Knockout Saco Pancada Pop 90
Saco de pancada pop 90 knockout 9 de agosto de 2016. Saco de pancada pop 50 knockout 9 de agosto de 2016. Mostrar todos. Publicado por mariussports em 9 de agosto de 2016. Lutas; Data 9 de agosto de 2016; Relacionados. 9 de agosto de 2016. Saco de pancada pop 120 knockout.
The one and only – ExpertBoxing’s complete list of punching combinations for boxers. This is just a list of combinations only.It’s not about what punches you throw, it’s how you throw the punches. A basic 1-2 punch combination becomes very deadly when you mix in some variations differing in speed, angle, footwork, timing, and power.If you need further explanation as to why and how you throw these combinations, please see my other articles on.For counter-punching routines, please click here.Mix & Match and Enjoy! (Last Updated 2/8/12). This pretty much irrelevent to the article but, here it goes. I’ve been boxing since I was 16, I had one fight, lost by decision.
I am 23 now, I have had multiple trainers since I began, although I have only had one fight, I want to compete again. Only this time I feel SO ready, afterall, I have been training for 7 years. My question is: do you think age is a limiting factor? Or do you feel that your age has nothing to do with competition, but rather the way you feel and what mental/physical condition you are in, is of utmost importance? I love your website btw thanks for all the great advice! Please email me back at your convenience. Johnny N,Love your site, I started Boxing Workouts in Nov 2012, I have follow alot of your teachings, how to’s & tips and I have progress greatly over a short period of time.
I totally got the Boxing Bug, My Wife supports me fully and we have even opened a boxing club & gym. We are setting up Coachs and programs to be in full swing very soon. Your site is wonderful and I have shared it with everyone I’ve worked with and plan to continue using it as both myself and our new Boxing Club grow.Thanks So Much,E.B.
McLlarky “C-4”Conway Boxing ClubNew Hampshire. Wow, what a great site. I am 51 years old and just started to fulfill my dream of learning how to box. I hope I did not start to late. I am in excellent physical shape and am a health fanatic. I already have been jumping rope religiously, and hoping this helps. On my off day of private lessons, I practice at home.
Can you give me some ideas what to practice, or should I just reinforce what I am learning in the gym? I have at home a speed bag, heavy bag, double end bag and a reflex bag. My trainer said do not be too concerned with the speed bag as yet.I want to learn boxing techniques at my lessons rather than the physical aspect of the sport ( I already train hard on my own time in a gym). My other question is I am going to buy another heavy bag to supplement the heavy bag I already havethinking of a wrecking ball or an angle bagwhat do you suggest?
Thank you so much. Hey Johnny,Great info on the combos. Few questions. The first one is I have all of the bags speed, heavy, double end, and slip bag. I’m 35 this year and really don’t have any desire to compete and am to old anyway.
I don’t mind a little sparring though many claim they will do some sparring and then back out and I have a tiny bit of sparring experience. I guess some people are just afraid to get hit lol.Anyway. I have a bad shoulder so heavy bag impacts don’t feel to good when it comes to that. I also don’t want to hang the bag up in our new house because it shakes the whole thing and will wake our baby up, so that one is out for me.
The speed bag has a platform I had built out of 3 pieces of plywood glued and screwed together, but is still loud. I guess those 900.00 platforms are the ones that make the least noise huh? The double end bag seems to be ok, but my question on that is everyone likes a different way that it is strung up.
I have mine set to where it will hit me in the face if I don’t get out of the way even at my own range, but every pro boxer I see has it set like super tight to where there is very little movement on it.Shadow boxing really is the only thing I have right now ( I use 2 lb dumbells as well) and just work common combinations. I am doing it for fitness, but to build speed and honestly for self defense. Is it enough to help with power as well as speed, reflexes, etc?My footwork is what isn’t very good though. Is there a video, or particular fight that shows pretty common footwork, but is pretty basic? There are literally NO boxing gyms around here so I have to try to learn the footwork all on my own.Any overall advice?Sorry for the long string of questions. JohnnyI am a 55 year old martial artist who has been studying traditional martial arts for over 40 years.
