Overexpression of the Kininogen-1 inhibits proliferation and induces apoptosis of glioma cells.

BACKGROUND: Glioma is the most common primary central nervous system tumor derived from glial cells. Kininogen-1 (KNG1) can exert antiangiogenic properties and inhibit proliferation of endothelial cells. The effect of KNG1 on the glioma is rarely reported, so our purpose in to explore its mechanism in glioma cells. METHODS: The differentially expressed genes (DEGs) were identified based on The Cancer Genome Atlas (TCGA) database. The KNG1-vector was transfected into the two glioma cells. The viability, apoptosis and cell cycle of glioma cells and microvessel density (MVD) were detected by cell counting kit-8 assay, flow cytometry and immunohistochemistry, respectively. The expression were measured by quantitative real-time PCR and Western blot, respectively. A tumor mouse model was established to determine apoptosis rate of brain tissue by terminal deoxynucleotidyl transfer-mediated dUTP nick end labeling (TUNEL) analysis. RESULTS: KNG1 was identified as the core gene and lowly expressed in the glioma cells. Overexpression of KNG1 inhibited cell viability and angiogenesis of glioma cells. Overexpression of KNG1 promoted the apoptosis and G1 phase cell cycle arrest of glioma cells. Moreover, the expressions of VEGF, cyclinD1, ki67, caspase-3/9 and XIAP were regulated by overexpression of KNG1. In addition, overexpression of KNG1 inhibited the activity of PI3K/Akt. Furthermore, overexpression of KNG1 decreased the tumor growth and promoted the apoptosis of decreased by overexpression of KNG1 in vivo. . CONCLUSIONS: Overexpression of KNG1 suppresses glioma progression by inhibiting the proliferation and promoting apoptosis of glioma cells, providing a therapeutic strategy for the malignant glioma.

Rat aortic smooth muscle cells in culture express kallikrein, kininogen, and bradykininase activity.

We have studied rat vascular smooth muscle (VSM) cells in culture for the presence of key elements of the glandular kallikrein-kinin system. Direct radioimmunoassay (RIA) using antiserum against rat urinary kallikrein detected a glandular kallikrein-like enzyme (GKLE) in VSM cells and in media. VSM homogenates and culture media had kininogenase activity, generating kinins from dog kininogen. About half of the GKLE was enzymatically inactive which could be activated with trypsin. Kininogenase activity was inhibited completely by aprotinin but only 20% by soybean trypsin inhibitor (SBTI). Trypsin liberated kinins from homogenates and media, demonstrating that VSM cells contain kininogen. Homogenates and media rapidly degrade bradykinin. GKLE, kininogen, and bradykininase activity were all present in VSM cells grown in defined media that contain no serum, thus eliminating any contamination or artefacts from fetal calf serum in standard culture media. Blood vessels of the rat have been reported to contain GKLE. Our observations indicate that GKLE is synthesized by VSM cells, not deposited from plasma. Furthermore, VSM cells synthesize kininogen and bradykininase(s), the other key elements of the glandular kallikrein-kinin system. Thus it is possible that the system functions as an autocoid mechanism that regulates local vascular tone.

Molecular analysis of the differential hepatic expression of rat kininogen family genes.

Serum concentration of rat T1 kininogen increases 20- to 30-fold in response to acute inflammation, an induced hepatic synthesis regulated primarily at the transcriptional level. We have demonstrated by transient transfection analyses that rat T1 kininogen gene/chloramphenicol acetyltransferase (T1K/CAT) constructs are highly responsive to interleukin-6 and dexamethasone. In these studies we examined the regulation of a highly homologous K kininogen gene promoter and showed that it is minimally induced under identical conditions. The basal expression of the KK/CAT construct was, however, five- to sevenfold higher than that of the analogous T1K/CAT construct. Promoter-swapping experiments to examine the molecular basis of this differentially regulated basal expression showed that at least two K kininogen promoter regions are important for conferring its high basal expression: a distal 19-bp region (C box) constituted a binding site for C/EBP family proteins, and a proximal 66-bp region contained two adjacent binding sites for hepatocyte nuclear factor 3 (HNF-3). While the C box in the K kininogen promoter was able to interact with C/EBP transcription factors, the T1 kininogen promoter C box could not. In addition, HNF-3 binding sites of the K kininogen promoter demonstrated stronger affinities than those of the T1 kininogen promoter. Since C/EBP and HNF-3 are highly enriched in the liver and are known to enhance transcription of liver-specific genes, these differences in their binding activities thus accounted for the K kininogen gene's higher basal expression. Our studies demonstrated that evolutionary divergence of a few critical nucleotides may lead to subtle changes in the binding affinities of a transcription factor to its recognition site, profoundly altering expression of the downstream gene.

