Synopsis about the work on AQP1,2,3 in Buffaloes in different seasons. To study thermotolerance & adoptation.

1. Relative expression analysis of aquaporins AQP1,
AQP2 and AQP3 in digestive and renal system of
buffaloes during different seasons.
Major Advisor
Dr. Ashutosh
Senior Scientist
Speaker
Suprith.B
16-M-AP-05
(M. V. Sc Scholar)
Synopsis seminar on

3. Discovery of Water Channels
• In 1992, Agre and associates injected the newly cloned complementary RNA (cDNA) for CHIP28
(channel-like integral membrane protein of 28 kDa) into Xenopus laevis oocytes and watched them
swell in hypo-osmotic medium.
• Discovered the first water channel, named aquaporin-1 (AQP1), and the identity of the channels
responsible for water transport across lipid bilayers was solved .
Figure 1. Consequences of AQP1 expression in Xenopus laevis
oocytes (the original experiment performed by Agre and associates).
When shifted to hypoosmotic media, the control oocyte (right) is
unaffected, whereas the oocyte injected with AQP1 complementary
RNA (left) exhibited high water permeability and exploded. Reprinted
with permission from [3] Preston G. M., Carroll T. P., Guggino
W. B. and Agre P. (1992) Appearance of water channels in
Xenopus oocytes expressing red cell CHIP28 protein. Science 256:
385–387. Copyright 1992 AAAS. www.sciencemag.org.

4. Currently, at least 13 isoforms of AQPs (AQP0–12) have been identified in mammals.
Into three groups. Specifically:
(1) Classical AQPs (AQP0, AQP1, AQP2, AQP4, AQP5, AQP6 and AQP8) are
selectively permeable to water.
(2) Aquaglyceroporins (AQP3, AQP7, AQP9 and AQP10) are permeable to
water,glycerol and urea.
(3) Non-classical AQPs (AQP11 and AQP12) .
• Until now, 11 isoforms of AQPs (AQP1–11) have been found to be expressed in the stomach and
intestines of mammals. It is true that multiple isoforms of AQPs are commonly co-expressed in
specific digestive organs or cells.
Alan et al., 2014)
Cont….

6. Zhu et al., 2016

7. AQP1
AQP1 constitutes approximately 1% of total cortical protein and has an important
function in the kidney. AQP1 is abundantly expressed in the proximal tubule and
descending thin limb of Henle .
Expression levels of AQP1 independent in response to either water restriction or
vasopressin infusion.
Humans lacking AQP1 have a urinary concentrating deficiency when deprived of
water.
(King et al. 2001).
Nielsen et al.,1995

8. AQP2
The collecting duct is the major site for vasopressin mediated regulation of body water
homeostasis. AQP2 is the predominant vasopressin-regulated aquaporin.
Yang B et al.,2001
Mice expressing an AQP2 gene with a mutation that causes NDI
in humans have a severe urine concentrating defect.
Die by 6 days of age unless supplemental fluids are given
(Yang et al. 2001).

9. AQP3
• AQP mRNA expression in gastrointestinal tract showed ubiquitous expression of AQP1
and AQP3 mRNA along the gastro intestinal tract.
• AQP3 mRNA expression was intense in the lower portion of the stomach, middle portion
of the small intestine, ileum, and both proximal and distal colon, whereas the expression
was less in the esophagus, upper portion of stomach, and jejunum of Human..
• VIP(Vasoactive intestinal peptide) upregulated the expression of AQP3 mRNA and
protein, and a cAMP-dependent pathway mediated this effect.
Koyama et al.,1999
Thiagarajah et al., 2015
Itoh et al., 2003

10. Introduction
Aquaporins (AQPs) are a class of membrane water channels ,Primary function is to
facilitate the passive transport of water across the plasma membrane of the cell.
The movement of water across cell membranes is fundamental to life. Water constitutes
roughly 70% of the mass of most living organisms.
Orderly distribution of water is required to maintain proper fluid balance within different
anatomic compartments.
Fluid transfer such as secretion and absorption is one of the major functions of the
digestive system.
The kidney is the main organ regulating body water homeostasis, filtering
approximately 180 l of blood per day in Human. Most of the filtered water is reabsorbed
in the proximal tubule,thin descending limb of Henle and collecting duct.
Pancreas Water can be secreted as digestive juices and then be absorbed by the
gastrointestinal epithelia.
Nejsum et al., 2005

11. • The water buffalo is ranked highly in the tropics and subtropics since it thrives
in hot conditions.
• The genus Bubalis surpasses the cattle genus Bos in its ability to adapt to the
hot, humid areas of muddy and swampy lands.
• It is because of its morphological and anatomical characteristics that the
buffalo is so well suited to hot and humid climates and muddy terrain.
Shafie et al., 1985

