The Renal System and function - Literature Review

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2.1 Conceptual Literature

Kidney Function

The kidney, which is the main concern site of the study, performs various functions. The kidney has the ability to keep the internal environment or internal milieu of the body constant within the physiologic limits (Shourouk, 2000). This ability is commonly achieved by specific function, Excretory function and production of urine, regulation of the acid-base balance of the body, hemopoietic function and endocrine function.

It is considered that the primary method of eliminating waste materials of metabolism that are no longer needed by the body is by means of our kidneys. Urea (metabolism of amino acids), creatinine (muscle), uric acid (from nucleic acids), end products of haemoglobin breakdown (ex bilirubin) and metabolites of hormones are some of the waste materials that should be eliminated in the body (Guyton, 2006). The kidney is also responsible in disposing toxins and other foreign substances such as drugs, pesticides and specific food additives.

The regulation of the acid-base balance of the body is also performed by the kidneys. When acid level increases (acidosis) the kidney prevents it through different mechanism such as reabsorption of bicarbonate filtered from the blood and consumption and excretion of H+ (Shorouk, 2000).

The hemopoietic function involves the role of the kidney in erythropoiesis in the bone marrow in which maintains the development of the red blood cells in the bone marrow.

Lastly, hormones and vasoactive substances constitute the kidneys by activating, degrading, and forming them. Insulin, Renin, Prostaglandins, Glucagon, Vasopressin, ANP and Aldosterone are some of the commonly affected hormones (Shorouk, 2000).

Kidney Diseases

The National Kidney and Transplant Institute conducted a study that concluded that almost 7,000 Filipinos every year are killed by a kidney-related disease. According to one of the nephrology experts, Dr. Romina Rusillon, many Filipinos commonly suffers from end-stage renal disease and and underwent dialysis.

Serious kidney diseases are divided into two groups: (1) the acute renal failure, wherein the kidneys stop working almost entirely and but may recover nearly its normal function and (2) chronic renal failure that includes continuous loss of function of nephrons and usually kills the kidney function (Guyton, 2006).

Under such categories, produces specific kidney diseases that involves kidney blood vessels, glomeruli, tubules, renal interstitium, and the parts of the urinary tract.

Spirulina platensis and its morphology

Spirulina platensis, is a planktonic cyanobacteria that are commonly found in the tropical and subtropical water bodies in which these water bodies are characterized by high levels of carbonate and bicarbonate (Vonshak, 1997). Today, cultivation of such species is made into food supplements such as dietary capsules.

Spirulina is considered as a one of a kind Superfood (Moorhead & Capelli, 1993). This is because of the endless benefits it can bring.

The filamentous cyanobacteria are very recognizable and evident because of its multicellular cylindrical trichomes. Using light microscopy, the non-heterocystous filaments are easily visible. These filaments produces a gliding motility, they are usually free-floating, granulated and are solitary. The Spirulina platensis are known for its short trichomes with 5-7 loose coils

The morphological attribute of the Spirulina are very sensitive to temperature and chemical condition. Any alteration of such could cause a difference of the geometry o (Vonshak, 1997).

Zea Mays L.

In the Philippines, Zea Mays L. (Corn) is one of the most cultivated crops in the Philippines (Co & Taguba, 1984). Corn can be categorize into two: (1) Dent corn (Zea mays indenata) used in livestock feeding, industrial products and processed food and (2) Flint corn (Zea mays indurate) that is very similar to dent corn but is distinguished by hard outer shells and kernel with colors that ranges from white to red.

Zea Mays L. is from the family Poaceae (Gramineae), which are characterized by having a fruit as a grain and has a grass spikelet structure (Simpson, 2006). The family of the Poaceae group is also distributed worldwide and considered as the most economically important group of plants.

Most of the common parts utilized in a corn are its corn hairs and cob. It is used to treat initial stages of Renal calculi nephritis, Chronic nephritis, edema, diabetes, and primary hypertension (Co & Taguba, 1984).

Nonsteroidal Anti-Inflammatory Drugs

The acts of NSAIDs are primarily inhibits the cyclooxygenase that will catalyse the initial and very first step of prostanoid biosynthesis that usually leads to decrease of prostaglandin production (Lippincott’s, 2009). NSAIDs are considered antipyretic, analgesic and anti-inflammatory but differs in their specific individual activities.

