Dynamic changes of urinary proteins in a rat model of acute hypercoagulable state induced by tranexamic acid

The hypercoagulable state leads to the development of thrombotic diseases, but it is difficult to diagnose due to the lack of available biomarkers. This study aimed to investigate systematic changes of the urinary proteome in the acute hypercoagulable state. A rat model of the acute hypercoagulable state was induced by an antifibrinolytic agent tranexamic acid and urine samples were collected for proteomic analysis by liquid chromatography-tandem mass spectrometry. A total of 28 differential proteins were detected in the urinary proteome of the model rats, of which 12 had been previously considered as candidate biomarkers such as myoglobin, and 10 had been considered stable in healthy human urine. Of the 28 differentially expressed proteins 18 had counterparts in humans. Of these 18 proteins, 10 were members of the human core urinary proteome distributed in a variety of human tissues but concentrated in the urinary and digestive systems. Fumarylacetoacetase was verified as a potential marker of the acute hypercoagulable state by Western blot analysis. In conclusion, urine proteome analysis is a powerful approach to identify potential biomarkers of acute hypercoagulable state.     

The renal expression of heat shock protein 47 and collagens in acute and chronic experimental diabetes in rats

Glomerulosclerosis and tubulointerstitial fibrosis are the main structural changes found in the later stages of diabetic nephropathy, which is clinically characterized by proteinuria, and progressive renal insufficiency. Heat shock protein (HSP) 47, a collagen-binding stress protein, has a specific role in the intracellular processing of procollagen molecules during collagen synthesis. It is implicated in the pathogenesis of various fibrotic diseases. However, the expression and significance of HSP47 in acute and chronic phases of diabetic nephropathy is not yet known. In this study, we studied the expression of HSP47 in the kidneys obtained from streptozotocin-induced diabetic rats, in both short- and long-term diabetes. To determine the renal expression of HSP47, and collagens (type III and IV) in acute (days 1, 3 and 14) and chronic (weeks 4, 12 and 24) diabetes, we have performed a time-course study using streptozotocin-induced diabetic rats. The expression pattern of alpha-smooth muscle actin (to identify mesangial cell damage), vimentin (to identify tubular epithelial cell damage), and desmin (to identify glomerular epithelial cell damage) was also determined in kidneys of these diabetic rats. Antibodies specific for HSP47, type III and type IV collagens, alpha-smooth muscle actin, vimentin, and desmin were used to assess the relative expression of their proteins in paraffin-embedded kidney sections by immunohistochemistry. Compared to control rat kidneys, no significant changes in the expression of HSP47 was found in the kidneys of acute diabetic rats. However a significant increase in the expression of HSP47 was noted in the kidneys of chronic diabetic rats; increased expression of HSP47 correlated with an increased renal deposition of types III and IV collagens. Similarly, compared to kidneys of control and acute diabetic rats, an increased expression of alpha-smooth muscle actin (in mesangial cells), vimentin (in tubular epithelial cells), and desmin (in glomerular epithelial cells) was detected in the kidneys of chronic diabetic rats; by dual immunostaining, these phenotypically-altered renal cells in kidneys of chronic diabetic rats were found to be HSP47-producing cells. Importantly, HSP47 up-regulation coincided with the initiation and progression of renal fibrosis, as determined by the expression and deposition of collagens. Our results strongly support a pathological role for HSP47 in the later stages (sclerotic phase) of streptozotocin-induced diabetic nephropathy, which is associated with glomerulosclerosis and tubulointerstitial fibrosis.     

Multicentric validation of proteomic biomarkers in urine specific for diabetic nephropathy

Background

Urine proteome analysis is rapidly emerging as a tool for diagnosis and prognosis in disease states. For diagnosis of diabetic nephropathy (DN), urinary proteome analysis was successfully applied in a pilot study. The validity of the previously established proteomic biomarkers with respect to the diagnostic and prognostic potential was assessed on a separate set of patients recruited at three different European centers. In this case-control study of 148 Caucasian patients with diabetes mellitus type 2 and duration ≥5 years, cases of DN were defined as albuminuria >300 mg/d and diabetic retinopathy (n = 66). Controls were matched for gender and diabetes duration (n = 82).