12. Wang et al., 2005

13. Major physiological functions of aquaporins
a | In kidney tubules and microvessels, a high
transepithelial water permeability in the
proximal tubule, thin descending limb of the loop
of Henle, vasa recta and collecting duct is
required for urine-concentrating function.
b | During fluid secretion by epithelial
cells, a high transepithelial water
permeability facilitates the active
secretion of near-isosmolar fluid.
Verkman et al.,2014

14. c | Aquaporin (AQP)-facilitated cell
migration involves the entry of water into
protruding lamellipodia in migrating cells.
d | Water permeation across the
blood–brain
barrier and the blood–CSF
(cerebrospinal fluid) barrier (that is,
the ependyma) facilitates the
movement of water into and out of the
brain.
Verkman et al.,2014

15. e | AQP4-facilitated water
transport in astrocytes during
potassium reuptake following
neuroexcitation causes
contraction of the extracellular
space, maintaining the driving force
for potassium reuptake.
f | AQP3 facilitates skin hydration
by maintaining high glycerol
levels in the stratum
corneum, which acts as a
humectant to retain water.Verkman et al.,2014

16. g | AQP3 maintains high cellular
glycerol levels for the generation of ATP and
lipid biosynthesis, which leads to cellular
proliferation.
h | AQP7 facilitates the exit of glycerol
from adipocytes, preventing the
intracellular accumulation of glycerol
and triglyceride. FFA, free fatty acid.
Verkman et al.,2014

18. AQP4 and AQP5 play important roles in water transport in the gastrointestinal tract. Co-
expression of several AQP family members in the large intestine makes it difficult to clarify the
specific function of each AQP in Buffalo Calves .
 AQP10 mRNA in the duodenum and jejunum of cattle could not be detected by RT-PCR due
to the fact that AQP10 is a pseudogene in cattle and in their relatives (sheep and goat).
In contrast to the findings in humans, rats and mice, there are limited reports regarding the
expression of water channel proteins in dairy animals.
Studies are undoubtedly needed to ascertain the role of each aquaporin in their possible
modulation in relation to feeding and the effect of single factors.
Luca et al., 2014
Pelagali et al., 2015
Tanaka et al., 2015
Review of Literature

19. Moreover,AQP1, AQP3, AQP7, AQP10 and AQP11 are abundantly expressed in the
small intestine, while AQP1, AQP3, AQP4 and AQP8 are the predominant isoforms
in the colon of rat.
pathological conditions related to water intake and outlet such as diarrhea or
malabsorption may be related with expression or the amounts of AQP family
members in the gastrointestinal tract.
The possible role(s) and involvement of AQP in physiological and pathological
conditions of the gastrointestinal tract remain to be studied.
Laforenza et al., 2012

20. The reduced mRNA expression of several AQPs (AQP1, AQP3, AQP7 and AQP8) in
human intestinal mucosa has been demonstrated at the early stage of inflammatory
bowel diseases (IBD), including Crohn’s disease and ulcerative colitis .
Notably, heat stroke has been shown to induce jejunum barrier damage and cell
apoptosis via the increased expression of AQP1, AQP3, AQP7, AQP8 and AQP11
mRNA .
• Enteritis, either in murine rodents or human, may be associated with alterations in
electrolyte and water transport mediated by a downregulation in AQPs’ expression.
Ricanek et al., 2015
Wang et al., 2015
Ricanek et al., 2015

21. The exposure to mercury, which inhibits of AQPs, significantly downregulated gastric AQP3
and AQP4 mRNA and their protein expression, as well as the AQP3 and AQP7 protein
expression in the small and large intestines of rats, thus leading to the accumulation of
intestinal fluids and, finally, diarrhea.
These results suggested that these AQPs may be potential targets for the prevention and
treatment of diarrhea in human and animals.
Anti-diarrhea effects of some substances, including emodin and berberine ,and the laxative
drugs magnesium sulfate(MgSO4) may involve the alteration of AQPs to regulate water
transport and absorption possibly, via the regulation of the cAMP-dependent PKA/p-CREB
signal pathway.
Bottino et al., 2016