Your website offers great advice and I wish I had discovered it sooner. Despite my years of training, my fighting style is very static because it is the result of too much basic training in static positions and not enough fluidity. Inspired by your advice I am favouring SLOW shadow boxing and pad work combinations in lieu of my tradional karate training.There is life in the old dog yet!Rushman1959. Hello JohnnyThankyou for this great information I have always liked boxing and enjoy training at it.I see in your discussions that you recommend learning boxing until it is ‘automatic’ for us.
I have a question then about the list of combinations above.Question: What is the minimum combination list to know ‘automatically’ before taking one’s first Amatuer fight? You have said a lot about amateur preparation but I can’t find a minimum list specified anywhere.The reason I ask is some coaching manuals recommend only basic 3 and 4 combination lists for the first fight, as they reckon defences, fitness and endurance are key.What do you think is best and the minimum combo list to drill and ‘ work up’?RegardsMarc.
There is no minimum or maximum. You learn as much as you want or need. Some areas are more competitive than others.
Your question is a bit like asking me, “How prepared do you have to be to get into the college of your choice?” The answer isit depends.My best advice for you is to train in a gym that has other amateur fighters and to see what they do. That will give you a good luck of how good you have to be in order to compete. As for coaching manuals, the majority of them are quite run down and don’t explain the many details you need.
ThankyouYes O.K, but Johnny you post the information not myself. Now, to explain my question.It is well known that in other M.A, to fight the open senior tournaments people first achieve black belt status, 3 to 4 years of constant training, as you know. One can start fighting earlier but they can only fight at their belt level.
Black belt requirements are pretty-well universal.This is why I questioned your list and minimum requirements for the boxing amateurs. I agree it would change from area to area and also through the decades, but generally the amateurs are keenly contested and usually have people who are from ‘not bad’ to ‘very good’. Manuals.Personally I reckon I could specify a list of minimum requirements to learn ‘on automatic’ before recommending anyone for the ring, as per the black belt requirement in M.A, and I reckon so could you.So it is not a bad question really.RegardsMarc. It’s hard for me to compare in the context of other sports because boxing competition is really quite different. There is no minimum requirement. You could be a day one beginner and enter a fight.
It’s possible to find a 5-year boxer doing his first fight just as it is a 5-week boxer doing his first fight. Quite often, these two can even be found facing each other in the same match.If you wanted a sports comparison, it would be something like entering a marathon.
There is no minimum skill or requirement to enter a marathon. You could be one of those doing-it-just-for-fun guys or one of those serious I-want-to-win guys.As I understand, your original question to me was what was the minimum list of combinations to know before entering a fight. And that’s like me saying, you should know a minimum number of running techniques or breathing techniques before entering a marathon.My answer to you remains just as I have first stated it. Look around and see how good the guys are and make sure you are at a comparable level, otherwise you can be very seriously hurt. Some areas have relatively inexperienced competitors and everybody appears untrained. Other areas can be full of ringers and dangerous competitors who look as good as the pros. For me to give you some kind of universal combination list to pass on to beginners would be the most irresponsible thing I could do as a boxing teacher.
Boxing is quite unforgiving as a sport and quality truly matters more than quantity. Besidesthe ability to memorize punching combinations doesn’t correlate well to overall fighting ability.
Comments.Hey Jhonny! Nice article! By the way have you ever tried or seen 'Fortis' boxing glove? They look cheap to. By Carlos.Any thoughts on Fight2Finish?I've been seeing their stuff at Los Angeles Pro Boxing Supplies stores.
By MR.My theory is, people who have sex regular are the people who succeed. The reason behind this theory is as.
By mal.Great post by Kes.Inexperienced fighters will often play macho rather than play smart. When hurt by a punch rather than taking an.
By the ghost.great article Johnny! By Sam.Great stuff! Good training guide for beginners.
I am a veteran boxer in my late 40s and I can confirm. By Gebbie Redman.Hey Johnny I use weights. I never shadow box with them.
What I do is I'll spend a few minutes.
Previously we found thymosin β4 (Tβ4) is up-regulated in glomerulosclerosis and required for angiotensin II-induced expression of plasminogen activator inhibitor-1 (PAI-1) in glomerular endothelial cells. Tβ4 has beneficial effects in dermal and corneal wound healing and heart disease yet its effects in kidney disease are unknown. Here we studied renal fibrosis in wild type and PAI-1 knockout mice following unilateral ureteral obstruction to explore the impact of Tβ4 and its prolyl oligopeptidase tetrapeptide degradation product, Ac-SDKP, in renal fibrosis. Additionally, we explored interactions of Tβ4 with PAI-1. Treatment with Ac-SDKP significantly decreased fibrosis in both wild type and PAI-1 knockout mice, as observed by decreased collagen and fibronectin deposition, fewer myofibroblasts and macrophages, and suppressed pro-fibrotic factors.