Methodology/Principal Findings

Proteome analysis was performed blinded using high-resolution capillary electrophoresis coupled with mass spectrometry (CE-MS). Data were evaluated employing the previously developed model for DN. Upon unblinding, the model for DN showed 93.8% sensitivity and 91.4% specificity, with an AUC of 0.948 (95% CI 0.898-0.978). Of 65 previously identified peptides, 60 were significantly different between cases and controls of this study. In <10% of cases and controls classification by proteome analysis not entirely resulted in the expected clinical outcome. Analysis of patient''s subsequent clinical course revealed later progression to DN in some of the false positive classified DN control patients.

Conclusions

These data provide the first independent confirmation that profiling of the urinary proteome by CE-MS can adequately identify subjects with DN, supporting the generalizability of this approach. The data further establish urinary collagen fragments as biomarkers for diabetes-induced renal damage that may serve as earlier and more specific biomarkers than the currently used urinary albumin.     

6-Keto-prostaglandin F1 alpha is not added to urine during passage through the rat urinary bladder in vivo

Studies were conducted to determine whether prostaglandins are added to the urine during its passage through the rat urinary bladder in vivo. Control rats and rats with chronic streptozotocin-induced diabetes were anesthetized with Inactin, 100 mg/kg i.p., and urine was collected simultaneously from both kidneys. Urine from the left kidney was collected directly from the renal pelvis via a ureteral cannula, while urine from the right kidney was collected via a cannula in the urinary bladder. Prostaglandins in the urine were measured by radioimmunoassay. No difference in urinary concentration or rate of excretion of 6-keto-PGF1 alpha or PGE2 was seen between ureteral urine and bladder urine from either normal or diabetic rats. The results of this study indicate that in vivo there is no intralumenal addition of either 6-keto-PGF1 alpha or PGE2 to the urine by the ureteral bladder of rats.     

Progression of early phase diabetic nephropathy in streptozotocin-induced diabetic rats: evaluation of various kidney-related parameters

Diabetic nephropathy (DN) is one of the serious secondary complications of diabetes, which results in end-stage renal failure. Reports on the progressive nature of early phase DN especially with respect to kidney parameters such as kidney weight, type IV collagen excretion, total kidney and urinary glycosaminoglycans (GAGs) are few. This work was undertaken to determine systematically the progression of early phase DN in relation to various kidney-related parameters for a period of four months. Experimentally-induced diabetic rats were grouped based on fasting blood glucose levels. Various basic and kidney-related parameters such as kidney weight, microalbuminuria, urinary excretion of GAGs and type IV collagen, total kidney GAGs, histopathology, glomerular area and glomerular volume were examined in control and diabetic rats. There was a progressive increase in fasting blood sugar, urine sugar, kidney weight, microalbuminuria, urine glycosaminoglycans, urine type IV collagen, glomerular area and glomerular volume but there was a progressive decrease in kidney glycosaminoglycans. Glomerular sclerotic condition was aggravated with the increase in duration of diabetes from 1 to 4 months. Onset of DN in rats begins subtly after one month of diabetes but gets vitiated and more pronounced at the end of four months.     

Gender dimorphism in regulation of plasma proteins in streptozotocin‐induced diabetic rats

In the present study, we examined differentially regulated plasma proteins between healthy control and streptozotocin (STZ)‐induced male and female diabetic rats by 2DE‐based proteomic analysis. Animal experiments revealed that significantly lower plasma insulin levels were observed in female diabetic rats, consequently resulting in higher blood glucose levels in female diabetic rats. Importantly, plasma levels of sex hormones were significantly altered in a gender‐dependent manner before and after STZ treatment. Results of the animal experiment indicated the existence of sexual dimorphism in the regulation of plasma proteins between healthy control and diabetic rats. Plasma proteome analysis enabled us to identify a total of 38 proteins showing sexual dimorphic regulation patterns. In addition, for the first time, we identified several differentially regulated plasma proteins between healthy control and diabetic rats, including apolipoprotein E, fetuin B, α‐1‐acid glycoprotein, β‐2‐glycoprotein 1, 3‐hydroxyanthranilate 3,4‐dioxygenase, and serum amyloid P‐component. To the best of our knowledge, this is the first proteomic approach to address sexual dimorphism in diabetic animals. These proteomic data on gender‐dimorphic regulation of plasma proteins provide valuable information that can be used for evidence‐based gender‐specific clinical treatment of diabetes.     

