What is Acute Renal Failure?

What is acute renal failure?

It is the RAPID  DECLINE in Glomerular Filtration Rate(GFR) over hours and days causing deterioration in renal function  resulting to build-up of nitrogenous wastes. It causes a sudden retention of endogenous and exogenous substances (e.g. Nitrogenous wastes). Reduction in urine flow rate aka oliguria <400ml/day and electrolyte and acid-base abnormalities are its common symptoms/effects.

Classification

Acute renal failure is classified into three namely Prerenal, Intrarenal, and  Postrenal renal failure. Prerenal ARF or azotemia causes renal hypoperfusion, resulting in decreased function without frank parenchymal damage  which accounts for approximately ~55% of ARF cases. Intrinsic ARF directly involve the renal parenchyma ~40% of the cases. Postrenal ARF is associated with urinary tract obstruction~5% of the cases.

 Prerenal Acute renal failure

    It is the most common cause of community-acquired ARF due to history of poor fluid intake, treatment with Non-Steroidal Anti-inflammatory Drug (NSAID), ACE inhibitors/ARBs and  worsening heart failure. Common features include blood volume depletion due to absolute/postural hypotension, low jugular venous pressure and  dry mucus or it can be due to  decreased effective circulatory volume(e.g., heart failure or liver disease) and  decreased cardiac output.

—  I. Hypovolemia

—    A. Increased extracellular fluid losses: hemorrhage

—    B. Gastrointestinal fluid loss: vomiting, diarrhea 

—    C. Renal fluid loss: diuretics, osmotic diuresis, hypoadrenalism

—    D. Extravascular sequestration: burns, pancreatitis, severe hypoalbuminemia (hypoproteinemia)

—    E. Decreased intake: dehydration, altered mental status

   II. Altered renal hemodynamics resulting in hypoperfusion

—    A. Low cardiac output state: diseases of the myocardium, valves, and pericardium (including tamponade); pulmonary hypertension or massive pulmonary embolism leading to right and left heart failure; impaired venous return (e.g., abdominal compartment syndrome or positive pressure ventilation)

—    B. Systemic vasodilation: sepsis, antihypertensives, afterload reducers, anaphylaxis 

—    C. Renal vasoconstriction: hypercalcemia, catecholamines, calcineurin inhibitors, amphotericin B

—    D. Impairment of renal autoregulatory responses: cyclooxygenase inhibitors (e.g., nonsteroidal anti-inflammatory drugs), angiotensin-converting enzyme inhibitors, or angiotensin II receptor blockers

—    E. Hepatorenal syndrome

Causes of Intrarenal acute renal failure

II. Parenchymal (Intrarenal)

     1. Specific

         a. Glomerulonephritis

         b. Interstitial nephritis

         c. Toxin, dye-induced

       2.Nonspecific

         a. Acute tubular necrosis

         b. Acute cortical  necrosis  

I would like to give particular attention on acute tubular necrosis .It is the death of the tubules inside of our kidney and this could be detrimental to the overall function of our kidney.  What  causes acute tubular necrosis?

—  A.      Ischemia: causes are the same as for prerenal ARF, but generally the insult is more severe and/or more prolonged

—   B.    Infection, with or without sepsis 

—   C.   Toxins:

—          1. Exogenous: radiocontrast, calcineurin inhibitors, antibiotics (e.g., aminoglycosides), chemotherapy (e.g., cisplatin), antifungals (e.g., amphotericin B), ethylene glycol

—          2. Endogenous: rhabdomyolysis (rapid breakdown of muscles), hemolysis

This illustration tells you how severe beating/ hazing or any intense muscular injury could lead to acute renal failure which could be fatal.

Causes of Postrenal Acute Renal Failure

III. Postrenal  -obstruction to the flow of urine

       1. Calculus in patients with solitary kidney

       2. Bilateral ureteral obstruction

       3. outlet obstruction

       4. Leak, posttraumatic

MANAGEMENT

Prerenal ARF

>Hypotonic solutions (e.g., 0.45% saline) are usually recommended as initial replacement in patients with prerenal ARF due to increased urinary or gastrointestinal fluid losses(Fluid replacement).

>isotonic saline may be more appropriate in severe cases

> Cardiac failure may require aggressive management with ionotropic agents, preload and afterload reducing agents, antiarrhythmic drugs, and mechanical aids such as intraaortic balloon pumps

INTRINSIC ARF

>Many different approaches to attenuate injury or hasten recovery  for ischemic  and nephrotoxic AKI failed.....

>Acute glomerulonephritis or vasculitis may respond to immunosuppressive agents (glucocorticoids, alkylating agents, and/or plasmapheresis, depending on the primary pathology).

POSTRENAL ARF

> requires close collaboration between nephrologist, urologist, and radiologist.

>Obstruction of the urethra or bladder neck is usually managed initially by transurethral or suprapubic placement of a bladder catheter, which provides temporary relief while the obstructing lesion is identified and treated definitively.

>Similarly, ureteric obstruction may be treated initially by percutaneous catheterization of the dilated renal pelvis or ureter.

>Obstructing lesions can often be removed percutaneously (e.g., calculus, sloughed papilla) or bypassed by insertion of a ureteric stent (e.g., carcinoma).

