Kidney Homogenate Supernatant Research Articles

Effect of subacute poisoning with lambdacyhalothrin on vascular endothelial growth factor 2 receptor in mice kidneys

BackgroundPyrethroids are used for insect control. They act on voltage-gated sodium channels in neurons. Lambdacyhalothrin (LCH) is a type II pyrethroid producing choreoatetosis and salivation in rodents. Vascular endothelial growth factor (VEGF) expression in the kidney is high in the glomerular podocytes and kidney tubules. VEGF receptor 2 (VEFGR2) is the main mediator in angiogenesis and it regulates blood vessel permeability. Lack of VEGF in podocytes impairs filtration. The nuclear factor κB (NFκB) is widely known as an important mediator of inflammation.The aim of the study was to check if subacute oral intoxication with 0.1LD50 of LCH affects kidney size, function and VEGFR2 and NFκB in mice kidneys.MethodsA total of 32 Albino Swiss mice was used: females controls, males controls, females receiving 2 mg/kg LCH, males receiving 2 mg/kg LCH orally for 7 days. On day 8 animals were sacrificed, blood and kidneys were obtained. Kidney mass was determined, creatinine concentration was measured in blood sera, VEGFR2 and NFκB in kidney homogenate supernatant with ELISA kit.ResultsThere was no statistically significant differences in kidney mass, creatinine concentration in blood sera nor NFκB but mean VEGFR2 concentration in the kidneys of females exposed to LCH was 128.01 ng/ml and showed statistically significant difference in comparison with control females. There was no statistically significant difference between VEGFR2 concentration in the kidneys of males exposed to LCH and control males.ConclusionThe VEGFR2 increases in the course of LCH intoxication in females probably due to the protective effect of oestrogens.

Bone marrow derived mesenchymal stem cells pretreated with erythropoietin accelerate the repair of acute kidney injury

BackgroundMesenchymal stem cells (MSCs) represent a promising treatment option for acute kidney injury (AKI). The main drawbacks of MSCs therapy, including the lack of specific homing after systemic infusion and early cell death in the inflammatory microenvironment, directly affect the therapeutic efficacy of MSCs. Erythropoietin (EPO)-preconditioning of MSCs promotes their therapeutic effect, however, the underlying mechanism remains unknown. In this study, we sought to investigate the efficacy and mechanism of EPO in bone marrow derived mesenchymal stem cells (BMSCs) for AKI treatment.ResultsWe found that incubation of BMSCs with ischemia/reperfusion(I/R)-induced AKI kidney homogenate supernatant (KHS) caused apoptosis in BMSCs, which was decreased by EPO pretreatment, indicating that EPO protected the cells from apoptosis. Further, we showed that EPO up-regulated silent information regulator 1 (SIRT1) and Bcl-2 expression and down-regulated p53 expression. This effect was partially reversed by SIRT1 siRNA intervention. The anti-apoptotic effect of EPO in pretreated BMSCs may be mediated through the SIRT1 pathway. In a rat AKI model, 24 h after intravenous infusion, GFP-BMSCs were predominantly located in the lungs. However, EPO pretreatment reduced the lung entrapment of BMSCs and increased their distribution in the target organs. AKI rats infused with EPO-BMSCs had significantly lower levels of serum IL-1β and TNF-α, and a significantly higher level of IL-10 as compared to rats infused with untreated BMSCs. The administration of EPO-BMSCs after reperfusion reduced serum creatinine, blood urea nitrogen, and pathological scores in I/R-AKI rats more effectively than BMSCs treatment did.ConclusionsOur data suggest that EPO pretreatment enhances the efficacy of BMSCs to improve the renal function and pathological presentation of I/R-AKI rats.