In contrast, Tβ4 plus a prolyl oligopeptidase inhibitor significantly increased fibrosis in wild type mice. Tβ4 alone also promoted repair and reduced late fibrosis in wild type mice. Importantly, both pro-fibrotic effects of Tβ4 plus the prolyl oligopeptidase inhibitor, and late reparative effects of Tβ4 alone, were absent in PAI-1 knockout mice. Thus, Tβ4 combined with prolyl oligopeptidase inhibition, is consistently pro-fibrotic, but by itself, has anti-fibrotic effects in late stage fibrosis, while Ac-SDKP has consistent anti-fibrotic effects in both early and late stages of kidney injury.
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These effects of Tβ4 are dependent on PAI-1. IntroductionThymosin β4 is a highly conserved G-actin sequestering protein that has a wide range of extracellular functions, including cell migration, angiogenesis, and extracellular matrix (ECM) synthesis, some of which might be receptor-mediated –.
Thymosin β4 is degraded by a two-step process to the anti-fibrotic tetrapeptide Ac-SDKP, with the initial step mediated by unknown protease(s) and then by prolyl oligopeptidase (POP). Moreover, POP can regulate the activity of the first step enzyme and thus auto-regulate the release of Ac-SDKP. Ac-SDKP is further inactivated by angiotensin-converting enzyme (ACE).
The beneficial effects of ACE inhibitors may, at least partially, relate to increased Ac-SDKP levels –.Thymosin β4 accelerates dermal wound healing with increased cell migration, tissue remodeling and accelerated collagen deposition, –. In contrast, thymosin β4 is anti-fibrotic in cultured human hepatic stellate cells,. Thymosin β4 induces adult epicardium progenitor mobilization and neovascularization and phase II clinical trials to test for improved cardiac function after myocardial infarction (MI) are planned –.Whether thymosin β4 has beneficial or fibrotic effects in the kidney has not been established. Previously, we observed that thymosin β4 is increased in early glomerulosclerosis, and is required for angiotensin II (AII)-induced plasminogen activator inhibitor-1 (PAI-1) expression in glomerular endothelial cells. PAI-1 inhibits matrix degradation and also alters cell migration,. Accumulation of matrix and inflammation versus preservation of parenchymal cells may differentially affect repair versus fibrosis at early and late stages after injury. We investigated the effects of thymosin β4 and Ac-SDKP on renal interstitial fibrosis induced by the unilateral ureteral obstruction (UUO) model.
Complete UUO starts a rapid sequence of events in the obstructed kidney, including reduced renal blood flow and glomerular filtration rate within 24 hours. This is followed by interstitial inflammation, tubular dilation, tubular atrophy and fibrosis within several days. The obstructed kidney progresses to a severely hydronephrotic kidney with marked loss of renal parenchyma and reaches end stage over 1- 2 weeks. Therefore, we chose two time points, day 5 and day 14 after UUO, to assess early and late repair vs.
Injury, as previously reported,. Our results demonstrate fibrosis is increased by thymosin β4 plus POP inhibitor at both early and late stages, while both thymosin β4 and Ac-SDKP ameliorate late matrix accumulation. The effects of thymosin β4 to enhance fibrosis when POP is inhibited, and to promote repair when given alone, are both PAI-1 dependent. These data suggest that thymosin β4 and in particular its degradation product Ac-SDKP are anti-fibrotic in the kidney. Thymosin β4 has divergent effects in early vs. Late stage injury, while exogenous Ac-SDKP consistently promotes repairAt day 5, tubular dilatation, tubular atrophy, and interstitial collagen accumulation were present in obstructed kidneys in WT mice. Tubulointerstitial fibrosis assessed by Sirius red morphometry was significantly increased by 17% in obstructed kidneys treated with thymosin β4 plus POP inhibitor vs.
