular injury in patients with diabetic kidney disease(DKD) may be accompanied by glomerular and microvascular diseases. It plays a critical role in the progression of renal damage in DKD, and is now known as diabetic tubulopathy(DT). To explore the multi-targeted therapeutic effects and pharmacological mechanisms in vivo of total flavones of Abelmoschus manihot(TFA), an extract from traditional Chinese medicine for treating kidney disease, in attenuating DT, the authors randomly divided all rats into four groups: a normal control group(normal group), a DT model group(model group), a DT model+TFA-treated group(TFA group) and a DT model+rosiglitazone(ROS)-treated group(ROS group). The DT rat model was established based on the DKD rat model by means of integrated measures. After successful modeling, the rats in the four groups were continuously given double-distilled water, TFA suspension, and ROS suspension, respectively by gavage every day. After 6 weeks of treatment, all rats were sacrificed, and the samples of their urine, blood, and kidneys were collected. The effects of TFA and ROS on various indicators related to urine and blood biochemistry, renal tubular injury, renal tubular epithelial cell apoptosis and endoplasmic reticulum stress(ERS), as well as the activation of the protein kinase R-like endoplasmic reticulum kinase(PERK)-eukaryotic translation initiation factor 2α(eIF2α)-activating transcription factor 4(ATF4)-C/EBP homologous protein(CHOP) signaling pathway in the kidney of the DT model rats were investigated. The results indicated that hypertrophy of renal tubular epithelial cells, renal tubular hyperplasia and occlusion, as well as interstitial extracellular matrix and collagen deposition occurred in the DT model rats. Moreover, significant changes were found in the expression degree and the protein expression level of renal tubular injury markers. In addition, there was an abnormal increase in tubular urine proteins. After TFA or ROS treatment, urine protein, the characteristics of renal tubular injury, renal tubular epithelial cell apoptosis and ERS, as well as the activation of the PERK-eIF2α-ATF4-CHOP signaling pathway in the kidney of the DT model rats were improved to varying degrees. Therein, TFA was superior to ROS in affecting the pathological changes in renal tubule/interstitium. In short, with the DT model rats, this study demonstrated that TFA could attenuate DT by multiple targets through inhibiting renal tubular ERS-induced cell apoptosis in vivo, and its effect and mechanism were related to suppressing the activation of the PERK-eIF2α-ATF4-CHOP signaling pathway in the kidney. These findings provided preliminary pharmacological evidence for the application of TFA in the clinical treatment of DT.
Rats ; Animals ; Abelmoschus ; Reactive Oxygen Species/metabolism* ; Flavones/pharmacology* ; Endoplasmic Reticulum Stress ; Diabetic Nephropathies/drug therapy* ; Apoptosis ; Diabetes Mellitus

Objective To investigate the effect of 1, 25-(OH)₂-VitD3 (VitD3) on renal tubuleinterstitial fibrosis in diabetic kidney disease. Methods NRK-52E renal tubular epithelial cells were divided into control group (5.5 mmol/L glucose medium treatment), high glucose group (25 mmol/L glucose medium treatment) and high glucose with added VitD3 group (25 mmol/L glucose medium combined with 10^-8 mmol/L VitD3). The mRNA and protein expression of Snail1, SMAD3, SMAD4, α-SMA and E-cadherin in NRK-52E cells were detected by real-time quantitative PCR and Western blot analysis respectively. The expression and localization of Snail1, SMAD3 and SMAD4 were detected by immunofluorescence cytochemical staining. The binding of Snail1 with SMAD3/SMAD4 complex to the promoter of Coxsackie-adenovirus receptor (CAR) was detected by chromatin immunoprecipitation. The interaction among Snail1, SMAD3/SMAD4 and E-cadherin were detected by luciferase assay. Small interfering RNA (siRNA) was used to inhibit the expression of Snail1 and SMAD4, and the expression of mRNA of E-cadherin was detected by real-time quantitative PCR. SD rats were randomly divided into control group, DKD group and VitD3-treated group. DKD model was established by injection of streptozotocin (STZ) in DKD group and VitD3-treated group. After DKD modeling, VitD3-treated group was given VitD3 (60 ng/kg) intragastric administration. Control group and DKD group were given normal saline intragastric administration. In the DKD group and VitD3-treated group, insulin (1-2 U/kg) was injected subcutaneously to control blood glucose for 8 weeks. The mRNA and protein levels of Snail1, SMAD3, SMAD4, α-SMA and E-cadherin in renal tissues were detected by real-time quantitative PCR and Western blot analysis respectively. Immunohistochemistry was used to detect the expression and localization of Snail1, SMAD3, SMAD4, α-SMA and E-cadherin in renal tissue. Results Compared with the control group, the mRNA and protein expressions of Snail1, SMAD3, SMAD4 and α-SMA in NRK-52E cells cultured with high glucose and in DKD renal tissues were up-regulated, while E-cadherin expression was down-regulated. After the intervention of VitD3, the expression levels of Snail1, SMAD3, SMAD4, α-SMA and E-cadherin in the DKD model improved to be close to those in the control group. Chromatin immunoprecipitation showed that Snail1 and SMAD3/SMAD4 bound to CAR promoter IV, while VitD3 prevented Snail1 and SMAD3/SMAD4 from binding to CAR promoter IV. Luciferase assay confirmed the interaction among Snail1, SMAD3/SMAD4 and E-cadherin. After the mRNA of Snail1 and SMAD4 was inhibited by siRNA, the expression of E-cadherin induced by high glucose was up-regulated. Conclusion VitD3 could inhibit the formation of Snail1-SMAD3/SMAD4 complex and alleviate the renal tubulointerstitial fibrosis in DKD.
Animals ; Rats ; Cadherins/genetics* ; Diabetes Mellitus/pathology* ; Diabetic Nephropathies/pathology* ; Epithelial-Mesenchymal Transition ; Fibrosis/pathology* ; Glucose/pharmacology* ; Kidney/pathology* ; Rats, Sprague-Dawley ; RNA, Messenger ; RNA, Small Interfering ; Transforming Growth Factor beta1/metabolism* ; Vitamin D/pharmacology*