Nonsteroidal anti-inflammatory drugs, despite of many therapeutic activities, could bring unwanted or severe effects such as (ICPH, 2009):

  • Gastric ulceration
  • Intestinal ulceration
  • Anemia
  • Resultant Blood Loss
  • Disturbances in platelet function
  • Water retention
  • Increased blood pressure
  • Delayed onset and prolongation of gestation
  • Changes and abnormalities in renal function

2.2 Related Studies

In search for different remedies for renal problems, studies have shown variations of plant species that provide nephroprotective potentials.

A latest study evaluated the combined effects of corn silk and binahong leaves in rat kidney failure by Sukandar, Sigit, and Adiwibowo (2013). The inducing agent used was gentamicin. According to Lakshmi et. Al (2012), getamicin, together with streptomycin, is an aminoglycoside antibiotic which is used for bacterial infections. But lately, their nephrotoxicity has been reported to occur on about 15-30% of all the renal failure treated subjects. In this study, both the extract of corn silk and binahong leaves were orally administered to the rats after 7 days of intraperitoneal treatment of gentamicin and piroxicam. The results suggested that the combination of these two plants would achieve more effective results than the half dose of each plant extract introduced individually. The extracts reduced the serum creatinine level and blood urea nitrogen level (BUN) as well as the depletion of kidney index by means of lowering the lipid peroxidation. In addition, an increased in antioxidant enzymes marked an increase which further contribute to the improvement of kidney. Furthermore, better reduction of kidney damage in histological examination was observed.

Al-Jawad et al. (2012) assessed the potentials of corn silk extract against nephrocalcinosis. Using a rabbit model, oxalic acid was used to induce nephrocalcinosis using a dose of 333mg/kg. Blood samples were collected from the ears of the rabbits for biochemical analysis and renal functions to assess the normal condition of the kidney of the kidneys of the test organisms. After ten days of experimentation, significant decreases in the serum creatinine (85.9 ± 0.2 vs. 97.3 ± 0.5) mmol/l, blood urea nitrogen (5.2 ± 0.08 vs. 7.3 ± 0.2) mmol/l and decrease in serum Na+ levels (137 ± 0.2 vs. 142.16 ± 0.7) mmol/l were produced. Also, it caused an increase of urinary Ca +2 levels compared to the normal values complementing its reduction in renal tissues. Furthermore, a reduction of the concentration in renal parenchyma after the histological examination was evaluated. \They also stated that components of corn such as flavonoids, volatile oil, saponins and allantoin besides the minerals typically found in the plant.

Another study which evaluated the potentials of corn silk was conducted by Shamsky, Al-Chalabi I and Al-Amery (2012). They compared the effects of corn silk with the alkalinizergent or Uralyt-U decomposing kidney stones. Uralyt-U which is alkalinize class of drug which is used to modify urine pH. In this experiment, the corn silk extract served as a drink for the patients in Al-Yarmook Teaching Hospital in Iraq. Sixty male patients with kidney stones were taken into test. The first half were asked to take one cup of Uralyt-U three times a day and the other half were advised to take two cups of corn silk extract daily. The corn silk showed an essential effect in the contraction of ureter smooth muscles causing an increase urinary output, and instead of destroying the kidney stones the corn silk extract regulate the increase of passage of these stones throughout the ureter.

A work by Kumari, Babitha, Jaffar, and Prasad (2011) showed the different potentials of Spirulina in the world of medicine. The study presented the effects of this cyanobacterium against allergies, rhinitis and diabetes. Also, its anticancer potential was stated here. Spirulina’s components which may provide a greater effectiveness against renal problems is its antioxidant activities such as phenolic compounds inhibiting free radicals and reactive oxygen giving way to ruptured tissues.

Another study evaluated the nephroprotective ability of a species of Spirulina against oxidative stress and renal disfunction caused by gentamicin. This work of Kuhad et al. (2005) investigated the renoprotective effect of Spirulina fusiformis. The administration of this algae (500, 1000, 1500 mg/kg) with the gentamicin (100 mg/kg) was assessed by measuring the serum creatinine, blood urea nitrogen, creatinine clearance and serum nitrite levels. After the introduction of the algae extract, significant reductions in the biomarkers were observed. The results clearly exhibited the therapeutic potential of this Spirulina species.