Proteomics analysis reveals diabetic kidney as a ketogenic organ in type 2 diabetes

Diabetic nephropathy (DN) is the leading cause of end-stage renal disease. To date, the molecular mechanisms of DN remain largely unclear. The present study aimed to identify and characterize novel proteins involved in the development of DN by a proteomic approach. Proteomic analysis revealed that 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase 2 (HMGCS2), the key enzyme in ketogenesis, was increased fourfold in the kidneys of type 2 diabetic db/db mice. Consistently, the activity of HMGCS2 in kidneys and 24-h urinary excretion of the ketone body β-hydroxybutyrate (β-HB) were significantly increased in db/db mice. Immunohistochemistry, immunofluorescence, and real-time PCR studies further demonstrated that HMGCS2 was highly expressed in renal glomeruli of db/db mice, with weak expression in the kidneys of control mice. Because filtered ketone bodies are mainly reabsorbed in the proximal tubules, we used RPTC cells, a rat proximal tubule cell line, to examine the effect of the increased level of ketone bodies. Treating cultured RPTC cells with 1 mM β-HB significantly induced transforming growth factor-β1 expression, with a marked increase in collagen I expression. β-HB treatment also resulted in a marked increase in vimentin protein expression and a significant reduction in E-cadherin protein levels, suggesting an enhanced epithelial-to-mesenchymal transition in RPTCs. Collectively, these findings demonstrate that diabetic kidneys exhibit excess ketogenic activity resulting from increased HMGCS2 expression. Enhanced ketone body production in the diabetic kidney may represent a novel mechanism involved in the pathogenesis of DN.     

The diabetic rat kidney mediates inosituria and selective urinary partitioning of D-chiro-inositol

Diabetic nephropathy is a serious complication of diabetes mellitus with a pressing need for effective metabolic markers to detect renal impairment. Of potential significance are the inositol compounds, myo-inositol (MI), and the less abundant stereoisomer, D-chiro-inositol (DCI), which are excreted at increased levels in the urine in diabetes mellitus, a phenomenon known as inosituria. There is also a selective urinary excretion of DCI compared to MI. As the biological origins of altered inositol metabolism in diabetes mellitus are unknown, the aim of this study was to determine whether the diabetic kidney was directly responsible. Kidneys isolated from four-week streptozotocin-induced diabetic rats were characterized by a 3-fold reduction in glomerular filtration rate (GFR) compared to matched non-diabetic kidneys. When perfused with fixed quantities of MI (50 µM) and DCI (5 µM) under normoglycemic conditions (5 mM glucose), GFR-normalized urinary excretion of MI was increased by 1.7-fold in diabetic vs. non-diabetic kidneys. By comparison, GFR-normalized urinary excretion of DCI was increased by 4-fold. Perfusion conditions replicating hyperglycemia (20 mM glucose) potentiated DCI but not MI urinary excretion in both non-diabetic and diabetic kidneys. Overall, there was a 2.4-fold increase in DCI urinary excretion compared to MI in diabetic kidneys that was independent of glucose ambience. This increased urinary excretion of DCI and MI in diabetic kidneys occurred despite increased renal expression of the inositol transporters, sodium myo-inositol transporter subtype 1 and 2 (SMIT1 and SMIT2). These findings show that the diabetic kidney primarily mediates inosituria and altered urinary partitioning of MI and DCI. Urinary inositol levels might therefore serve as an indicator of impaired renal function in diabetes mellitus with wider implications for monitoring chronic kidney disease.     

6-keto-prostaglandin F1α is not added to urine during passage through the rat urinary bladder

Studies were conducted to determine whether prostaglandins are added to the urine during its passage through the rat urinary blader . Control rats and rats with chronic streptozotocin-induced diabetes were anesthetized with Inactin, 100 mg/kg i.p., and urine was collected simultaneously from both kidneys. Urine from the left kidney was collected directly from the renal pelvis via a ureteral cannula, while urine from the right kidney was collected via a cannula in the urinary bladder. Prostaglandins in the urine were measured by radioimmunoassay. No difference in urinary concentration or rate of excretion of 6-keto-PGF_1α or PGE₂ was seen between ureteral urine and bladder urine from either normal or diabetic rats. The results of this study indicate that there is no intralumenal addition of either 6-keto-PGF_1α or PGE₂ to the urine by the ureteral bladder of rats.     