Untreated UUO. Neither thymosin β4 alone nor POP inhibitor alone significantly affected early fibrosis, although thymosin β4 numerically decreased this parameter. In contrast, fibrosis was significantly less by 16% in obstructed kidneys treated with Ac-SDKP vs.
Untreated UUO. Neither thymosin β4 nor Ac-SDKP administration affected POP activity.
POP activity was decreased in mice receiving POP inhibitor, and consequently, Ac-SDKP levels were decreased. However, treatment with thymosin β4 alone did not change Ac-SDKP levels. Early tubulointerstitial fibrosis in wild type and PAI-1−/− mice. (A) Tubulointerstitial fibrosis in wild type mice at day 5 after UUO was significantly increased by thymosin β4 plus POP inhibitor (Tβ4+POPi) but was significantly decreased by Ac-SDKP (Control, n=8; POPi, n=6; Tβ4+POPi, n=6; Tβ4 n=6; Ac-SDKP, n=7;.p. Late tubulointerstitial fibrosis in wild type and PAI-1−/− mice.
(A) Tubulointerstitial fibrosis in wild type mice at day 14 after UUO was significantly increased by thymosin β4 plus POP inhibitor (Tβ4+POPi) but was significantly decreased by thymosin β4 alone or Ac-SDKP (Control, n=10; Tβ4+POPi, n=7; Tβ4 n=5; Ac-SDKP, n=6;.p. Thymosin β4 mRNAPOP ActivityAc-SDKPThymosin β4/GAPDH(pmol/min.mg tissue)(pmol/mg tissue)TreatmentObsCLObsCLObsCLControl10.81±0.891.29±0.27 †27.60±2.2640.10±2.97 †1.40±0.192.41±0.22 †POPi10.97±1.331.34±0.24 †2.06±0.26.12.68±1.30., †0.66±0.08.1.34±0.12., †Tβ4+POPi9.14±0.681.27±0.26 †5.73±1.59.18.35±1.42., †0.86±0.09.1.48±0.26., †Tβ413.76±2.880.86±0.22 †21.18±1.6146.88±3.51 †1.09±0.121.94±0.19 †Ac-SDKP6.49±0.61.0.57±0.03., †31.88±1.1825.40±2.63., †2.41±0.08.4.06±0.34., †Normal0.73±0.15 #31.39±3.20 ††2.13±0.31 #. Anti-fibrotic effects of Ac-SDKP are linked to decreased fibronectin, macrophages, myofibroblasts, and profibrotic factorsAt day 5 in WT mice, fibronectin was significantly increased by 35% in obstructed kidneys treated with thymosin β4 plus POP inhibitor vs. Untreated UUO, but decreased by 40% in response to Ac-SDKP (, ). Since monocyte/macrophage infiltration occurs early after UUO, we evaluated F4/80 positive macrophages. At day 5, F4/80 positive macrophages were significantly decreased by 52% in obstructed kidneys treated with Ac-SDKP vs.
Untreated UUO (, ). Further, α-SMA-positive myofibroblasts, a key contributor to extracellular matrix, were also significantly decreased by 65% at day 5 in response to Ac-SDKP. In contrast, thymosin β4 plus POP inhibitor tended to increase α-SMA- positive cells (, ). Thymosin β4 whole kidney protein was similar in all day 5 obstructed groups regardless of treatment. Thymosin β4 mRNA at day 5 in obstructed kidneys was significantly reduced by 41% in response to Ac-SDKP, but not by other interventions.
PAI-1 whole kidney protein, a key profibrotic factor, was significantly decreased by 52% in response to Ac-SDKP treatment in obstructed kidneys at day 5. Phosphorylated Smad3, a marker of TGF-β1 signaling activation, was not detected by either immunostaining or Western blot in obstructed kidneys at day 5 (data not shown). These data show that various indicators of early fibrosis are reduced at day 5 by Ac-SDKP.
Fibronectin accumulation, monocyte/macrophage infiltration and myofibroblast expression at early stage of fibrosis at day 5 after UUO in wild type mice. (A) Fibronectin (FN), one of the major extracellular matrices, was significantly increased by thymosin β4 plus POP inhibitor (Tβ4+POPi) but was significantly decreased by Ac-SDKP. Neither thymosin β4 (Tβ4) nor POP inhibitor (POPi) changed fibronectin accumulation. (B) F4/80 positive infiltrating cells were significantly decreased by Ac-SDKP, but not by other treatments.