Alleviation of apoptosis of bone marrow-derived mesenchymal stem cells in the acute injured kidney by heme oxygenase-1 gene modification

Bone marrow-derived mesenchymal stem cells (BMSCs) transplantation is beneficial for the treatment of acute kidney injury (AKI), but the poor survival of BMSCs limits the repair effect. The oxidative stress in the AKI microenvironment is regarded as the main reason. Considering the potent anti-oxidant ability of heme oxygenase-1 (HO-1), HO-1 overexpression in BMSCs can be expected to improve the survival of BMSCs and correspondingly enhance the AKI repair effect. Here, BMSCs are transfected with pLV-HO-1/eGFP and pLV–eGFP by the lentivirus vector to get HO-1-BMSCs and eGFP-BMSCs, respectively. Ischemia/reperfusion-AKI kidney homogenate supernatant (KHS) is prepared for treating BMSCs, eGFP-BMSCs and HO-1-BMSCs. AKI-KHS results in a high inhibitory rate of BMSCs growth and a high proportion of TUNEL positive BMSCs, while HO-1 overexpression inverses this phenomenon and re-establishes the antioxidant and oxidant balance in HO-1-BMSCs. Phosphorylations of p53 and p38 mitogen-activated protein kinases (p38 MAPK) in HO-1-BMSCs decrease. Lower levels of monocyte chemotactic protein 1, tumor necrosis factor-α and interleukin 1β are also observed in supernatant of HO-1-BMSCs. The in vivo study shows that HO-1 overexpression sharply decreases the apoptosis of BMSCs in the injured kidney, and correspondingly the renal function of the AKI rats improves significantly. In conclusion, BMSCs with HO-1 overexpression suggests a better survival in the I/R-AKI microenvironment and a better kidney repair effect. The anti-oxidant effect via the inactivations of the downstream p53 and p38MAPK in BMSCs and the anti-inflammation could be the mechanisms. It provides a novel approach for the cell-based AKI-therapy.

Effect of erythropoietin on mesenchymal stem cell differentiation and secretion in vitro in an acute kidney injury microenvironment

We investigated the effect of erythropoietin (EPO) on differentiation and secretion of bone marrow-derived mesenchymal stem cells in an acute kidney injury microenvironment. Acute kidney injury mouse models were prepared. Both renal cortices were then immediately collected to produce the ischemia/reperfusion kidney homogenate supernatant. The morphological and ultrastructural changes in the cells were observed using an inverted microscope and a transmission electron microscope. Cytokeratin-18 was detected using flow cytometry. Bone morphogenetic protein-7 levels, hepatocyte growth factor, and vascular endothelial growth factor in the culture medium were detected using an enzyme-linked immunosorbent assay. The cells had high CD29 and CD44 expression, as well as low CD34 and CD45 expression. More round and oval cells with cobble-like appearances were observed after EPO treatment. In addition, an increase in the number of rough endoplasmic reticula, lysosomes, and mitochondria was observed in the cytoplasm; the intercellular junction peculiar to epithelial cells was also seen on the cell surface. After treatment with ischemia/reperfusion kidney homogenate supernatant, cytokeratin-18 expression increased significantly and EPO could magnify its expression. Bone morphogenetic protein-7 levels, hepatocyte growth factor, and vascular endothelial growth factor levels after treatment with ischemia/reperfusion kidney homogenate supernatant significantly decreased, whereas EPO increased the cytokine secretion. The acute kidney injury microenvironment can induce the bone marrow-derived mesenchymal stem cells to partially differentiate into renal tubular epithelium-shaped cells, but weaken their secretion function. EPO intervention can boost up their differentiation function and reverse their low secretion effect.

Effect and mechanism of erythropoietin on mesenchymal stem cell proliferation in vitro under the acute kidney injury microenvironment

Erythropoietin (EPO) can stimulate the proliferation and protraction of endothelial progenitor cells, and plays an important role in the proliferation and differentiation of marrow-derived mesenchymal stem cells (mMSCs) under the acute kidney injury (AKI) microenvironment. In the present study, C57BL/6 mice mMSCs were isolated, and AKI mice models were prepared. The renal cortex was obtained to prepare the ischemia/reperfusion (I/R) kidney homogenate supernatant. P3-mMSCs were treated by different methods: one group was added only I/R kidney homogenate supernatant, and another contained different concentrations of EPO (1, 5, 10, 50 IU/mL) in I/R kidney homogenate supernatant. The proliferation and apoptosis of mMSCs were detected by CCK-8 and TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling), respectively. Expression of erythropoietin receptor (EPOR) and protein of the signal pathway related to proliferation/apoptosis were also examined. The results showed that the proliferation ability of mMSCs treated with I/R kidney homogenate supernatant decreased significantly, while the apoptosis percentage was significantly higher than that of the control. After intervention of EPO, their proliferation enhanced and the apoptosis percentage decreased. EPOR expression was positive in P3-mMSCs. EPO decreased the expression of caspase-3 of mMSCs under the AKI microenvironment in a dose- and time-dependent manner, but increased the Bcl-2 expression. The expression of phosphor-Janus kinase 2, phosphor-signal transducer and activator of transcription (pSTAT-5) increased significantly in 10 IU/mL EPO cultured for five days. Our results show that EPO can promote proliferation of mMSCs in vitro under the AKI microenvironment, which is mediated by EPOR and related with the proliferation/apoptosis signal pathway.