>Most patients experience an appropriate diuresis for several days following relief of obstruction. 

Science, Nature and Troubleshootings: Troubleshooting and Doing Gene Expression Analysis

http://en.wikipedia.org/wiki/Gene_expression Science, Nature and Troubleshootings: Troubleshooting and Doing Gene Expression Analysis

How To Do Real Time Gene Expression Work for Beginners?

This gene  expression experiment was done using  rice Oryza sativa seeds at various stages of seed  development. The following picture will show you the flow of the work.

Shoot and seed samples of the rice plant were collected and  immediately  frozen in liquid nitrogen. Futhermore, RNA extraction was done using Trizol method with slight modifications like twice the addition of Lithium Chloride precipitation. After that RNA quality and quantity was verified using gel electrophoresis  and nanodrop spectrophotometer. Two intact bands(18s and 28s) of RNA were observed  indicating a good quality RNA. Next the crude RNA samples were treated with Promega RQ1 RNase free DNase.

 For the purpose of clarity, I posted here the picture of the different stages of grain development in rice.

Here are the different tissues we considered for the experiment.

DOWNSTREAM APPLICATIONS
    cDNA synthesis

  Afterwards, cDNA was synthesized   from  the DNase treated RNA using the Roche 1^st Transcript cDNA synthesis kit. Here are the components of the reaction: template mixture( oligo dT, random hexamer, DNase treated  RNA) and reverse transcriptase mixture( RT buffer, RNase inhibitor, Deoxynucleotide mix and reverse transcriptase).
Here is the procedure how to do it? First, denature the template mixture for 10min at 65 degrees centigrade and put  immediately in ice for 5mins. Then followed by addition of  the RT mixture to the template mixture. Incubate for 10 min  at  25 degrees centigrade then   followed by 30 min at 65 degrees centigrade.

PRIMER TEST

Primer design and primer check took most of  my time in doing  this work. For the purpose of protecting my Intellectual Property rights, I would not reveal the identity of the genes which I designed and tested. Our research will be published officially this year.

So here is the point in this work.
First, you have to design your primers using bioinformatic softwares. I would discuss in detail how to design primers in my next post. After designing my primers and ordering them, we then test our primers with cDNA using the conventional PCR like G-storm cycler to check PCR product size and the number of band(s) that appeared on the gel. If the primers showed to have one distinct band  and  appropriate product size on the gel, I can now proceed in performing the real time PCR primer check (qRT-PCR). qRT-PCR is highly sensitive and very accurate. In the illustration, after performing melting peak analysis the primers which showed to have two peaks were discarded and  we JUST considered  the primers with ONLY ONE MELTING PEAK . So in short, primers working with conventional PCR will not necessarily work with qRT-PCR. Primer design and specificity is the first priority. 

SELECTION OF REFERENCE GENES

From an original choice of  four reference genes(housekeeping genes), we selected three reference genes to normalize the expression of our target genes based on its expression profile using its Crossing point. The more uniform the crossing point(Ct or Cp) to each other the better it is. Gene stability was checked across the different rice genotypes and its accompanied rice tissues.

We tested the reference genes stability at the different stages of grain development and across different genotypes of rice. We used the geNorm software to evaluate our four reference genes which debunks or removes reference genes with M(relative stability)>1.5 which in this case it was EP1(Ref4). Bioinformatic softwares like geNorm will make the work very easy. Just click on the link.

NOTE:

Contamination should be avoided with all means possible. Always work in  a room designated for RNA extraction only and a room with no possible contamination of plasmids.

 Relative expression computation

Computation of relative expression could be done by means of relative quantification with of course the use of internal reference genes(control genes) and the other method utilizes the power of calculus with is the absolute of quantification method. Pfaffl 2001 suggests the used of an internal calibrator/control sample to normalize the expression of the target sample using the formula of delta delta Ct(relative expression).Relative expression computation can be done in Microsoft excel.  Another method used to compute was through delta Ct method wherein all Ct values of samples or reference genes were converted to raw expression values with used of a control sample or a sample with highest(earliest Ct). These raw expression of the target genes would be normalise by the geomean of AT LEAST three reference genes in order to correct its raw expression (GeNoRM, Vandonsempele, 2000).

Here is the sample gene expression  data we got using relative quantification method  with the use of  at least THREE reference  genes.

Reference:
Pfaffl M.W. , 2001. A new mathematical model for relative quantification in real-time RT–PCR 

Vandosempele et al 2002

 

   

    

Troubleshooting RNA Extraction in Seeds Oryza sativa

Extracting RNA from the seeds was never easy as mentioned by one of the PostDocs of the International Rice Research Institute(IRRI). As a researcher of IRRI,  I've worked  with those samples close to three years and indeed STARCH is a big problem in the RNA extraction. Face with this problem, I developed or optimised a protocol on how to extract the RNA with the LEAST amount of STARCH possible. By the way, starch causes one  of  the major problem in downstream applications like conventional PCR and especially real time PCR. One of the procedures, I emphasized is the used of doubly repeated LiCl precipitation. If you want some advice or  if you want a copy of the protocol, please feel free to email me at magponcio@gmail.com.