Two-dimensional fluorescence difference gel electrophoresis analysis of the urine proteome in human diabetic nephropathy

Urinary proteins may provide clues regarding pathogenesis of kidney disease as well as providing markers of disease activity. We employed two-dimensional differential in-gel electrophoretic analysis (2-D DIGE) to assess multiple urine samples in patients with diabetic nephropathy. Patient samples were collected as timed overnight collections. All the patients had longstanding diabetes, impaired renal function, and overt proteinuria. Control and patient urinary protein were analyzed by 2-D DIGE and DeCyder analysis. Ninety-nine spots were significantly regulated in the urine proteome of the diabetic samples, with 63 up- and 36 down-regulated. One spot corresponding to a pI 5-6 and a molecular weight between 45 and 66 kDa was consistently up-regulated by 19-fold across individuals in the diabetic group. Surface-enhanced laser desorption/ionization-time of flight analysis of in-gel tryptic digest of this spot identified this protein as alpha 1 antitrypsin (AAT). ELISA of urine samples from a separate group of patients and controls confirmed a marked increase of AAT in diabetic patients. Immunostaining of human diabetic kidneys revealed up-regulation of AAT in areas of renal fibrosis. In conclusion, we developed a method to analyze numerous urine samples from patients and allowed for detection and identification of regulated urine protein spots.     

Differential expression of kidney proteins in streptozotocin-induced diabetic rats in response to hypoglycemic fungal polysaccharides

Diabetic nephropathy remains a major cause of morbidity and mortality in the diabetic population and is the leading cause of end-stage renal failure. Despite current therapeutics including intensified glycemic control and blood pressure lowering agents, renal disease continues to progress relentlessly in diabetic patients, albeit at a lower rate. Since synthetic drugs for diabetes are known to have side effects, fungal mushrooms as a natural product come into preventing the development of diabetes. Our previous report showed the hypoglycemic effect of extracellular fungal polysaccharides (EPS) in streptozotocin (STZ)-induced diabetic rats. In this study, we analyzed the differential expression patterns of rat kidney proteins from normal, STZ-induced diabetic, and EPS-treated diabetic rats, to discover diabetes-associated proteins in rat kidney. The results of proteomic analysis revealed that up to 500 protein spots were visualized, of which 291 spots were differentially expressed in the three experimental groups. Eventually, 51 spots were statistically significant and were identified by peptide mass fingerprinting. Among the differentially expressed renal proteins, 10 were increased and 16 were decreased significantly in diabetic rat kidney. The levels of different proteins, altered after diabetes induction, were returned to approximately those of the healthy rats by EPS treatment. A histopathological examination showed that EPS administration restored the impaired kidney to almost normal architecture. The study of protein expression in the normal and diabetic kidney tissues enabled us to find several diabetic nephropathy-specific proteins, such as phospholipids scramblase 3 and tropomyosin 3, which have not been mentioned yet in connection with diabetes.     

Resveratrol ameliorates early diabetic nephropathy associated with suppression of augmented TGF-β/smad and ERK1/2 signaling in streptozotocin-induced diabetic rats

Diabetic nephropathy (DN) is the major cause of end-stage renal disease. The early changes in DN are characterized by an increased in kidney size, glomerular volume, and kidney function, followed by the accumulation of glomerular extracellular matrix, increased urinary albumin excretion (UAE), glomerular sclerosis, and tubular fibrosis. Resveratrol (RSV) has been shown to ameliorate hyperglycemia and hyperlipidemia in streptozotocin-induced diabetic rats. In the present study, we examined the beneficial effects of RSV on DN and explored the possible mechanism of RSV action.Male Sprague–Dawley rats were injected with streptozotocin at 65 mg/kg body weight. The induction of diabetes mellitus (DM) was confirmed by a fasting plasma glucose level ≥300 mg/dL and symptoms of polyphagia and polydipsia. The DM rats were treated with or without RSV at 0.75 mg/kg body weight 3 times a day for 8 weeks. Animals were sacrificed and kidney histology was examined by microscopy. Urinary albumin excretion, glomerular hypertrophy and expressions of fibronectin, collagen IV, and TGF-β in the glomeruli were alleviated in RSV-treated DM rats, but not in untreated DM rats. In addition, RSV treatment reduced the thickness of the glomerular basement membrane (GBM) to the original thickness and increased nephrin expressions to normal levels in DM rats. Moreover, RSV inhibited phosphorylation of smad2, smad3 and ERK1/2 in diabetic rat kidneys. This is the first report showing that RSV alleviates early glomerulosclerosis in DN through TGF-β/smad and ERK1/2 inhibition. In addition, podocyte injuries of diabetic kidneys are lessened by RSV.     