Characterization of a major form of human isatin reductase and the reduced metabolite.

Isatin, an endogenous indole, has been shown to inhibit monoamine oxidase, and exhibit various pharmacological actions. However, the metabolism of isatin in humans remains unknown. We have found high isatin reductase activity in the 105,000 g supernatants of human liver and kidney homogenates, and have purified and characterized a major form of the enzyme in the two tissues. The hepatic and renal enzymes showed the same properties, including an M(r) of 31 kDa, substrate specificity for carbonyl compounds and inhibitor sensitivity, which were also identical to those of recombinant human carbonyl reductase. The identity of the isatin reductase with carbonyl reductase was immunologically demonstrated with an antibody against the recombinant carbonyl reductase. About 90% of the soluble isatin reductase activity in the liver and kidney was immunoprecipitated by the antibody. The Km (10 microm) and k(cat)/K(m) (1.7 s(-1) x microm(-1)) values for isatin at pH 7.0 were comparable to those for phenanthrenequinone, the best xenobiotic substrate of carbonyl reductase. The reduced product of isatin was chemically identified with 3-hydroxy-2-oxoindole, which is also excreted in human urine. The inhibitory potency of the reduced product for monoamine oxidase A and B was significantly lower than that of isatin. The results indicate that the novel metabolic pathway of isatin in humans is mediated mainly by carbonyl reductase, which may play a critical role in controlling the biological activity of isatin.

An ATP-inhibited soluble 5'-nucleotidase of rat kidney.

Hydrolysis of 5'-AMP by 5'-nucleotidase is a possible source of adenosine in the kidney. A renal membrane-bound ecto-5'-nucleotidase has been previously described. The present study deals with the catalytic properties of a 5'-AMP phosphohydrolase partially purified from high-speed supernatants of rat kidney homogenates. It exhibits phosphatase activity toward 5'-AMP, 5'-IMP, and 5'-GMP, but not toward 2'- and 3'-AMP and corresponds therefore to a 5'-nucleotidase. The hydrolysis of 5'-AMP by the soluble 5'-nucleotidase requires divalent cations. Maximal activity is reached with 10 microM of either Mn2+ or Co2+, whereas half-maximal activity is obtained with approximately 400 microM Mg2+. The soluble 5'-nucleotidase exhibits Michaelis-Menten kinetics with a Km of 9.5 microM for 5'-AMP. In the presence of 1 mM of free Mg2+, physiological concentrations of ATP provoke an increase of the Km for 5'-AMP and a decrease of Vmax. An increase of the pH of 0.4 units in the pH range 6.4-7.4 roughly doubles the rate of hydrolysis of 5'-AMP. The effects of ATP and of the pH are compatible with a role of the renal soluble 5'-nucleotidase in the hydrolysis of 5'-AMP and in the production of adenosine during hypoxia.

Localization and properties of NAD-dependent 15 hydroxyprostaglandin dehydrogenase activity in spontaneously hypertensive rat kidney.

To evaluate the pathophysiological significance of renal NAD-dependent 15 hydroxyprostaglandin dehydrogenase (PGDH) in young spontaneously hypertensive rats (SHR), the intranephron distribution of PGDH was compared with age-matched Wistar Kyoto rats (WKY). With supernatants of kidney homogenates, PGDH activities in 3 week-old SHR were significantly lower than those in WKY, and no difference in PGDH activities was found between male and female in both WKY and SHR. By using microdissected nephron segments of 3 week-old WKY and SHR, PGDH activities in both groups were found to be exclusively distributed in the proximal tubules, within which the activity of the straight portion (PST) was several fold higher than that of the convoluted one (PCT). In 3 week-old SHR, a significant decrease of PGDH activity was observed only in PST, possibly indicating a decrease of enzyme content in PST because of similar Km values in both groups. These results may suggest that intraproximal PG concentration could be regulated by the change of PGDH in PST of young SHR.