A second expressed kininogen gene in mice.

We isolated PCR, RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE-PCR)-, and RT-PCR-generated clones from mouse kininogen family transcripts. DNA sequencing indicated that the clones were from two distinct genes. One set (K1) is from the previously reported mouse kininogen gene. The second set (K2) has an open reading frame, is 93% identical to K1 in the overlapping nucleotide sequence, and, unlike T-kininogens in the rat, encodes a bradykinin motif identical to K1. We discovered that K2 exists with two different 5' ends. We used RT-PCR to determine the distribution and relative abundance of K1 and K2 mRNA in mouse tissues. K2 is transcribed and K1 and K2 are generally both expressed in the same tissues; however, they differ in their regulation of the alternative splicing event that yields either low-molecular-weight kininogen (LMWK) or high-molecular-weight kininogen (HMWK). For example, in the liver K1 is expressed as both HMWK and LMWK, whereas K2 is only expressed as LMWK. Conversely, in the kidney K2 is strongly expressed as both HMWK and LMWK, whereas K1 is not expressed as HMWK and expressed only very weakly as LMWK.

Gene expression in clonally derived cell lines produced by in vitro transformation of rat fetal hepatocytes: isolation of cell lines which retain liver-specific markers.

The pattern of gene expression in fetal hepatocytes transformed in culture with a hepatocarcinogen (FRL cells) is studied with respect to a range of markers which are either developmentally regulated and/or shown to be expressed at high levels in hepatoma cells. The relative abundance of the respective mRNAs is determined and immunocytochemistry is used to detect the respective proteins in cultured cells. When compared with its normal counterpart, FRL cells retain the expression of transferrin, alpha 1-acid glycoprotein, gamma-glutamyltranspeptidase, and tyrosine aminotransferase at near normal levels, while expression of the liver-specific isoenzymes of pyruvate kinase (L form) and aldolase (B form) is reduced. The cell lines are different in that they fail to express albumin, alpha-fetoprotein, thiostatin and alpha 2-macroglobulin, and they express high levels of M2-pyruvate kinase and aldolase A, markers often found in abundance in hepatoma cells. Therefore transformation has resulted in different effects on different genes. Furthermore, it is of interest to find that the cells coexpress both forms of the pyruvate kinase isoenzymes which does not occur in the normal developing hepatocyte. These results indicate that it is possible to use this model to study changes which accompany transformation of fetal rat hepatocytes. The resulting cell lines have a stable phenotype and retain the changes which result from transformation even after extended passaging. This facilitates comparisons between the precursor cell and the tumor cell, both of which can be maintained under controlled conditions which exist in culture.

Murine fibroblasts synthesize and secrete kininogen in response to cyclic-AMP, prostaglandin E2 and tumor necrosis factor.

Fibroblasts prepared from the meninges of newborn mice or from mouse embryos, as well as fibroblast L929 cells, secreted an immunoreactive material (ir-kininogen) against rabbit anti-mouse low-molecular-weight kininogen antibody in response to dibutyryl cAMP. Western blots using a bradykinin-directed monoclonal, as well as a polyclonal anti-mouse low-molecular-weight kininogen antibody, showed that ir-kininogen had a molecular weight of 80,000 and that it contained a kinin moiety. N-terminal amino acid sequence of the ir-kininogen was consistent with that of mouse L-kininogen. The ir-kininogen produced by fibroblasts released a kinin by incubating with trypsin and mouse submandibular gland kallikrein, and it was identified as bradykinin by means of high-performance liquid chromatography, indicating that mouse fibroblasts produce and secrete a kininogen. Forskolin, prostaglandin E2 and tumor necrosis factor alpha stimulated the production of ir-kininogen by meningeal fibroblasts, whereas neither dibutyryl cAMP nor these agents influenced kininogen production by mouse hepatocytes in primary cultures. These results demonstrated that fibroblasts are a source of kininogen in the mouse, and that it is regulated by the inflammatory mediators, prostaglandin E2 and tumor necrosis factor. Therefore locally produced kininogen is implicated in pathogenesis of inflammation.

