Kidney - Science topic

Hemodynamic factors in acute renal failure (ARF). Renal blood flow is not luxurious, it results from the metabolic demands of renal medulla and cortex. Within both areas, effective shuntdiffusion for blood gases is present. In the outer medulla, the oxygen supply of the thick ascending limb (TAL)-segments especially those of outer cortical nephrons is limited and tubular demands are regulated by the tubuloglomerular feedback. Thus inflowing tubular fluid into Henle´s loop can be adapted to the oxygen supply of the TAL-segment and its proximal tubule (PCT). This adaption works oscillating, experimentally in the rat with a frequency of

30 mHz (2/min) and can be measured by oscillating of proximal tubular pressures, with a phase lag of distal sodium concentration and by oscillating oxygen pressures measured at the renal surface. A change between aerobic supply to glycolysis at the TAL-segment led via a changed fluid sodium concentration at the macula dense to an adjustment of oxygen consumption considering also the fact, that proximal tubules practically have no anaerobic metabolic reserves. If under ischemic and hypoxic conditions reduction of transport capacity is inadequate, deterioration of proximal tubuli and of TAL-segments may be programmed as the kidney´s answer: acute renal failure (ARF).

Abstract in: Hämodynamische Faktoren beim akuten Nierenversagen (ANV)

H. J. Schurek und O. Johns

Nieren- und Hochdruckkrankheiten 20, 2/1991, p 56-60

I will and it will give you more informations about the disease....

Dear Amal ... we are following an established protocol thta worked perfectly fine in our hand for primary tubular cells (https://www.hindawi.com/journals/omcl/2008/471575/).

I am not sure what kind of medium you used to grow these cells, but I would suggest adding epidermal growth factor to help the tubular cells to attach and grow. You may also try coating your plates with 1% Collagen-1 to help the attachment of the cells.

The image is not clear, but it is normal to have cell debris with the isolated cells, but most importantly you should notice cell proliferation by the second or third day of culture. The protocol provided is detailed and contains the supplements that should be added to the culture medium (we use DMEM/F12). I hope this answer is somehow helpful.

Sébastien Soubeyrand I also use pjun abs from Cell Signaling. This time, I didn't include a phosphatase inhibitor, so that might be the reason. Following your advice, I'm going to try Westernblot again. Thank you for your answer.

Looks you want RNA from outer layer of kidney. Is it not possible for you to do microdissection under microscope and dissect the ourer layer. I will also recommend to do microdissection when whole kidney is under RNALATER solution, that will protect from RNAases

There are multiple different ways to define AKI. A generally accepted definition is that put forth by the Kidney Disease Improving Global Outcomes group. They define AKI in 2 ways - by change in function (serum creatinine) and/or by urine output. This is well described by the Kidney Disease Improving Global Outcomes group. The KDIGO guidelines stage AKI by severity which is predictive of important kidney outcomes AKI can also be defined categorically by where the injury is happening relative to the kidney (pre-renal, renal, post-renal). Defining in this way can be helpful in terms of differential diagnosis/identifying cause(s) of AKI. A more practical, clinical approach to AKI is that of functional vs tissue level injury. Similar ways of thinking of this could be mild vs severe, quickly reversible vs prolonged or irreversible, early vs late, etc. The mainstay of AKI management is determination of whether or not urine is being made at a reasonable amount. If anuria/oligoanuria is observed, more than likely renal replacement therapy will be indicated. Diuretic stress test using loop diuretics can help to determine if oliguria/anuria present. If a response is observed with high dose loop diuretics, the injury is likely less severe and recovery without RRT may occur. AKI management involves determine cause(s), assessing severity, managing possible trouble to occur from low kidney function (hyperkalemia, pulmonary edema) as well as preventing further injury. Optimizing hemodynamics, avoiding nephrotoxins, and renally dosing medications is critical. These patients should be evaluated by nephrologists in the hospital and followed up in the ambulatory setting. AKI is an important clinical event portending significant kidney, cardiovascular and mortality outcomes. Ongoing efforts to prevent and diagnose AKI earlier are being made to reduce the burden it this common serious entity.