Identification of intracellular degradation intermediates of aldolase B by antiserum to the denatured enzyme.

A method has been developed that enables us to identify intracellular degradation intermediates of fructose-bisphosphate aldolase B (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13). This method is based on the use of antibody against thoroughly denatured purified aldolase. This antibody has been shown to recognize only denatured molecules, and it did not interact with "native" enzyme. supernatants (24,000 X g for 30 min) of liver and kidney homogenates were incubated with antiserum to denatured enzyme. The antigen-antibody precipitates thus formed were subjected to NaDodSO4/PAGE, followed by electrotransfer to nitrocellulose paper and immunodecoration with antiserum to denatured enzyme and 125I-labeled protein A. Seven peptides with molecular weights ranging from 38,000 (that of the intact subunit) to 18,000, which cross-reacted antigenically with denatured fructose-bisphosphate aldolase, could be identified in liver. The longest three peptides were also present in kidney. The possibility that these peptides were artifacts of homogenization was ruled out as follows: 125I-labeled tagged purified native aldolase was added to the buffer prior to liver homogenization. The homogenates were than subjected to NaDodSO4/PAGE followed by autoradiography, and the labeled enzyme was shown to remain intact. This method is suggested for general use in the search for degradation products of other cellular proteins.

Maleate effects on kidney peptidases and proteinuria of male and female rats

The effects of maleate on membrane-bound and lysosomal peptidases were studied histochemically in the kidney and biochemically in the kidney and the urine of male and female rats 6 h after the administration of two different doses of sodium maleate (150 and 300 mg/kg body weight). Additionally, the proteinuria of experimental animals was electrophoretically analysed to detect maleate-induced alterations in the urinary protein composition. The histochemistry of the brush-border peptidases (aminopeptidase A, gamma-glutamyltransferase) showed dose-dependent maleate effects in the late pars convoluta and the pars recta of the proximal tubule (blurring of the brush-border enzyme reaction pattern). The female animals were more severely affected by both maleate doses. After maleate treatment, enzyme-activity measurements in the kidney homogenate supernatant and urine indicated dose-dependent structural destruction of the proximal tubule, especially of brush-border membranes, and revealed an increase in enzyme excretion. Both the maleate-induced enzyme excretion and proteinuria were more pronouncedly increased in females than in males. Electrophoretic analysis of urinary proteins revealed alterations in the urinary-protein composition after maleate treatment, which favoured the excretion of proteins with a molecular weight higher than 20,000 daltons. Again, sex-related differences in the maleate effects were demonstrated. The results indicate that maleate causes alterations in the brush-border membranes and, especially at higher doses, results in cellular destruction selectively in the late proximal tubule of rat kidneys. Selectivity was also encountered in the maleate effects on urinary-protein composition, suggesting that the tubular alterations lead to an inhibition of the reabsorption of mainly high-molecular-weight proteins. Although the nature of the effects was independent of sex, it appears that females are less well protected against tubular damage caused by maleate.

RENAL HANDLING OF HOMOLOGOUS RADIOIODINATED THYROTROPHIN IN THE RAT

Previously, TSH metabolism and tissue distribution in the rat were studied using heterologous hormone and/or unphysiological doses. We injected iv [131I]rTSH (S.A. 100 micronCi/microgram) in physiological doses (10 ng) to KI blocked male rats. Two to 180 min after which TCA precipitable 131I was measured in the kidney, liver, muscle, fat and testis. The highest TCA precipitable radioactivity concentration, peak value 12% at 30 min; organ/blood ratio, 16, was found in the kidney. With the kidney validated as the major site of localization of TSH, the renal localization and handling of [125I]rTSH was studied by autoradiography. Radioactivity was confined to the cortex. Two to 30 min samples showed 125I in Bowman's space and luminal parts of the proximal tubular cells while samples from 60 to 180 min revealed activity in contraluminal areas, suggesting glomerular filtration and tubular re-absorption. Gel-filtration of supernatants of kidney homogenates revealed progressive diminution of the TSH peak and the appearance of a smaller MW peak. We conclude that (1) the kidney is the major site of localization and metabolism of homologous TSH, (2) the renal handling of TSH involves both glomerular filtration and tubular re-absorption.

