In Renal Protection in Chronic Kidney

The kidneys play an important role in the body by ridding it of wastes products from the blood. They are also involved in regulating blood pressure, electrolyte balance, and red blood cell production. Many Americans suffer from kidney failure and many more are added to the ongoing waitlist for organ donations each year. and becoming matched with an organ is half the battle. Once kidney function starts to decline, the likelihood that other organs will eventually shut down as well doubles. Currently, there aren’t many treatments to significantly slow down or stop the rapid decline of a person’s health when they are dealing with kidney failure and studies like the one done in Renal protection in chronic kidney disease: hypoxia-inducible factor activation vs. angiotensin II blockade can help find new ways to treat kidney disease[3]. In Renal protection in chronic kidney disease: hypoxia-inducible factor activation vs. angiotensin II blockade by by Aihua Deng MAK, Mary Ann K. Arndt, Joseph Satriano, et al., the researchers focused on using a simulated kidney with signs of chronic kidney disease (CKD) using Winstar rat to show how uncommon methodologies could stop the effects of CKD. One of these effects being Hypoxia or a condition that develops when sufficient oxygen is unable to reach the tissues. Researches used two methods: cobalt chloride (CoCl2) and dimethyloxalyglycine (DMOG) in order to initiate Hypoxia Inducing Factors (HIF) and associated proteins. Researchers later combined these methodologies with angiotensin (Ang II) blockade and observed oxygen consumption and other renal hemodynamics to assess whether these methods were effective in treating the simulated kidney or not. Coupled with this, in this study, “hypoxia inducing factor (HIF) proteins were assessed using Western blotting and PCR”. The study was broken up into a week by week basis where researchers recorded what was observed and how these methods affected kidney functioning.

In conclusion, the researchers found that using ang II blockade treatment and HIF therapies were successful in returning oxygen consumption levels and functioning in the tissue back to normal rates. II. Hypothesis The researchers in this article have chosen to examine this topic because “they wanted to identify proteins linked to HIF to serve as indications of any beneficial effects when combining multiple methodologies like ANG II blockade, Cobalt Chloride, and dimethyloxalyglycine.” In this article, the hypothesis was that “induction of HIF, as demonstrated in kidney tissue by Western blot and indexed by the expression of VEGF, HO-1, Epo, and GLUT1 mediates the beneficial effects of ANG II blockade in CKD.” The most important part of this study/ hypothesis is specifically looking at the expression of certain proteins in the body to serve as a way to combat CKD and how their expression correlates with how well a kidney is functioning. Researchers looked at oxygen consumption and renal blood function (RBF) as a basis for this measurement. This area of research is important because kidney failure has increasing epidemic rates while there are some treatments and alternatives like transplants available, these treatments are high risk with high fail rates or are a means to an end like dialysis.

There is an acute organ transplant rejection rate of 30-50 percent and dialysis patients have reported feeling washed out and unable to work [3]. Patients even suffered from disabling bone disease, dementia caused by aluminum intoxication, and severe fatigue from uncontrollable anemia. Coupled with this, screening for early detection of kidney injury has increased with blood tests but early preventative measures are hard to come by for certain cultural groups like African Americans who some are from low income backgrounds so seeking out expensive medical care before reaching an ultimately fatal prognosis is unlikely.

Previous studies like the one conducted in Heme oxygenase-1 is Upregulated in the Kidney of Angiotensin II-induced Hypertensive Rats: Possible Role in Renoprotection, researchers studied the kidneys of rats with hypertension. The rats were given dosages of ang II blockade or noepinephrine for approximately a week to see how it would affect heme oxygenase-1 (HO-1). Researchers found that the increasing Ang II in the kidneys of rats with hypertension did “provide protection against Ang II induced injury” [2]. This study was conducted ten years prior to the article of focus and helped future researchers use methodologies like angiotensin II to further study the physiology of the kidneys. III.Methods One part of the materials and methods that I found interesting and to exhibit the part of the study that was important to its overall purpose was the “renal function measurement and oxygen consumption calculation.” During this part of the study, male Wistar rats that weighed between 225 grams and 250 grams were used and randomly placed into four groups. These groups were composed of a control group, one group designated as “1-wk A/I group”, one group designated as “1-wk A/I ANG II blockade group”, one group designated as “1-wk A/I cobalt chloride group”, and one group designated as “1-wk A/I DMOG group.” Cobalt chloride and DMOG were injected into the rats for a series of eight days. Renal function and oxygen consumption were measured by putting the rats under anesthesia with Inactin and placing them on a temperature controlled table of 37 degrees Celsius. “After cannulation of trachea, left jugular vein, left femoral artery, and urinary bladder, the left renal blood flow (RBF, ml/min) was monitored with a perivascular ultrasonic transit time flow probe. Once the rats were stabilized for 60 minutes, researchers recorded their systemic blood pressure and RBF.” Blood samples were taken using a color spectrophotometer. “Oxygen content (O2ct) was calculated by the formula: O2ct(ml/ml blood) (1.39 tHb O2Hb% Po2 0.003) ⁄ 100. The total left kidney oxygen consumption (QO2 ) was calculated as RBF times arterial minus renal venous O2ct.” This fit into the experimental design because it initially tests how well the kidneys and other organs are functioning in the body after protein treatments to see how effective these treatments are to the rats. These findings help later support or disprove the hypothesis because if the oxygen consumption values increase then the hypothesis can be accepted but if not then the hypothesis is rejected. IV. Results **thought i was supposed to include a part from the paper with the word for word results, not stated in the word document explaination but in the rubric, it states include the graph and its caption, thats what I did but I didn’t make it clear that thats where it was from. The part of the results section that I chose to analyze in detail was the results that went with the renal function measurement and oxygen consumption calculation. This part of the results was called “Improvement of renal hemodynamics by cobalt treatment and ANG II blockade, shown by figure 1.” The normal A/I kidneys exhibited significant decreases in both RBF and GFR (Fig. 1).