Erin Denny I believe that if the neuro-retinal rim concept was correct, then I should have found an optic disc histology depicting the ‘donut’ appearance. I still have not come across any such image in the textbooks during my 60 years in ophthalmology. Thank you for the helpful information. I appreciate your kind response.

If you have not done it, the first thing you need to do is work with known samples. We never test a new technique on valuable samples. Once you are familiar and comfortable with the new technique, and know you can reliably get data, then go on to process the precious samples.

It seems you are comfortable with the SP3 protocol, so it makes sense the problem was earlier on in the process. All I can suggest is what you probably already know, go back and try again with "test" samples.

I have not used the Covaris reagents, but we routinely process FFPE samples and deparaffinisation is a standard protocol that works well in our hands. The only thing to watch out for is that all the wax is removed. Some tissue sections have a huge amount of wax and if it's not all removed it does cause problems further down the line.

Good luck

What is the turnover of your protein in vivo?

Very uncommon in my opinion except if the cow is in poor body condition (or maybe a small breed).

Péter Degrell

Thanks for your response.

It could be done if you "calibrate" strain gauge measurement using known force applied on the hand rim, and then you could measure real applied force during the movement.

Also, you should have measurement of the rotational velocity, or RPM of the wheel.

Upper gel:

In samples 3 & 4 there is obviously no or very little DNA.

In my opinion all other DNAs are not properly dissolved and the gel is overloaded; and you might have RNA contamination.

Ensure your DNAs are completely dissolved and as suggested by others determine the concentration and reload equal amounts.

And, as also suggested by others, you should always seek advice from your supervisor or a senior lab member.

Edema is a functional condition and it is not easy, to capture this condition in the histological preparation. The question is how to freeze this functional state; certainly not with the usual histological techniques. In the case of the kidneys, normothermic perfusion fixation with glutaraldehyde, a colloid-osmotically adequate additive such as hydroxyethyl starch, is recommended.

HJ Schurek, W Kriz: Morphologic and functional evidence for oxygen deficiency in the isolated perfused rat kidney. Laboratory Investigation 53: 145-155, 1985

You probably can try some chemical reagents such as detergents, chaotropic agents, or solvents, or using protease enzymes, and possibly combine these with mechanical methods such as homogenizer: https://lab.plygenind.com/how-to-homogenize-animal-tissue-for-dna-extraction-a-practical-guide

Dear colleague,

Sorry for the late answer, I don't connect here frequently.

If I look at 177Lu S values from OLINDA V1, for UB content to UB wall:

- 5 YO: 1.85 10-4 mGY/MBq.s

- 10 YO: 1.17 10-4 mGY/MBq.s

This is normal, as the model is smaller, since most of the irradiation is from non penetrating radiations, therefore the same energy is absorbed in a smaller volume, and therefore the absorbed dose increases.

This is a general trend, probably less pronounced for hollow organs but this remains to be seen.

Then obvioulsy the cumulated activity should taken into account, as theS value basically considers the same number of decay in the 2 models, which may not be true...

Best regards,

Manuel

Hello Luminaa Anandh

You will have to use a non-denaturing lysis buffer since you will be assessing the activity of an enzyme. The non-denaturing buffer will contain mild non-ionic detergent and a combination of various salts and agents to enhance extraction and stability of proteins. Non-ionic detergent like NP-40 or Triton X-100 are surfactants, basically soaps, that disrupt the fatty acid bilayer. NP-40 can be used to solubilize proteins but not denature them. The non-denaturing lysis buffer should also contain low salt concentration, usually 120 mM NaCl. Some salt is needed to prevent non-specific protein interactions. EDTA which is a common additive that has multiple functions including protease inhibition and protection against oxidative damage may be added to a final concentration of 5mM. Tris is another additive used to buffer the pH and prevent protein denaturation. It is always better to add protease inhibitors in the lysis buffer prior to use in order to protect proteins.