22. Upregulation of AQP2 in the distal colon was found in cirrhotic rats with ascites, and its
expression is inhibited by Tolvaptan, which probably leads to decreased water
reabsorption and induces diarrhea in cirrhotic rats with ascites.
AQP gene transfer has been considered for treating salivary gland hypofunction,
which is a common side effect of radiation therapy ,Drugs that stimulate saliva secretion
by targeting acinar cells are generally ineffective.
Vasopressin increases water permeability of kidney collecting duct by inducing
translocation of aquaporin-CD water channels to plasma membrane.
AQP8 exhibits a preference for neutral NH3 molecules over water, suggesting a
physiological role in maintenance of acid-base equilibrium. In physiological
concentrations AQP8 may augment the basal NH3 conductivity 3- to 5-fold.
Chen et al., 2016
Nielsen et al., 1995
Saparov et al., 2006

23. • Cisplatin-induced polyuria is associated with a significant decrease in the expression
of collecting water channels AQP2 and AQP3 and PCT water channel AQP1 in the
inner medulla.
• Secretin induces the apical insertion of aquaporin-1 water channels in rat
cholangiocytes, It has provided first cohesive and comprehensive molecular
explanation for hormone-induced ductal bile secretion.
• The total retention time for digesta in the large intestine was approximately 30, 20,
and 9 h in the pigs, sheep, and cows respectively. The rate of water absorption from
the large intestine was most rapid in the cows and slowest in the pigs.
Kishore et al., 2000
Marinelli, et al., 1999
Hecker et al.,1995

24. Kishore et al.,2000

25. Koyama et al., 1999

26. Relative gene expression of aquaporin genes (AQP1, AQP3 and AQP5) and to
compare the metabolic profile and physiological adaptations of the native goats in
different agro-climatic regions.
 Presence of AQP1, AQP3 and AQP5 in the skin fibroblasts of the Sirohi and the
Barbari goats were observed in vitro studies and they play a major role in
Thermoregulation.
 Relative expression of AQP3 and AQP5 significantly increased with increased
temperatures and hyperosmotic stress in skin fibroblasts under in vitro .
 The Aldosterone, ADH and Angiotensin-II levels were significantly increased in
Sirohi breed of the arid region during summer.
 Sirohi breed had lower magnitude of increase in all physiological, hematological,
plasma electrolytes and biochemical components indicating its high drought and
thermotolerance.
WORK DONE AT NDRI:
Francis Femi, 2017

27. HORMONAL VARIATION IN SUMMER AND WINTER
• 47± 0.42ng/L WINTER
• 122.37±0.49 ng/L SUMMER
ALDOSTERONE
• 1.87± 0.03 ng/L WINTER
• 10.02±0.14ng/L SUMMER
ADH
• 20.72 ± 0.72ng/L WINTER
• 98.70±0.79ng/L SUMMER
ANGIOTENSINOGEN 2
BLOOD ION VARIATION IN SUMMER AND WINTER
•139.72 ± 0.90mmol/L WINTER
•147.14 ±1.01mmol/L SUMMER
Plasma Sodium
•5.12±0.09)mmol/L WINTER
•4.05± 0.10 mmol/L SUMMER
Plasma Potassium
•26.40 ±0.89mg/dL WINTER
•36.33 ±0.89mg/dL SUMMER
Blood Urea
Francis Femi, 2017

28. Aquaporins in Diseases
• Diahorea
• IBD
• Edema
• Mastitis
• Delayed wound healing/gangrene
• Metritis
• Parasitic infestation/ Worm load
• NMO (Neuromyelitis optica): an autoimmune disease caused by AQP4-specific antibodies.
• NMO is an autoimmune inflammatory disease of the CNS that leads to paralysis and loss of vision.
• NDI(Nephrogenic diabetes insipidus): a genetic disorder caused by loss-of-function AQP2 mutations.
• Loss-of-function mutations in AQP2 cause NDI, an autosomal hereditary disease that is characterized by severe polyuria and
polydipsia.
Verkman et al.,2014

29. Gaps in Knowledge
1. Role of Aquaporin Water Channels in GIT & Renal System is still unknown in Dairy Animals.
2. Aquaporins are majorly concerned about health and disease conditions .Ex- Oedema, Diarrhoea,
Cataract, Diabetes Insipidus,etc…
3.Aquaporins involvement in secretion, absorption, reabsorption, gas transport & solute transport in
different tissues need to be studied.
4.Buffalo is wallowing animal and have less number of sweat glands, information related to
presence of aquaporins in different systems for Fluid homeostasis & thermoregulation is not
available.