(C) α-SMA-positive myofibroblasts, a key contributor to extracellular matrix, tended to be increased by thymosin β4 plus POP inhibitor (Tβ4+POPi), and were significantly decreased by Ac-SDKP (×200, immunostaining as indicated). Both profibrotic and reparative effects of thymosin β4 in fibrosis are PAI-1 dependentPAI-1 is increased in fibrotic kidney diseases, and may contribute to scarring both by decreasing extracellular matrix proteolysis and affecting infiltrating cells,. We therefore assessed impact of PAI-1 deficiency on effects of thymosin β4 and Ac-SDKP in both early and late fibrosis. At both stages (day 5 and 14 after UUO), tubulointerstitial fibrosis was similarly increased in PAI-1−/− vs.
WT compared to non-obstructed kidneys ( and ). Ac-SDKP significantly decreased fibrosis in both WT and PAI-1−/− mice at early and late stages.
However, at both early and late stages, PAI-1−/− mice had no further increase in fibrosis with thymosin β4 plus POP inhibitor treatment, in contrast to WT ( and ). Thymosin β4 alone did not affect early fibrosis in PAI-1−/− mice, as observed in WT mice. However, late stage fibrosis was not decreased by thymosin β4 alone in PAI-1−/− mice, in contrast to less fibrosis in WT treated with thymosin β4 ( and ).We next examined POP enzyme activity and Ac-SDKP levels in the obstructed kidneys. POP activity was decreased at day 5 and day 14 in both WT and PAI-1−/− mice in response to the combination treatment compared to untreated UUO. Neither Ac-SDKP nor thymosin β4 changed POP enzyme activity in either WT or PAI-1−/− mice. Ac-SDKP kidney levels were similarly reduced by the combination treatment and increased by exogenous Ac-SDKP in both WT and PAI-1−/− mice. At day 14, in WT, but not in PAI-1−/− mice, treatment with thymosin β4 alone increased Ac-SDKP levels vs.
Untreated UUO, but not as much as with exogenous Ac-SDKP treatment.Early injury markers at day 5, i.e. Fibronectin, macrophages and myofibroblasts, were decreased in PAI-1−/− mice treated with Ac-SDKP.
Knockout Saco Pancada Pop 90s
Ac-SDKP treatment also significantly decreased macrophages at late stage day 14 in both WT and PAI-1−/− mice. Thymosin β4 and phosphorylated Smad 3 immunostaining expressions were similar in WT and PAI-1−/− mice at day 14 in obstructed kidneys in all groups (data not shown). Neither thymosin β4 alone nor combination treatment affected macrophage number in WT or PAI-1−/− mice. Further, at day 14, Ac-SDKP treatment tended to reduce collagen I and phosphorylated Smad 3 protein in PAI-1−/− mice. DiscussionIn the current study, we examined the roles of thymosin β4 and Ac-SDKP in modulating early versus late tubulointerstitial fibrosis and investigated possible mechanisms. Ac-SDKP has protective effects in hypertensive kidney disease models of glomerulosclerosis –.
Endogenous thymosin β4 is up-regulated in the kidney ischemia-reperfusion model, but the role of exogenous thymosin β4 in the kidney has not been defined. We now demonstrate that thymosin β4 is up-regulated in kidneys after obstructive injury, along with increased expression in macrophages, fibroblasts, and occasional tubular cells. We also show that exogenous administration of thymosin β4 plus POP inhibitor, which prevents metabolism of thymosin β4 to Ac-SDKP, exacerbated both early and late interstitial fibrosis in WT mice.
In contrast, Ac-SDKP treatment decreased both early and late fibrosis, with less total collagen and fibronectin deposition, decreased myofibroblasts and monocyte/macrophages, and suppressed profibrotic factors, PAI-1 and TGF-β1. Late phase fibrosis was also decreased by thymosin β4 alone in WT mice. These effects of thymosin β4, namely to enhance fibrosis when POP is inhibited, and to promote repair when given alone, were both PAI-1 dependent. Taken together, these data indicate that thymosin β4 has divergent effects on early vs.