Diabetic nephropathy is a microvascular complication of diabetes. Its etiology involves metabolic disorder-induced endothelial dysfunction. Endothelium-derived nitric oxide (NO) plays an important role in a number of physiological processes, including glomerular filtration and endothelial protection. NO dysregulation is an important pathogenic basis of diabetic nephropathy. Hyperglycemia and dyslipidemia can lead to oxidative stress, chronic inflammation and insulin resistance, thus affecting NO homeostasis regulated by endothelial nitric oxide synthase (eNOS) and a conglomerate of related proteins and factors. The reaction of NO and superoxide (O₂^.-) to form peroxynitrite (ONOO^-) is the most important pathological NO pathway in diabetic nephropathy. ONOO^- is a hyper-reactive oxidant and nitrating agent in vivo which can cause the uncoupling of eNOS. The uncoupled eNOS does not produce NO but produces superoxide. Thus, eNOS uncoupling is a critical contributor of NO dysregulation. Understanding the regulatory mechanism of NO and the effects of various pathological conditions on it could reveal the pathophysiology of diabetic nephropathy, potential drug targets and mechanisms of action. We believe that increasing the stability and activity of eNOS dimers, promoting NO synthesis and increasing NO/ONOO^- ratio could guide the development of drugs to treat diabetic nephropathy. We will illustrate these actions with some clinically used drugs as examples in the present review.
Diabetes Mellitus ; Diabetic Nephropathies/drug therapy* ; Endothelium, Vascular ; Humans ; Nitric Oxide/metabolism* ; Nitric Oxide Synthase Type III/therapeutic use* ; Oxidative Stress ; Peroxynitrous Acid/therapeutic use*

Diabetic nephropathy (DN) is one of the most common complications of diabetes mellitus, which is characterized in renal tubulointerstitial fibrosis (TIF). The current study was designed to investigate the protective effect of Jujuboside A (Ju A) on TIF in type 2 diabetes (T2DM) mice, and explore its underlying anti-fibrosis mechanism. A mouse T2DM model was established using high fat diet (HFD) feeding combined with intraperitoneal injection of streptozotocin (STZ). Then, diabetic mice were treated with Ju A (10, 20 and 40 mg·kg^-1·d^-1, i.g.) for 12 weeks. Results showed that administration of Ju A not only down-regulated fasting blood glucose (FBG) levels, but also improved hyperlipidemia and renal function in diabetic mice. Moreover, the reduced ECM accumulation was observed in the renal cortex of Ju A treated diabetic mice, while the TIF progression was also attenuated by Ju A through blocking the epithelial-to-mesenchymal transition (EMT) of renal tubular epithelial cells (RTECs). Further mechanism studies showed that Ju A treatment effectively down-regulated the protein expression and subsequent nuclear translocation of Yin Yang 1 (YY1) in the renal cortex of diabetic mice, and reduced the levels of transforming growth factor-β1 (TGF-β1) in the serum and renal cortex of Ju A treated mice. According to invitro studies, the up-regulated YY1/TGF-β1 signaling pathway was restored by Ju A in high glucose (HG) cultured HK-2 cells. Taken together, these findings demonstrated that Ju A can ameliorate the TIF of DN through down-regulating the YY1/TGF-β1 signaling pathway.
Animals ; Blood Glucose ; Diabetes Mellitus, Experimental/metabolism* ; Diabetes Mellitus, Type 2/drug therapy* ; Diabetic Nephropathies/metabolism* ; Fibrosis ; Mice ; Saponins ; Signal Transduction ; Streptozocin ; Transforming Growth Factor beta1/metabolism*