Species differences in the conjugation of 4-hydroxy-3-methoxyphenylethanol with glucuronic acid and sulphuric acid.

The biosynthesis of the glucuronide and sulphate conjugates of 4-hydroxy-3-methoxyphenylethanol was demonstrated in vitro by using the high-speed supernatant and microsomal fractions of liver respectively. These two conjugates were also produced simultaneously by using the post-mitochondrial fraction of rat, rabbit or guinea-pig liver. In contrast only the glucuronide was synthesized by human liver and only the sulphate by mouse and cat livers. Neither of these conjugates was formed by the kidney or the small or large intestine of the rat. A high sulphate-conjugating activity was observed in mouse kidney; the rate of sulphation of 4-hydroxy-3-methoxyphenylethanol with kidney homogenate and high-speed supernatant preparations was 1.8 times greater than with liver preparations. The sulpho-conjugates of 4-hydroxy-3-methoxyphenylethanol and 4-hydroxy-3-methoxy-phenylglycol were also formed by enzyme preparations of rabbit adrenal and rat brain; the glycol was the better substrate in the latter system. Mouse brain did not possess any sulphotransferase activity. For the conjugation of 4-hydroxy-3-methoxyphenylethanol by rabbit liver, the Km for UDP-glucuronic acid was 0.22 mM and that for Na2SO4 was 3.45 mM. The sulphotransferase has a greater affinity for 4-hydroxy-3-methoxyphenyl-ethanol than has glucuronyltransferase, as indicated by their respective Km values of 0.036 and 1.3 mM. It was concluded that sulphate conjugation of 4-hydroxy-3-methoxyphenylethanol predominates in most species of animals.

Modulation of leucine transaminase activity by dietary means.

The specific activity of leucine transaminase was measured in supernatants of liver, skeletal muscle (gastrocnemius), and kidney homogenates obtained from fed, starved, and protein-deprived rats. After 12 h of starvation, there were slight reductions in leucine transaminase activity of both muscle and kidney tissues. When starvation was prolonged to 1 full day, the activity of this enzyme increased by approximately twofold in both muscle and kidney. Prolongation of fasting to 5 days resulted in an additional increase in specific activity of leucine transaminase in muscle. During the entire 5 days of starvation, leucine transaminase activity remained unaltered in liver of starved rats. Protein deprivation for 1 or 5 days resulted in significant reductions in specific activity of leucine transaminase in skeletal muscle. Protein deprivation did not produce a remarkable effect on the activity of this enzyme in kidney or liver tissue. The results of this study, together with those previously obtained, indicate that within our experimental conditions increased oxidation of leucine in skeletal muscle of starved rats is not initially related to an alteration in activity of leucine transaminase. When caloric deficiency is prolonged, the potential for transamination is also increased. These adaptive changes increase the ability of skeletal muscle to use leucine as a metabolic fuel.

Pressor activity in bovine kidney homogenate: enhancement by cadmium and zinc ions.

Cadmium and zinc ions added separately to male bovine kidney homogenate increased pressor activity fourfold to fivefold after at least ten hours of incubation. Dialysis of activated preparations first against Tris-saline pH 7.3 then against Tris-saline containing 0.001 M edetic acid (EDTA) led to removal of the added ions and to continued high activity indicating a removal of a pressor inhibitor rather than direct activation by the heavy metals. Dialysis of a fresh kidney homogenate supernatant against 0.001M cadmium chloride for five days and subsequent extraction of the resultant precipitate and diffusate with 2: 1 chloroform-methanol led to no diminution of pressor activity when the extracted material was added to separate samples of unactivated homogenate supernatant. The latter finding suggested that the postulated inhibitor is either made strongly unavailable for reuse or is degraded on removal.