You may use the following recipe for preparing non-denaturing lysis buffer.

NP-40/Triton X-100 lysis buffer:

50 mM Tris HCl, pH 8.5,

120 mM NaCl,

5mM EDTA,

1% detergent (NP40 / Triton X-100).

However, the concentration of salt and detergent could be altered according to your needs. Also, please note that protease inhibitors should be added fresh to the lysis buffer at the time of lysis.

Hope this helps!

Good Luck!

Hello,

If the kit is not available you can use a colorimetric method. This method is reproducible.The rate of lipoperoxidation (MDA level) in the homogenat in our case we evaluate the MDA in gastric mucous membrane. the MDA content was assessed by using the thiobarbituric acid reactive substances (TBARS) test (Ohkawa et al., 1979). 100 μL of the gastric homogenate was added to 750 μL of 8.1% SDS, 750 μL of 20% acetic acid and 750 μL of 0.8% TBA. The mixture was incubated in a water bath (Memmert, WB22, Germany) at 95 °C for 1 h. After cooling, the reactants were supplemented with 500 μL distilled water, 2.5 mL n-butanol-pyridine (15:1), shaken vigorously for 1 min and centrifuged at 4000×g for 10 min. Individual values were extrapolated from a TEP (1,1,3,3- tetraethoxypropane) standard curve (mmol/mL) and the results expressed as nmoles (TBARS) per mg of protein (nmol TBARS/mg prot).

I remain at your disposal for any other information of malondialdehyde (MDA) content.

13 should be 'efferent' not 'afferent' as it takes oxygenated haemolymph away from the ctenidium.

All good points mentioned so far. I’m not sure I completely understand the question. I think there are perhaps two questions at hand: one about kidney blood flow and one about impacts of living with one kidney.

Kidney transplantation and Living kidney donation provide a nice model to describe both. In living kidney donation, an open or laparoscopic nephrectomy is performed. The renal arteries/veins are ligated in the donor and they are left with one kidney.

The recipient of a kidney transplant ends up with a surgically created anastomosis of the donor renal arteries and veins to the iliac system. This is typically performed on the right side but can be on the left. Either way you could have a donated left kidney with left renal arteries and veins connected to the recipients iliac system on the right. Point being laterally doesn’t really matter: it’s more about the patency of the vessels and surgical technique.

These are generalities/always exceptions to the rules/I’m not a transplant surgeon.

To summarize- laterality is less important in kidney blood flow compared to vessel/anastomosis characteristics.

In terms of living with one kidney, it depends. Like others have mentioned, if congenitally absent or atrophies early in life, more likely that glomerular hypertrophy and secondary FSGS develop.

For people who are living donors, typically their remaining kidney compensates without developing secondary FSGS and their kidney function can end up being around 75% of their pre-donation GFR.

In conclusion- 1) Kidney blood flow is typically fine after nephrectomy and as transplant shows laterality of vessels is less important. 2) How someone does with one kidney is complicated and depends on timing/circumstances, but in the best of cases (living donation), people live relatively normal lives with kidney function that can be 75% of their pre-donation kidney function.

Thank you so much Tahib Habshi and Marcos Divino Ferreira, information you both shared is very useful. I am going to try both the method and see how it works one for already dissected kidney (stored) and the another for upcoming experiments.

5 years since this was asked and more research (see a select few articles below) seem to be using urine pO2 as a solid metric of kidney function and health. Worth keeping an eye on.

The cells appeared look like early dendritic cells, they would not properly grow until you do not add IL-4 and GM-CSF in the media.

from mouth to kidney, the way is long, every components have time to be ingested in intestines, by the body or by the bacterials, in muscles, in liver...so that at the end some characteristics may miss. and in addition, some components are part of sheet, sorry.

all the best for this new year

fred

Thanks alot🙏

Hi Sreyasi Das . If you are able to threshold your image, you can "Set Measurements" to include Feret's diameter. When you "Analyze Particles", this parameter would be part of your results.