Molecular cloning of cDNAs for mouse low-molecular-weight and high-molecular-weight prekininogens.

We isolated cDNAs encoding low-molecular-weight (L-) and high-molecular-weight (H-) prekininogens from a mouse liver cDNA library using rat T-kininogen cDNA and rat H-kininogen cDNA respectively, as probes. The signal peptide, the heavy chain, and the bradykinin moiety, which are common between the two prekininogens, consist of 20, 359, and 9 amino acids, respectively, while the light chains of the L- and H-prekininogens are composed of 44 and 273 amino acids, respectively. All 19 cysteine residues present in both mouse prekininogens are located at the same positions relative to those of human origin. The light chain of H-prekininogen contains a characteristic 15-repeated His-Gly sequence and a conserved sequence for binding prekallikrein or factor XI. Northern blotting or reverse transcription-polymerase chain reaction followed by Southern blotting using mouse L- and H-kininogen cDNAs demonstrated that both L- and H-kininogens are predominantly expressed in the liver and kidney. L-Kininogen mRNA was also expressed in other tissues, such as the adrenal gland, brain, spinal cord, testis, lung, heart, and skin, while levels of H-kininogen mRNA in these tissues were too low to detect, suggesting that L-kininogen is synthesized in various tissues of mouse, while H-kininogen is exclusively synthesized in the liver and kidney. A genomic Southern blot using H-prekininogen cDNA revealed that the L- and H-prekininogen mRNAs in mouse are probably encoded by a single gene, as is the case in both human and bovine.

Rat fibroblasts synthesize T-kininogen in response to cyclic-AMP, prostaglandin E2 and cytokines.

T-Kininogen is a plasma protein characterized as a kinin-precursor, a cysteine protease inhibitor and an acute phase protein in the rat. Rat fibroblasts prepared from meninges or embryos and 3Y1-B clone 1-6 cells, a rat fibroblast cell line, secreted T-kininogen. Incubating these cells with 1 mM Bt2cAMP or a combination with 1 microM dexamethasone resulted in a marked increase in T-kininogen secretion, as well as in the incorporation of radioactive methionine into newly synthesized T-kininogen. Secretion of T-kininogen by meningeal fibroblasts was stimulated by forskolin, prostaglandin E2, bradykinin and cytokines, such as tumor necrosis factor alpha, interleukin-1 alpha (IL-1) and IL-6. Expression of T-kininogen mRNA was demonstrated in meningeal fibroblasts by Northern blot hybridization using T-kininogen cDNA as a probe, and the expression was stimulated by Bt2cAMP, prostaglandin E2, and the cytokines described above. In contrast, expression of T-kininogen mRNA in rat hepatocytes was not altered by Bt2cAMP, prostaglandin E2, tumor necrosis factor and IL-1, whereas it was greatly stimulated by IL-6, suggesting the differential regulation of T-kininogen gene expression in fibroblasts and hepatocytes. These results demonstrated for the first time, that rat fibroblasts express the T-kininogen gene, and that the expression is regulated by inflammatory mediators and cytokines.

Kininogen expression by rat vascular smooth muscle cells: stimulation by lipopolysaccharide and angiotensin II.

To identify the presence of a local kallikrein-kinin system in vascular wall, we have studied whether rat vascular smooth muscle cells (VSMC) express kininogen in vitro and in vivo. Western blots using anti-T-kininogen antibody revealed the presence of T-kininogen in conditioned medium of cultured VSMC. T-Kininogen secretion by VSMC was markedly enhanced by the addition of lipopolysaccharide (LPS), angiotensin II (AII) and phorbol 12-myristate 13-acetate (PMA) to the culture. Experiments using specific inhibitors for protein kinases and on the PMA-induced down-regulation of protein kinase C suggested that a protein kinase C-dependent or unidentified pathway is involved in AII or LPS action, respectively. The intravenous injection of LPS (0.5 mg/kg) resulted in an increase in T-kininogen mRNA levels in the vascular smooth muscle of rat aorta, peaking at 16 h. Polyacrylamide gel electrophoresis of cDNA products generated by reverse transcription-polymerase chain reaction (RT-PCR) from aortic mRNA using primers specific for either T- or low-molecular-weight kininogen revealed that rat vascular smooth muscle expressed T-kininogen gene but not low-molecular-weight kininogen gene, and that LPS exclusively stimulated T-kininogen expression. The mRNA for high-molecular-weight kininogen was undetectable in either aortic smooth muscle or cultured VSMC by means of RT-PCR analysis. RT-PCR using specific primers for rat tissue kallikrein genes showed that aortic smooth muscle expressed KLK1 (true kallikrein) mRNA, but not KLK10 (T-kininogenase) mRNA. These results demonstrated that rat VSMC are a source of T-kininogen but not of low-molecular-weight- or high-molecular-weight kininogen, in contrast to the expression of true kallikrein but not of T-kininogenase by these cells.