Another species of Spirulina was tested against hyperlipidemia and oxidative-stress liver and kidney damage. Canchichuamin et al. (2010) studied the effects of Spirulina maxima in reducing the renal damage induced by lead acetate. The results evaluated the alterations on the plasma and lipid level on the liver. Also, the antioxidant level both kidney and liver showed distinguished changes. The Spirulina maxima succeeded in improving the body weight and biological markers. They also mentioned the antihyperlilipdemic and antioxidant effects of this species due to the components it has such as protein, iron, gamma-linolenic fatty acid, carotenoids, phycocyanin, etc.

Further works related with Spirulina species was conducted by Akhter et al. (2010). In this study, cisplatin was used to induce interstitial nephritis in Long Evans rats. A single dose of 6 mg/kg cisplatin was given intraperitoneally to produce nephrotoxicity. On the other hand, a dose of 1000 mg/kg of Spirulina powder was administered through intragastric feeding and a dose of 50 mg/kg vitamin C was injected subcutaneously in the abdominal wall of the rats. The findings showed that both vitamin C and Spirulina are effective in reducing the kidney impairment thus spirulina was observed to be more potent in repairing tissue damages in histological samples. Moreover, the protein content of Spirulina further contribute to the cisplatin-induced renal damage if removed.

Betton et al. (2011) evaluated the ability of N-phenylanthranilic acid in causing renal papillary necrosis in rats. Urine and blood samples were collected within 4, 8, and 15 days with corresponding dosages of 50, 350, 400, and 700 mg/kg a day. Histopathologic examination was also determined to assess the renal injury caused by NPAA. After different days of administration, nephrotoxic damage was seen in strains above 50 mg/kg dosage. A characterized degeneration and necrosis in the duct of the papilla indicated the kidney damage. The noticeable injury was more severe in the collecting duct in Han-Wistar rats than of the Sprague-Dawley rats. Hence, the study showed that urinary RPA-1 and clusterin served as biomarker in the absence of BUN and serum creatinine level test in the implication of NPAA-induced duct injury.

Chapter 3


3.1 Research Design

The research study considered a randomized complete block design wherein it will identify the nephroprotective potential of Zea mays L Silk and Athrospira platensis against Mefenamic-induced nephrotoxicity in Male Rattus norvegicus (WISTAR RATS). Twelve (24) Wistar Rats were divided into six (6) treatment groups. T0 (-) being the positive control group wherein administration of the mefenamic-acid for 7 days was done. T0 (-) being the negative control group with no treatment. T1 took 4 weeks administration of grinded powder of corn silk (50%). T2 also took 4 weeks administration of Spirulina platensis (50%) powdered tablets. T5 received 50% synergism of both for four (4) weeks. T6 received 75% synergism for four (4) weeks.

3.2 Research Setting

The administration of the treatment for the rat models was exclusively done in _________________________. The rats were placed in steel cages with dimensions of each and will have daily access to food and water. The main site of the treatment room was kept in a very good condition that includes a ventilated air system and installed decent light-bulbs. The room temperature was also maintained and monitored between 28-30°C.

For the other experimental procedures, such as the dissection of the rats prior the treatment was done in the PCH Biology laboratory room in De La Salle University - Dasmariñas. The preparation of the Zea Mays L. (CORN) Silk powder was done in the PCH Chemistry laboratory room in De La Salle University - Dasmariñas.

3.3Research Procedure

3.3.1Procurement of test organisms

Twenty-four (24) Wistar rats, weighing 100-150 grams, were purchased from Standard and Testing Division-Industrial Technology Development Institute DOST compound, Taguig City. All Wistar rats procured were within the reproductive stage composing of only male rats.

3.3.2Maintenance and Acclimatization of test organisms

Caged rats were acclimatized in good condition within seven (7) days. A well-ventilated area with a temperature of about 28-30°C was provided to sustain living condition for the rats. The cages were kept in sanitized condition by brushing to ward off contaminants. Proper disposal of feces and food waste were also observed. The rats were fed with rat pellets and provided with tap water which was replaced daily.