Recent article on Amyloidosis caused by Spike Protein

https://wmcresearch.substack.com/p/severe-covid-and-long-covid-as-rapid?

Paraffin embedded sections are more stable than the cryosections. for the preparation of the H&E I prefer to use Paraffin sections. It will give great results.

The cryosections can be better for the detection of specific antigens. That cant be detected by the Paraffin sections...

Hi Lucia Marinas del Rey, in my experience of IHC the usual culprit is generally the antibody, antigen retrieval, incubation periods, or antigen degradation due to over processing. Since the antibody you selected has some pretty good reviews, I would recommend trying out different antigen retrieval times and methods. Heat mediated antigen retrieval generally works the best in my experience, but enzymatic could be another option. Increasing the antibody incubation period may also help if you haven't tried that yet. If you can provide a bit more info on what is unsuccessful about your staining (e.g., no stain, non-specific staining, etc.) I may be able to help more. Don't give up, IHC is a pain!

Erythrocytes and hemoglobin from lysed erythrocytes exhibit strong autofluorescence, esp., in the green channel (Fluorescein and alike). If you're making thin sections, you might get away without perfusion.

Zarna Patel Hello Zarna, thank you for the recommendation! I will carry out lysis with ATL.

I have the same problem as Kyaw San Lin! can I ask if you tried extracting RNA from these frozen samples multiple times or only once? Did all the samples degrade because the RNAlater didn't penetrate into the cells? And what was your RNA extraction method?

Thanks Rodrigo, it was the idea I had. We made immunostaining for macrophagues but not PAS. We will try. Thank you

Atef Erasha

Thank you for your reply.

I'll try my best!

You can review papers and follow the the staining method preferred.

Hi

I think the eosinophilic material in Bowman's space is protein debris. I advise looking for signs of proteinuria such as hyaline casts. Indeed, occasionally within the renal tubules there appear to be small amounts of eosinophilic (proteinaceous) material...

TNF, CRP must be taken into account for anticancer activity

CT to detect size and place renal ston if a big size need to surgical intervention such as Lapraroscopic nephrolithotomy . if smal size can be treat with pharmalogic intervention depending on type of stons causes..Calcium stones. To help prevent calcium stones from forming, your doctor may prescribe a thiazide diuretic or a phosphate-containing preparation.

I labeled section.

I think

red: bladder

skyblue: spine

purple + blue arrow: prostate tumor

Is it right?...

IT IS DIFFICULT TO DISCRIMINATE FROM RADIOLOGICAL IMAGES. YOU CAN COMPARE PATIENTS WHO HAVE BIOPSY FOR ANY REASON.

Not very likely. But if there is systemic spread then may be possible.

Yes, the management of UTUC and bladder Ca depends on many patient-related factors, including age, grade, stage, multifocality, bilaterality or status of contralateral kidney function….

For high grade, high stage, AND unilateral UTUC, radical Nephroureterectomy is recommended.

For low grade, superficial and single tumor, solitary kidney or bilateral involvement,…nephron sparing approach including ureteric resection and reimplantation is preferable.

But in this index case, we know the status bladder Ca (high grade pT1) and that of ureteric tumor was not mentioned.

If high grade or stage tumor, Radical cystectomy with Nephroureterectomy can be an option. But we need to consider the mentioned factors to decide.

You could check in the qiagen webpage for RNA extraction protocols/kits. They usually divide the protocols depending on the tissue you're working with, so maybe you need a slighlty different protocol for your tissues.

Good luck!

Thank you Ahmed. The question was about posterior retroperitoneoscopic kidney tumour resection. I noticed there has been one in literature (back in 2006) for a tumour in the posterior surface of the kidney. I did use this procedure today for a tumour in the anterior surface of the kidney, went good.

I am also searching for the same kidney fibroblast cell line but I haven't any luck in it yet. Please help if anyone found already?