Detection of renal and urinary tract proteins in urine before and after space flight

The urine protein composition (proteome) of healthy humans was analyzed using proteomic techniques to obtain data under normal physiological conditions and after six-month space flights. It was shown that, after long space flights, specific minor proteins are revealed in cosmonauts’ urine that can be identified as proteins coming from kidneys and urinary tracts.     

大鼠急性低氧模型尿液蛋白质组的变化

本研究旨在探究急性低氧对大鼠尿液蛋白质组造成的影响。在该项研究中,大鼠被放置于模拟海拔5 000 m高原环境的低氧舱内24 h。在低氧后0、12、24 h收集尿液样本,并使用液相色谱-串联质谱技术（LC-MS/MS）对尿蛋白进行分析。与低氧0 h相比,低氧12 h组共鉴定到144个差异蛋白,低氧24 h组共鉴定到129个差异蛋白。功能分析显示,差异蛋白参与了一系列与低氧应激有关的生物学通路,如抗氧化应激、糖酵解、补体和凝血级联反应等。研究结果表明,尿液蛋白质组可以反映急性低氧刺激后的显著变化。这些发现可能提供了一种判断机体缺氧状态的方法,有助于辅助检测缺氧状态。     

Differential expression of microRNAs associated with neurodegenerative diseases and diabetic nephropathy in protein l-isoaspartyl methyltransferase-deficient mice

Protein l -isoaspartyl methyltransferase (PIMT/PCMT1), an enzyme repairing isoaspartate residues in peptides and proteins that result from the spontaneous decomposition of normal l -aspartyl and l -asparaginyl residues during aging, has been revealed to be involved in neurodegenerative diseases (NDDs) and diabetes. However, the molecular mechanisms for a putative association of PIMT dysfunction with these diseases have not been clarified. Our study aimed to identify differentially expressed microRNAs (miRNAs) in the brain and kidneys of PIMT-deficient mice and uncover the epigenetic mechanism of PIMT-involved NDDs and diabetic nephropathy (DN). Differentially expressed miRNAs by sequencing underwent target prediction and enrichment analysis in the brain and kidney of PIMT knockout (KO) mice and age-matched wild-type (WT) littermates. Sequence analysis revealed 40 differentially expressed miRNAs in the PIMT KO mouse brain including 25 upregulated miRNAs and 15 downregulated miRNAs. In the PIMT KO mouse kidney, there were 80 differentially expressed miRNAs including 40 upregulated miRNAs and 40 downregulated miRNAs. Enrichment analysis and a systematic literature review of differentially expressed miRNAs indicated the involvement of PIMT deficiency in the pathogenesis in NDDs and DN. Some overlapped differentially expressed miRNAs between the brain and kidney were quantitatively assessed in the brain, kidney, and serum-derived exosomes, respectively. Despite being preliminary, these results may aid in investigating the pathological hallmarks and identify the potential therapeutic targets and biomarkers for PIMT dysfunction-related NDDs and DN.     

Prevention of diabetic nephropathy in rats through enhanced renal antioxidative capacity by inhibition of the proteasome