Malignant hypertension: a syndrome accompanied by plasmatic diminution of low and high molecular weight kininogens.

Total kininogen (Kgn), kallikrein, and prekallikrein were measured in patients with malignant hypertension (MH), essential hypertension (EH), normotensive control (NC), and hypertension and chronic renal failure (HRF). These components of the kallikrein-kinin system were related to the levels of creatinine and fibrinogen. High molecular weight Kgn and low molecular weight Kgn were also measured in blood samples from a peripheral vein, arterial blood, and suprahepatic vein in NC, EH, and MH. Results showed that total Kgn levels were diminished in MH and this diminution could not be ascribed to decreases in renal function, hematocrit, or fibrinogen levels. Appropriate antihypertensive treatment for over 1 year did not normalize Kgn levels in 10 of 11 patients. High molecular weight Kgn and low molecular weight Kgn were both diminished in MH (0.26 +/- 0.04 nmol bradykinin/ml and 0.93 +/- 0.12 nmol lysyl-bradykinin/ml, respectively) as compared to NC (0.39 +/- 0.07 and 1.92 +/- 0.16) and EH (0.51 +/- 0.07 and 1.65 +/- 0.13). Higher concentrations of high molecular weight Kgn were demonstrated in the suprahepatic vein as compared to arterial blood, demonstrating its synthesis by the liver. However, patients with MH had a diminished capacity to synthetize high molecular weight Kgn. A decrease in synthesis of high molecular weight Kgn may be a partial explanation for low levels of total Kgn. It is suggested that a lack of Kgn may play a role in the pathogenesis of MH.

Primary structure of a gene encoding rat T-kininogen.

A gene encoding the acute-phase reactant, T-kininogen, has been isolated from a rat genomic library. The nucleotide sequence of all exons as well as of a large portion of the introns was determined. The gene, T-KG, is approximately 24 kb in length and contains 11 exons. The promoter appears to be rather unusual in that there are no consensus CCAAT or TATAA boxes or SP1-binding sites. An A-rich region present upstream of the transcription start point may function as a TATAA-equivalent sequence. In addition, several potential glucocorticoid regulatory elements and a sequence similar to the AP-1 binding site are present in the 5' region of the gene. The intron/exon boundaries in this T-KG gene are identical to those seen in the human K-kininogen gene. This is consistent with the inclusion of T-KG in the cystatin supergene family of cysteine protease inhibitors.

The high-molecular-mass kininogen deficient rat expresses all kininogen mRNA species, but does not export the high-molecular-mass kininogen synthesized.

The Katholiek substrain of Brown Norway (BN/Kat) rats exhibits a very low level of circulating high-molecular-mass (HMW) kininogen and a partial deficiency in plasma prekallikrein. Northern blot analysis of liver RNA revealed that HMW kininogen and prekallikrein mRNAs are present in these rats with a similar size and abundance compared to control Brown Norway (BN/Orl) rats. The low-molecular-mass kininogen mRNA, encoded by the same kininogen gene as HMW kininogen mRNA by alternative splicing, is detected in both strains by dideoxynucleotide limited primer extension analysis. Measurement of HMW kininogen by radioimmunoassay was performed in liver subcellular fractions. It reveals that, in contrast to its absence in the cytosolic fraction, HMW kininogen in deficients rats is slightly more abundant in the microsomal fraction, than in control rats. These observations exclude both an abnormality at the level of gene transcription and a major structural modification of the transcribed RNA and of the synthesized HMW kininogen. They favour the hypothesis of an abnormal intracellular transport of the HMW kininogen in deficient rats.