The Wistar rats were acclimatized one week prior to the experimentation. This was done to monitor the good condition of the test organisms including their health status, defecation, daily behavior and response, etc.

3.3.3Procurement and Preparation of the extracts

Corn silks/husks were collected from street vendors along Sta. Rosa-Tagaytay Road, Brgy. Pulong, Sta. Cruz City, Sta. Rosa. Separately, Spirulina tablets were purchased online. The dried corn silks and Spirulina tablets were pulverized using mortar and pestle (Shamkhy, 2012; Besa, 2012). Both powdered corn silk and Spirulina tablets were dissolved in water. T2, T3, T4, and T5 were prepared with the following concentrations: 50% Spirulina (0.50 g of Spirulina powder + 0.50 ml of distilled water), 50% corn silk (0.50 g of powderized corn silk + 0.50 ml of distilled water), 50% synergisim (0.25 g of Spirulina powder + 0.25 g of powderized corn silk + 0.50 ml distilled water), 75% synergisim (0.375 g of Spirulina powder + 0.375 g of powderized corn silk + 0.25 ml distilled water), respectively.

3.3.4Induction of N-Phenylanthranilic Acid-mefenamic

Administration of N-Phenylanthranilic Acid was done for seven (7) consecutive days to induce nephrotoxicity. The drug was induced through intraperitoneal injection.

3.3.5Administration of the Corn silk and Spirulina concentrations

The first treatment group (T0(-) ) was administered with saline solution. The second treatment group (T1(-) ) was administered with 500 mg/kg/day of NPAA-mefenamic for seven (7) days as suggested to be the maximum tolerable dosage (Betton et al., 2012). T2, T3, T4, and T5 groups were administered using the prepared concentrations. Using gavage method, treatments with Spirulina, corn silk and synergism of these two were supplied to the Wistar rats for twenty-eight (28) days.

3.3.6Histopathologic Examination (Sukandar et al., 2013)

Assessment of kidney damage was done through histopathologic examination by selection of rat per treatment possessing the most severe sign of nephrotoxicity in terms of visible behavior after the induction of mefenamic acid. Both of the kidneys were removed and fixed in buffered formalin following the standard histologic technique done Sukandar et al. (2013). The preservation of the rat kidneys was done in Paulo Campos Hall, De La Salle University-Dasmariñas, City of Dasmariñas ,Cavite. The samples were processed at The Research Institute for Tropical Medicine in Alabang, Metro Manila. Histopathologic assessment was done in Biology Research Laboratory in De La Salle University-Dasmariñas.

3.3.7Blood Collection

Cardiac puncture was used to collect blood samples from the Wistar rats. Three (3) ml of blood was obtained and centrifuged at 1500 rpm after the twenty-four (24) days of treatment trials. The serum acquired was stored in a temperature of about -20°C to -37°C for serum creatinine and blood urea nitrogen tests.

3.3.8Determinaton of Serum Creatinine, Blood Urea Nitrogen (BUN) and Kidney Index

Serum creatinine and blood urea nitrogen (BUN) levels were measured using prepared kits. The absorbance was obtained using spectrophotometer. Kidney index was done by means of measuring the damaged kidney comparing to the normal one.

3.3.9 Disposal of test organisms

Test organisms were euthanized based on the guidelines provided by the Bioethics Committee of the University. Rats were properly terminated after all necessary data have been collected.

3.4 Data Gathering and Statistical Analysis

On the seventh day of every treatment week, blood samples were extracted from the control group. The samples were then used for Serum Creatinine Test and Blood Urea Nitrogen Test. The individual treatment means were identified and were compared to the normal values of BUN and Serum Creatinine in wistar rats (Arejola & Austria, 2009)

Twenty-four (24) hours after the 4th week of the treatment and administration, one from the control groups was sacrificed for histopathology examination. The kidneys were carefully weighed and fixed with 10% buffer formalin solution in later preparation for light microscopy. (Sukandar, 2013). Kidney samples were sent and processed in Research Institute for Tropical Medicine (RITM) in Alabang, Muntinlupa City. Images were gathered for comparison and detection of abnormalities.