Thank you very much for your input!

Thanks sir for you contribution in diagnosis of the problem

Not a personal recommendation, but Bioss says their rabbit pAb uses a mouse SGLT1 immunogen and is suitable for IF. They also offer a guarantee. Might be worth taking a look: https://www.biossusa.com/products/bs-1128r

Were you able to solve this? I have the same concern.

Hello Dr Hassan H Kaabi.

we have done an extensive study on the relation between periodontal disease and many systemic diseases and we found that periodontal disease affects kidneys

Periodontitis had a significant direct effect, an indirect effect through diabetes, on the incidence of Chronic Kidney disease. Awareness about systemic morbidities from periodontitis should be realized

The American Academy of Periodontology (AAP)

has published a statement on kidney disease and tooth loss (periodontal disease)

Researchers Caution that Tooth Loss May Increase Risk of Chronic Kidney Disease in U.S. Adults

as for your questions about the selection of patients

dr Grubb Vanessa Grubbs, MD, an assistant professor and neprology specialist in the UC San Francisco's School of Medicine who is determined to advance this research as part of her commitment to preventing the chronic health problems associated with kidney disease.

you might want to contact he she can tell you how she selected patients

Dr Grubbsince 2013 has published that paper and i have attached it, it really shows you how she selected her patients and you might want to look at it even copy the method

the title of the paper is The association of periodontal disease with kidney

i also attached a 2021 paper on the topic, good luck in your study that is a very important topic

sincerely

Dr.K.A.Galil.Professor of Medicine and Professor of Dentistry DDS.,D.Oral & Maxillofacial Surgery ,PH.D,FAGD.,FADI.,Cert.Periodontist(Uof Michigan) (Royal College Dent Surg.Ont) Departments of Periodontics,Orthodontics and Clinical Anatomy Schulich School Of Medicine and Dentistry. University of Western Ontario, London,Ontario.

Please see this reference :

Doaa M Mokhtar (2020) The structural and ultrastructural organization of the cellular constituents of the trunk kidney of grass carp (Ctenopharyngodon idella). Microscopy research and technique.

Jang Hyoseok

You can try and run PCR with a positive control (bacterial culture) the next time you run PCR. Then you'll know what to expect.

Hi Lingyun, It is very likely that you are losing the surface epitope recognized by your CD4 as a consequence of the digestion protocol. If you are doing intracellular flow for cytokines or transcription factors, you can add CD4 to the intracellular cocktail of Abs. Alternatively, you can rest the cells for a bit after digestion and before staining. Otherwise, you end up having to trust the CD3+CD8- fraction to be Th cell, but as CD8 is also somewhat vulnerable to some digestion protocols, that is less than ideal.

Yes, I thought for a long time that uninephrectomy was necessary, but it is not.

We have just been giving a Doca pellet and 1% salt to drink. Blood pressure increases similarly to the one kidney model. One does not see the massive renal hypertrophy which accelerates and complicates evaluation of the effects of hypertension on the progression of kidney disease.

Hi, basically frozen sections (FS) are not good for kidney evaluation especially if you are looking for glomerular changes other than global sclerosis. Thus, the use of FS will depend on the potential findings you are looking for.

Hi Ya Gong ,

I agree with all the points suggested by Emre Bektik .

If you have been following all the steps he recommended and still notice high cell death then maybe also check the E8 medium you are using. Does it contain the necessary supplements? Also the medium should not be too old or too cold when added to the cells. The medium stays good for about 14 days and not longer. In case you use water bath to warm your medium I could recommend to make small aliquots of the medium and use as much needed (instead of warming the entire medium bottle everyday).

Hope you are able to figure out better conditions for your iPSCs.

Good luck!

Best wishes

A good place to look for protocols to assay basic metabolic enzymes is at Worthington Biochemical's web site.

Here is an example of a protocol for hexokinase.