Stimulation of hepatic T-kininogen production by interferon.

T-kininogen is known to be an acute-phase reactant as well as a kininogen in rat plasma. Three kinds of cytokines, interleukin-1, tumor necrosis factor and interferon, were assayed for their abilities to stimulate hepatic production of T-kininogen. Of these cytokines, interferon was able to stimulate hepatic production of T-kininogen, but few effects were observed for interleukin-1 and tumor necrosis factor. In addition, the stimulatory effect of interferon was inhibited by tumor necrosis factor. Our data suggest that interferon is a candidate for the leukocyte-derived factor mediating the acute-phase response of T-kininogen.

T-kininogen is a biomarker of senescence in rats.

We have previously reported on the identification of T-kininogen (T-KG) as a gene whose expression is increased during senescence in male Sprague-Dawley (S-D) rats. Serum T-KG levels increase 2.5-4 months before the time of death for any given animal, irrespective of the actual age of the animal at the time of this event. Furthermore, dietary restriction (DR) delays, but does not prevent, the increase in serum T-KG levels. In the present study, we have assessed whether or not the age-related increase in T-KG is a common feature of senescence in other strains of rat. We have analyzed hepatic T-KG mRNA levels in male Fischer 344 rats (F344), as well as in male and female (Fischer 344 x Brown Norway)F1 rats (F1). In both of these strains, we observed a dramatic increase in hepatic T-KG mRNA levels when male rats approach senescence. The mRNA levels behave similarly in F1 and S-D rats, in that the increase occurs late in life, and it is either repressed or delayed by DR. In contrast, the increase in T-KG mRNA levels in F344 rats occurs earlier in life, and is not significantly affected by DR. Young female F1 rats fed ad libitum (AL) show a statistically significant (P = 0.0009) 2.6-fold higher level of T-KG mRNA, as compared to their male counterparts. Thus, while we still observe an age-related increase in this parameter in both AL and DR female F1 rats, the difference is statistically significant (P = 0.0001) only in DR animals. We conclude that the increase in T-KG gene expression is a common feature of senescence and that, at least in males of these commonly used rat strains, T-KG can be used as a reliable biomarker of aging. Since the increase in T-KG gene expression does not appear to correlate with inflammatory processes, and since different strains of animals succumb to different pathologies, these results further suggest that the increase in T-KG expression might be related to the process of aging per se, rather than to any given age-related pathology.

Synthesis of kininogen and degradation of bradykinin by PC12 cells.

1. In this study, the abilities of PC12 cells to synthesize and degrade kinins were investigated. Kinin formation was assessed as kinin and kininogen content of cells and supernatants in serum-free incubations by use of a bradykinin-specific radioimmunoassay. Expression of kininogen mRNA was demonstrated by reverse-transcriptase PCR. Kinin degradation pathways of intact PC12 cells were characterized by identification of the kinin fragments generated from tritiated bradykinin either in the absence or presence of the angiotensin I-converting enzyme inhibitor ramiprilat. 2. Kinin immunoreactivity in the supernatant of PC12 cell cultures accumulated in a time-dependent fashion during incubations in serum-free media. This effect was solely due to de novo synthesis and release of kininogen (35 pg bradykinin h-1 mg-1 protein) since it could be suppressed by cycloheximide. Continuous synthesis of kininogen was a specific property of PC12 cells, as it was not observed in cultured macro- or microvascular endothelial cells. PC12 cells contained only minor amounts of stored kininogen. The rate of kininogen synthesis was not affected by ramiprilat, bacterial lipopolysaccharide, nerve growth factor or dexamethasone, but was stimulated 1.4 fold when cells were pretreated for 1 day with 1 microM desoxycorticosterone. 3. By use of cDNA probes specific for kininogen subtype mRNAs, expression of low-molecular-weight kininogen and T-kininogen in PC12 cells was confirmed. Expression of high molecular weight kininogen mRNA was also shown, though only at the lowest limit of detection of the assay. 4. Degradation of tritiated bradykinin by PC12 cells occurred with a half-life of 48 min resulting in the main fragments [1-7]- and [1-5]-bradykinin. The degradation rate of bradykinin decreased to 15% in the presence of ramiprilat (250 nM). Apart from angiotensin I-converting enzyme direct cleavage of bradykinin to [1-7]- and [1-5]-bradykinin still occurred under this condition as a result of additional kininase activities. 5. Along with previous findings of B2-receptor-mediated catecholamine release, these results now confirm the hypothesis that a cellular kinin system is expressed in PC12 cells. The presence of such a system may reflect a role of kinins as local neuromodulatory mediators in the peripheral sympathetic system.