Saco De Pancada Knockout Pop 90
Late stages of injury, while Ac-SDKP promotes repair and decreases fibrosis at both early and late stages. Whether higher amounts of thymosin β4 could have similar effects on early fibrosis awaits further study.In dermal wound healing, thymosin β4-accelerated collagen/matrix deposition is beneficial. In kidneys, excess extracellular matrix accumulation is usually detrimental. However, accumulation of matrix and macrophages may be a provisional wound healing-like response, which may ultimately promote resolution of scarring to allow less parenchymal loss. We therefore further investigated whether effects of thymosin β4 differ in early vs.
Late stages of renal fibrosis. When thymosin β4 metabolism to Ac-SDKP was inhibited, both early and late tubulointerstitial fibrosis increased with more total collagen and fibronectin accumulation, but no change in macrophages.
We recognize that POP has numerous targets in addition to thymosin β4. Importantly, POP inhibitor by itself did not enhance early fibrosis, supporting that the effects of the combination of thymosin β4 and POP inhibitor are attributed to specific thymosin β4 activity. In contrast, Ac-SDKP treatment ameliorated both stages of fibrosis with less collagen and fibronectin accumulation. We also showed that short-term treatment with thymosin β4 with or without POP inhibitor, or Ac-SDKP, had minimal effects in the non-injured contralateral kidneys.We next investigated the possible mechanisms underlying effects of thymosin β4 and Ac-SDKP in renal fibrosis.
Myofibroblasts, one of the major contributors of interstitial extracellular matrix, may arise from different sources, including quiescent fibroblasts or pericytes –. We found that α-SMA expression, a marker of activated myofibroblasts, tended to be increased by the thymosin β4 and POP inhibitor combination, but was significantly decreased by Ac-SDKP. Macrophages have numerous functions, including removal of debris in injury, progression of fibrosis, and repair, and can be phenotypically switched in different environments. Macrophage infiltration was significantly decreased by Ac-SDKP treatment, but not affected by exogenous thymosin β4 with or without POP inhibitor at early or late stage of injury. Future investigation to determine the phenotypes, functions, and responses of these macrophages will be of interest.Effects on two key profibrotic factors, PAI-1 and TGF-β1, likely contributed to the changes in fibrosis.
At late stage in WT mice, treatment with thymosin β4 plus POP inhibitor resulted in significant upregulation of these molecules with increased fibrosis. In contrast, these factors were decreased with corresponding reduced fibrosis in response to Ac-SDKP or thymosin β4 alone. PAI-1 has effects both on proteolysis and cell migration, processes that are crucial for response to injury,. Ultimate effects on fibrosis are determined by a balance of matrix modulation and repair, and are likely time- and context-dependent.
We have previously shown that thymosin β4 was required for angiotensin II-induced PAI-1 expression in cultured glomerular endothelial cells. Thymosin β4 also increases PAI-1 expression in human umbilical vein endothelial cells,. We therefore examined PAI-1 dependence of thymosin β4 effects. Tubulointerstitial fibrosis was similar in PAI-1−/− and WT mice after 5 days and 14 days of UUO, and Ac-SDKP treatment decreased this fibrosis similarly in PAI-1−/− and WT mice.
However, in contrast to the increased early and late fibrosis in WT induced by exogenous thymosin β4 plus POP inhibitor combination, PAI-1−/− mice showed no increase in fibrosis with this intervention. Further, the decreased late fibrosis in response to thymosin β4 alone was present only in WT, but not in PAI-1−/− mice. These data show that both the pro-fibrotic effect of thymosin β4 combined with POP inhibitor at either early or late stage of fibrosis, and the reparative effects of thymosin β4 alone in late stage injury, were PAI-1 dependent. Of note, increased Ac-SDKP levels in thymosin β4-treated mice did not account for this protective effect. Thus, Ac-SDKP levels in thymosin β4-treated WT mice were numerically even lower than that in Ac-SDKP treated mice, although late fibrosis was even less with treatment with thymosin β4 alone than with Ac-SDKP treatment. Thymosin β4 induces vascular endothelial growth factor (VEGF) expression by an increase in the stability of hypoxia inducible factor (HIF)-1α protein,.
We postulate progressive fibrosis could result in increased hypoxia in the interstitium, and thymosin β4 beneficial effects could be contributed to by preserving peritubular capillaries. Whether thymosin β4 also affects macrophage phenotypic switch toward tissue repair and regeneration awaits further study. The mechanisms of beneficial effects of thymosin β4 in late kidney injury point to a novel reparative pathway for progressive kidney disease.In summary, our study shows that Ac-SDKP has anti-fibrotic effects in both early and late stages of renal fibrosis induced by UUO.