Pefusion using saline solution (room temperature recommended) gives excellent results.

looking at proteins for western blots, it is not a big deal to not perfuse. I never do it. The main key is to freeze the tissue as soon as possible after removing it from the rat. This is particularly important if you are looking at phosphorylated proteins which can degrade quickly. If you were performing confocal or immunofluorescence microscopy then it is very important to perfuse the kidney to remove red blood cells because red blood cells autofluorese and distort the quality of the images.

Hi Andrea,

I would recommend you delete all contacts and just use the two joints you already defined. You can define friction coefficient in the joints created. If you are not interested in components deformation you could also use Rigid Dynamics instead of Transient Structural. It is not very clear what you want to analyse. If you give more details, I can try to help you better.

I hope this information was helpful.

Best regards,

Tarsis

Kindly check http://www.bio.vu.nl/thb/course/ecol/MetzMyli2008.pdf

Md. Masudul Haque Understood, so you want to do quantitation. From my experience I know that different organs/tissues require different homogenization buffers, for example kidney i had to use a mannitol/sucrose/Mops/ Pmsf buffer, for brain i had to use HEPES/sucrose/pmsf, but since you are not interested in the proteins but the drug I think PBS should work fine. However, for elisa a lysis buffer step is always recommended to dissociate large protein complexes that may interfere with ELISA performance (sensitivity/specificity) even if you take the supernatant of a spun homogenate. Usually, the lysis buffer includes a non-ionic detergent such as Triton X-100 or NP-40 are best, or Sodium deoxycholate (Abcam protocol worked with me for elisa protein detection). But RnDsystems have a protocol specific for tissue homogenate using PBS which should work best with your protocol, note they use freeze-thaw cycles to break proteins apart (https://www.rndsystems.com/resources/protocols/elisa-sample-preparation-collection-guide). Good luck.

Dear Lolita Ray Oliver,

From what you have described, it appears that this 39-year-old female has had Systemic lupus erythematosus (SLE) for more than 4-years if her daughter got complications of this disease (Congenital lupus) and now most likely she has chronic kidney disease with acute kidney injury. If some person with SLE develops RPGN with stage III or IV lupus, doctors will not wait for one week if they already recommended dialysis. Possibly there is some reversible factor for which the doctors would like to wait for another week.

Was a renal biopsy done? If not it is likely patient might be recovering with intensification of the treatment of lupus nephritis.

Dear Jacob

I don't think that the specificity can be applied here. Missing some calyces couldn't be in any way considered as a false positive result.

It is a matter of defect in the accuracy. Although Sensetivity is not the proper test, but at least is more fitting than the specificity in your case.

Hi can you share the protocol how you have isolated the mitochondria?

I've used the In Situ Cell Death Detection Kit from Roche for immunofluorescence staining and it works very well in formalin-fixed paraffin-embedded heart and liver sections. I think you may consider change your TUNEL kit. If you want to use your own reagents and solutions instead of buying this kit, you may try following the steps described on this kit protocol. I enclose the protocol below.

Prof. @ Rasul Akbarov

You could Check the following paper, you might the answer of your question;

Link: https://www.sciencedirect.com/science/article/pii/S1359431120330210

To be honest, everything seems fine, I guess there is always some sort of problems without obvious explanation. Only thing I can advise is to gen new ones, it will save time and, in long term, cost,

Mathematical Modeling of Kidney Transport

By: Anita T. Layton

By: Aurélie Edwards

Thanks a lot Dr. Pant for your help. It will be a lit bit more helpful if there are videos showing the excision of such of organs especially the kidney and ovary from the fish body..

Yeah that's true. Thanks for your valuable suggestion Dr. @Xuhua Wang.

Any thing beyond the expiry date should not be used.. whether medicines,or any edible stuff or even your toiletries kit!

Protective effects of Rhizoma smilacis glabrae extracts on potassium oxonate- and monosodium urate-induced hyperuricemia and gout in mice.

Phytomedicine : international journal of phytotherapy and phytopharmacology 2019-4-22

dr suzan

Suzan S. Ibraheim