Plasma kininogen deficiency: associated defective secretion of kininogens by primary cultures of hepatocytes from brown Norway Katholiek rats.

To clarify the mechanism of plasma kininogen deficiency of Brown Norway Katholiek strain (B/N-Katholiek) rats, we compared synthesis and secretion of kininogens by primary cultures of hepatocytes from B/N-Katholiek and B/N-Kitasato (normal strain) rats. Pulse-and-chase experiments using [35S]methionine demonstrated that kininogen antigens with molecular masses of 100 and 66 kDa, corresponding to high- and low-molecular-weight kininogens (HK and LK), respectively, were detected in the hepatocytes of both strains. These proteins were then processed to 108- and 71-kDa forms, respectively, and secreted by the normal hepatocytes, while the latter forms were hardly secreted in the culture media of the deficient hepatocytes. However in the deficient cells, 100- and 66-kDa forms were accumulated, but 108- and 71-kDa bands were faint. A subcellular fractionation study showed that a relatively higher amount of the kininogen antigens was present in the lysosomal fraction of B/N-Katholiek hepatocytes than in that of B/N-Kitasato hepatocytes. From these results we postulate the cause of the secretion defect of B/N-Katholiek liver to be as follows. (i) B/N-Katholiek liver could synthesize the mature secretable forms of HK and LK, but they are too rapidly transported to the lysosomes, or (ii) the mature forms in B/N-Katholiek hepatocytes might be synthesized much more slowly than those in the normal cells. T-Kininogen was normally synthesized and secreted by the hepatocytes of B/N-Katholiek, suggesting that the secretion defect could be limited to HK and LK, at a common site.

Bradykinin decreases T-kininogen synthesis in a rat hepatoma cell line: evidence of bradykinin B2-type receptors.

Using the rat H4-II-E-C3 hepatoma cell line, we investigated the presence of [125I][Tyr8]BK binding sites and the direct modulation of T-kininogen synthesis, an acute phase protein of inflammation, by bradykinin (BK) analogues. H4-II-E-C3 membrane preparations exhibited [125I][Tyr8]BK binding sites with a Kd of 4 nM and a Bmax of 120 fmol/mg of protein. Des-Arg9-BK showed no affinity (Ki > 10(-4) M) for these sites. The B2 metabolism-resistant and selective agonist [Phe8 psi (CH2-NH)Arg9]BK decreased the T-kininogen concentration in H4-II-E-C3 medium by 23% (p < 0.05). This effect was reversed by coincubation with the B2 antagonist HOE140. The B1 agonist Sar[D-Phe8]des-Arg9-BK and the B1 antagonist Lys[Leu8]des-Arg9-BK did not modify T-kininogen concentrations. The interaction between cytokines and kinins in the modulation of T-kininogen synthesis was also studied. Preincubation of hepatoma cells for 1 h with interleukin-1 alpha (IL-1 alpha) alone reduced T-kininogen concentrations by 37%, and this effect was blocked by co-addition of HOE140. Preincubation with interleukin-6 (IL-6) increased T-kininogen levels by threefold. Coincubation in the presence of the B2 agonist decreased this augmentation by 24%. The latter effect was reversed by co-addition of HOE140. None of the cytokines tested induced a response to the B1 agonist or antagonist under the experimental conditions studied. Overall, these results support the presence of a functional B2 receptor on H4-II-E-C3 cells that modulates T-kininogen synthesis. We suggest that the receptor is involved in vivo in a retroaction loop between kinins and T-kininogen production during inflammation. We speculate that BK could be a mediator in the modulation of acute phase protein synthesis by the cytokines IL-1 alpha and IL-6.

A correlation between a proteomic evaluation and conventional measurements in the assessment of renal proximal tubular toxicity.