AimsOxidative stress may play an important role in the pathogenesis of diabetic nephropathy (DN). Recent studies have shown that the ubiquitin–proteasome pathway (UPP) and oxidative stress have interaction. We aimed to investigate whether inhibiting the proteasome has a preventive effect on DN through suppression of renal oxidative stress.Main methodsMale Sprague–Dawley rats were randomly divided into three groups: a normal control (NC) group, a streptozotocin-induced DN model group, and a DN plus MG132 (10 μg/kg) treatment group.Key findingsIncreased 24-h urinary protein excretion rate (UPER) and renal pathological changes were all improved after MG132 administration. Furthermore, enhanced renal 26S proteasome activity and concentration in DN rats were effectively reduced after MG132 administration. Increased p47phox and nitrotyrosine (NT) expressions in kidneys of DN rats were decreased after MG132 treatment. Renal mRNA and protein expressions of NF-E2 related factor 2 (Nrf2) were up-regulated by MG132 in comparison to DN alone. Decreased renal mRNA expression of superoxide dismutase 1 (SOD1), catalase (CAT) and glutathione peroxidase (GPx) in DN rats was heightened after MG132 intervention. Depressed activities of renal SOD, CAT and GPx in DN rats were also improved by MG132 treatment. Increased renal nuclear factor κB (NF-κB) activity was inhibited after MG132 administration in DN rats at the end of 12 weeks.SignificanceOur present data suggest that inhibition of the proteasome by low-dose MG132 has a preventive effect on DN development and progression in rats through the up-regulation of antioxidant genes.     

Enhanced activity of serum and urinary hyaluronidases in streptozotocin-induced diabetic Wistar and GK rats

Using streptozotocin-induced diabetic Wistar and GK rats as models of type 1 and type 2 diabetes, respectively, we investigated the changes in serum and urinary hyaluronidase activity with the pathological progress. The serum hyaluronidase levels of streptozotocin-induced rats started to increase on the third day after injection and thereafter maintained approximately threefold higher levels compared with control rats; those of GK rats were already higher ( approximately twofold) from the beginning of the experiment. The increases of serum hyaluronidase activity in both diabetic rats were similar to those of blood glucose level, indicating that diabetes mellitus was accompanied by enhanced activity of circulating hyaluronidase from the early phase of its development. In zymography, every serum from diabetic and control rats gave two hyaluronidase isomers, a major 73-kDa band (Hyal-1 type) and a minor 132-kDa band, suggesting that the increases in serum hyaluronidase activity were not due to the appearance of novel isomers. The hyaluronidase activity in 24-h urine of streptozotocin-induced rats was 3-, 7-, and 11-fold higher at the 8th, 15th, and 18th week than that of control rats, respectively, and the urinary hyaluronidase activity of GK rats was not significantly different from controls. There was a good correlation between the urinary hyaluronidase activity and the albumin excretion. Thus the increase in urinary hyaluronidase activity may reflect enhanced glomerular permeability in streptozotocin-induced diabetic rats and may be a useful marker for diabetic nephropathy. Relative resistance to SDS-denaturation in zymography of rat serum and urinary hyaluronidases compared with human serum hyaluronidase are also shown.     

The extracellular cAMP-adenosine pathway regulates expression of renal D1 dopamine receptors in diabetic rats

Activation of D1 dopamine receptors expressed in the kidneys promotes the excretion of sodium and regulates sodium levels during increases in dietary sodium intake. A decrease in the expression or function of D1 receptors results in increased sodium retention which can potentially lead to the development of hypertension. Studies have shown that in the absence of functional D1 receptors, in null mice, the systolic, diastolic, and mean arterial pressures are higher. Previous studies have shown that the expression and function of D1 receptors in the kidneys are decreased in animal models of diabetes. The mechanisms that down-regulate the expression of renal D1 receptor gene in diabetes are not well understood. Using primary renal cells and acutely isolated kidneys from the streptozotocin-induced rat diabetic model, we demonstrate that the renal D1 receptor expression is down-regulated by the extracellular cAMP-adenosine pathway in vitro and in vivo. In cultures of primary renal cells, a 3 mm, 60-h cAMP treatment down-regulated the expression of D1 receptors. In vivo, we determined that the plasma and urine cAMP levels as well as the expression of 5'-ectonucleotidase, tissue-nonspecific alkaline phosphatase, and adenosine A2a receptors are significantly increased in diabetic rats. Inhibitors of 5'-ectonucleotidase and tissue-nonspecific alkaline phosphatase, α,β-methyleneadenosine 5'-diphosphate, and levamisole, respectively, blocked the down-regulation of D1 receptors in the primary renal cells and in the kidney of diabetic animals. The results suggest that inhibitors of the extracellular cAMP-adenosine pathway reverse the down-regulation of renal D1 receptor in diabetes.