This reduction in GFR and RBF was not due to the reduction in renal mass in A/I kidney since both GFR and RBF increased significantly by ANG II blockade as well as by the incorporation of cobalt chloride therapy. When compared with the normal kidney total renal oxygen consumption (QO2 ) didn’t change. Elevation of QO2 /TNa was telling in that there was a decrease “renal metabolic efficiency” [1]. “This renal metabolic inefficiency was caused by an increased demand for oxygen in the untreated A/I kidney, as ANG II blockade and cobalt treatment significantly increased GFR to normal levels while QO2 remained unchanged, improving QO2 /TNa to normal values”. In the article,” it was concluded that all these renal effects produced by cobalt treatment are due to the activation of the HIF pathways”. To confirm this conclusion, researchers used another HIF-1 inducer, DMOG, in A/I rats, which produced similar renal protective effects as the cobalt treatments and as renal function improved and overall renal oxygen consumption decreased. V.Analysis The purpose of this study was to “identify proteins linked to HIF that serve as indicators as to whether the beneficial effects of combined ANG II blockade as a result from the expression of these proteins.” Coupled with this, the hypothesis in this article stated that “the induction of HIF, as demonstrated in kidney tissue by Western blot and indexed by the expression of VEGF, HO-1, Epo, and GLUT1 mediates the beneficial effects of ANG II blockade in CKD. They are linked because the purpose of the article is essentially to determine whether the prediction made by the researchers in the hypothesis is correct or not. The experimental design for this study and the hypothesis are linked because the study is broken up into sections: renal function and oxygen consumption, Murine cortical tubular cell culture, Immunoprecipitation of HIF, Immunoblotting Analysis, Quantitative RT-PCR, Cell Proliferation by 5- bromodeoxyuridine, and Statistical Analysis are all portions of the design that incorporate the different proteins to test their effects of the rats and that data is collected. The results and conclusion are linked because the results present a summary of the data while the conclusion explains why these findings are important to the overall study.

For example, the expression of other HIF-induced targets was also investigated at either protein level by Western blot or mRNA level for certain molecules by real-time PCR. As shown in Fig. 6, VEGF protein was constitutively expressed in normal kidney, tended to increase in untreated A/I kidneys, further increased in ANG II-blocked kidneys and demonstrated maximal expression in A/I kidneys treated with cobalt. HO-1 protein (Fig. 7) was undetectable in normal kidneys, induced in the untreated group, suppressed in the ANG II-inhibited group, and greatly induced in the cobalt-treated group. When GLUT1 was examined, the pattern was similar to that observed with VEGF, whereby it was modestly elevated in A/I kidneys and further increased in the cobalt and ANG II blockade kidneys (Fig. 8). The major findings of this study is that activation of the HIF pathway and ANG II blockade improved renal hemodynamics. It was demonstrated that HO-1 was substantially increased with cobalt treatment. Increased production of CO and bilirubin due to upregulation of HO-1 could contribute several effects, including enhanced renal vasodilation, reduced oxygen consumption, and reductions in inflammation. Combined ANG II blockade completely prevented any rise in HO-1 by eliminating the normally stimulatory effects of ANG II on HO-1 (1, 18).

Taken in the aggregate, it seems unlikely that the beneficial effects of cobalt treatment and HIF activation were mediated principally by induction of HO-1, since ANG II blockade also corrected the metabolic and hemodynamic effects in the absence of significant influences on HO-1. The levels of HO-1 during cobalt treatment were quite clearly increased and different from both the untreated and combined ANG II blockade animals. The changes in GLUT1 protein expression were similar with both treatments and are part of a variety of metabolic alterations that occur with HIF-induced activation of target proteins. The kidney is the primary organ for the production of Epo, and regulation of Epo expression is mainly at the transcriptional level. It is well documented that Epo mRNA is upregulated by HIF in response to hypoxia (4, 41, 42). Recent studies (23, 27) have shown that hypoxic induction of Epo is suppressed by inflammatory cytokines via activation of other inhibitory transcription factors. The strengths of this article is that it is concise and thorough on the approach to studying how different proteins can help mediate a potential treatment for CKD. The weaknesses of this article were that when I was reading, it was clear at the beginning the involvement of the rats in the experiment but it wasn’t always clear later in the article when reading through the results and discussion. In their design approach because it was so intricate, it makes it hard for someone to recreate the same study without many changes and/or costly items