4-Aminophenol (4-AP), D-serine, and cisplatin are established rodent nephrotoxins that damage proximal tubules within the renal cortex. Using high throughput 2D gel proteomics to profile protein changes in the plasma of compound-treated animals, we identified several markers of kidney toxicity. Male F344 and Alpk rats were treated with increasing doses of 4-AP, D-serine, or cisplatin, and plasma samples were collected over time. Control groups received saline or nontoxic isomers, L-serine, and transplatin. Plasma proteins that displayed dose- and temporal-dependent regulation in each study were further characterized by mass spectrometry to elucidate the protein identity. Several isoforms of the rat-specific T-kininogen protein were identified in each study. T-kininogen was elevated in the plasma of 4-AP-, D-serine-, and cisplatin-treated animals at early time points, returning to baseline levels 3 weeks after treatment. The protein was not elevated in the plasma of control animals or those treated with nontoxic compounds. We propose that T-kininogen may be required to counteract apoptosis in proximal tubular cells in order to minimize tissue damage following a toxic insult. In addition, T-kininogen may be required to stimulate localized inflammation to aid tissue repair. We also identified several isoforms of the inter-alpha inhibitor H4P heavy chain in the 4-AP and D-serine studies. In each case, the protein expression levels in the blood samples paralleled the extent of kidney toxicity, highlighting the correlation between protein alterations and clinical chemistry endpoints. A further set of proteins correlating with kidney damage was found to be a component of the complement cascade and other blood clotting factors, indicating a contribution of the immune system to the observed toxicity. These observations underscore the value of proteomics in identifying new biomarkers and in the elucidation of mechanisms of toxicity.

Kinins are involved in the antiproteinuric effect of angiotensin-converting enzyme inhibition in experimental diabetic nephropathy.

The present study examined non-insulin-treated streptozotocin (STZ)-induced diabetic rats to determine the role of kinins in diabetic nephropathy. Their involvement in the renoprotective effect of the angiotensin-converting enzyme inhibitor (ACEI) ramipril was investigated using the bradykinin (BK) B(2)-receptor antagonist, icatibant (HOE 140), or a combination of the two drugs.Although, none of the treatments prevented the decline of the glomerular filtration rate (GFR) in diabetic rats, ramipril (3 mg/kg/day), but not icatibant (HOE 140; 500 microg/kg/day), prevented proteinuria in these animals. However, the antiproteinuric effect of ramipril was reduced by 45% when combined with icatibant. To explore whether the renal kallikrein-kinin system (KKS) belongs to the underlying mechanisms of these findings, we also determined urinary BK levels, renal kallikrein (KLK) and angiotensin-converting enzyme (ACE) activity as well as renal cortical mRNA levels of neutral endopeptidase 24.11 (NEP) and low-molecular weight (LMW) kininogen. STZ led to a reduction of renal KLK and ACE activity and NEP expression and to a three-fold increase of urinary BK excretion and renal kininogen expression. Icatibant given alone had no effect on these parameters. In contrast, ramipril treatment normalized urinary protein and BK excretion as well as kininogen mRNA expression without affecting NEP mRNA expression or KLK and ACE activity. Our data demonstrate that renal BK is increased in severe STZ-induced diabetes mellitus, but may affect glomerular regulation only to a minor degree under this condition. However, kinins are partly involved in the antiproteinuric action of ACEI at this stage of diabetic nephropathy.

Lack of expression of T-kininogen gene in the hearts of untreated and turpentine-injected rats.

Cystatins, inhibitors of cysteine proteinases, are present in rat heart. However, the controls of genes coding for various cystatins in the heart, and the cellular sites of expression of these genes are not known. With a sensitive reverse transcriptase--polymerase chain reaction T-kininogen mRNA was readily detected in the submandibular glands and livers, but not in the hearts, of control or turpentine-injected rats. Immunocytochemical observations employing a monoclonal antibody to bradykinin, which reacts with kininogens in general, revealed no specific staining in cardiac structures, but a weak staining was apparent in blood vessels and on the surface of endothelial cells of both control and turpentine-injected rats. The monoclonal antibody revealed the presence of kininogens in the acinar cells of the submandibular gland, and, in acute inflammation, in the hepatocytes. These findings suggest that the T-kininogen gene is not expressed in the heart, and the T-kininogen demonstrable in heart extracts derives from the blood. Circulating kininogens are likely bound to endothelial cells, and may be a local source of kinins. In addition, kininogens, as potent inhibitors of cysteine proteinases, may play a role in pathologic conditions of the heart by controlling the deleterious effects of cathepsins released from lysosomes or secreted by macrophages.