Thymosin β4 alone has context- and time-dependent effects on renal fibrosis and promotes PAI-1-dependent repair of late fibrosis. Experimental ProtocolMice (25∼30 g; Jackson, Indianapolis, IN), age 8 to 10 weeks, underwent UUO under sterile conditions as described previously. Mice were then randomized to groups as follows (n=6–8 in each group): untreated UUO control, UUO treated with prolyl oligopeptidase (POP) inhibitor (S17092, 40 mg/kg/d, mixed with peanut oil and administered by gavage), UUO with thymosin β4 (150 µg/d, i.p.), UUO with combination (thymosin β4 plus POP inhibitor), and UUO treated with Ac-SDKP (Calbiochem, Gibbstown, NJ, 1.6 mg/kg/d, delivered by micro-osmotic pump) (Alzet, model 1007D, DURECT Corp., Cupertino, CA). The doses and duration of POP inhibitor, thymosin β4 and Ac-SDKP were chosen based on previous reports and pilot studies,.
S17092 and thymosin β4 were kindly provided by Institut de Recherches Internationales Servier, France and RegeneRx Biopharmaceuticals, Inc. (Rockville, MD), respectively. Five or 14 days after UUO surgery, mice were sacrificed and kidneys were harvested for morphological, immunohistochemical, biochemical and molecular analyses. Morphology and ImmunohistochemistryQualitative tubulointerstitial injury was examined by Masson’s trichrome staining.
For quantitative assessment of fibrosis, 3-µm sections of tissues were stained with picrosirius red (0.1% Sirius red in saturated picric acid) for 16 h, followed by two changes of 0.5% acetic acid, dehydrated in three changes of 100% ethanol, and coverslip mounting. Sections were examined by polarized light microscopy, as reported by other investigators. For each kidney, 12 consecutive, non-overlapping cortical × 40 fields were captured by an Olympus BX-41 microscope equipped with a digital camera, and morphometric analysis performed using NIH image (version 1.63). Arteries and periadventitial areas were excluded. Results are expressed as percentage fibrosis of total tubulointerstitial area.Kidney sections were stained for fibronectin (1:400, Rockland Immunochemicals, Inc., Gilbertsville, PA), α-smooth muscle actin (α-SMA) (1:100, DAKO Corp., Carpinteria, CA), F4/80 (1:800, AbD Serotec, Raleigh, NC), thymosin β4 (1:300, Meridian Life Science, Inc., Saco, ME), phosphorylated Smad3 (1:100, Rockland Immunochemicals, Inc, Gilbertsville, PA) with Vectastain ABC kit (Vector Laboratories, Inc., Burlingame, CA) with diaminobenzidine as a chromogen. For assessment of fibronectin, α-SMA, and F4/80, 12 consecutive, non-overlapping cortical x40 fields were captured with digital images and the ratio of positively stained areas was analyzed using NIH image and expressed as percentages of total cortical area,.
Arteries were excluded for α-SMA staining analysis. All morphologic and morphometric assessments were examined without knowledge of the treatment protocol. Tissue Preparation and POP Activity AssayPOP activity was assayed using the fluorogenic substrate, Suc-Gly-Pro-7-amino-4-methylcourmarin (Z-Gly-Pro-AMC, Bachem, Torrance, CA) as previously described. In brief, frozen kidney samples (10mg) were homogenized in 90 µl assay buffer (0.1 M Na-K-phosphate buffer, pH 7.0), centrifuged at 13,000g at 4°C for 20 mins and the supernatant was stored at −80°C until determination of enzyme activity. Kidney homogenate supernatant (2 µl) was preincubated with 93 µl assay buffer at 30°C for 30 mins.
The reaction was initiated by adding 5 µl of substrate (200µM Z-Gly-Pro-AMC) with 60 mins incubation at 30°C. The reaction was stopped by adding 100 µl 1 M sodium acetate buffer (pH 4.2). Formation of the AMC product was measured fluorometrically (excitation 360 nm and emission 460 nm). Results are expressed as picomole AMC per minute per milligram of total protein.