30. Objectives
Objective
1
• To study the expression of AQP1 and AQP3 genes in
digestive system of buffaloes during different
seasons.
Objective
2
• To study the expression of AQP1 and AQP2 genes
in renal system of buffaloes during different
seasons.
• To study the endocrine and metabolic profile in
relation to aquaporin expression in digestive
system and renal system in buffalos.
Objective
3

31. Plan of
Work

32. Plan of Work
• Pre slaughter blood collection from Lairage/slaughter house.
• Kidney,GIT dissection and sample collection.
• Storage in n-Hexane @ -80ºC.
• Collection of monthly weather data.
• Isolation of RNA,C-DNA synthesis.
• Primer designing, Real Time- PCR.
• Blood parameters estimation.
• Hormones- ADH, ANG-II, Secretin, Gastrin, Motilin.
• Physiological responses and surface temperature.
• Electrolytes- Na+, K+, Cl-
• Statistical data analysis.

33. Buffalo
Summer group
(n=6)
Winter group
(n=6)
Site of Sample Collection: Slaughter
house/during postmortem of institute animals
Plan of Work
Location of experiment : NDRI, Karnal
Techniques used
RT-PCR
Immunohistochemistry
ELISA/RIA
Haematology/CBC

34. Hypothesi
s
AQP1,AQP3 in Digestive system and AQP1,AQP2 in Renal
system of Buffalo play a Major role in maintaining fluid
homeostasis, Digetive juice / enzyme secretion and thermoregulation in
body. Aquaporin expression of GIT and renal system varies
according to different Season.

35. Expected Outcome
What are the aquaporins expressed in digestive system and
renal system of buffalo.
Any variation in aquaporin expression between different
seasons.
Variation in aquaporin expression according to endocrinal
variation and blood electrolyte concentration.

36. “”NO H2O, NO LIFE. SO, SAVE H2O SAVE LIFE.”

37. References
• Agre, P., M. Bonhivers, and M. Borgnia, 1998: The aquaporins,blueprints for cellular plumbing systems. J.
Biol. Chem. 273,14659–14662.
• Chen, C.; Chen, R.P.; Lin, H.H.; Zhang,W.Y.; Huang, X.L.; Huang, Z.M. Tolvaptan regulates aquaporin-2 and
fecal water in cirrhotic rats with ascites. World J. Gastroenterol. 2016, 22, 3363–3371.
• De Luca, A.; Vassalotti, G.; Pelagalli, A.; Pero, M.E.; Squillacioti, C.; Mirabella, N.; Lombardi, P.; Avallone,
L.Expression and localization of aquaporin-1 along the intestine of colostrum suckling buffalo calves.Anat.
Histol. Embryol. 2015, 44, 391–400.
• Dicay, M.S.; Hirota, C.L.; Ronaghan, N.J.; Peplowski, M.A.; Zaheer, R.S.; Carati, C.A.; MacNaughton,W.K.
Interferon- suppresses intestinal epithelial aquaporin-1 expression via Janus kinase and STAT3 activation.
PLoS ONE 2015, 10, e0118713.
• Hecker, J.F.; Grovum, W.L. Rates of passage of digesta and water absorption along the larg intestines of cows
and pigs. Aust. J. Biol. Sci. 1975, 28, 161–167.
• Ishibashi, K., S. Sasaki, K. Fushimi, S. Uchida, M. Kuwahara, H. Saito, T. Furukawa, K. Nakajima, Y.
Yamaguchi, T. Gojobori, and F. Marumo. Molecular cloning and expression of a member of the aquaporin
family with permeability to glycerol and urea in addition to water expressed at the basolateralmembrane of
kidney collecting duct cells. Proc. Natl. Acad. Sci.USA 91: 6269–6273, 1994.
• Koyama, Y.; Yamamoto, T.; Tani, T.; Nihei, K.; Kondo, D.; Funaki, H.; Yaoita, E.; Kawasaki, K.; Sato, N.;
Hatakeyama, K.; Kihara, I. Expression and localization of aquaporins in rat gastrointestinal tract. Am. J.
Physiol. 1999, 276, C621–C627.

38. • Matsuzaki, T., Y. Tajika, N. Tserentsoodol, T. Suzuki, T. Aoki,H. Hagiwara, and K. Takata, 2002:
Aquaporins: a water channel family. Anat. Sci. Int. 77, 85–93.
• Miranda K, et al. Characterization of a novel organelle in Toxoplasma gondii with similar
composition and function to the plant vacuole. Mol Microbiol. 2010; 76:1358–1375.
• Marinelli, R. A., and N. F. LaRusso. Solute and water transport pathways in cholangiocytes. In:
Seminars in Liver Disease.New York: Thieme, 1996, p. 221–229
• Squillacioti, C.; De Luca, A.; Pero, M.E.; Vassalotti, G.; Lombardi, P.; Avallone, L.; Mirabella, N.;
Pelagalli, A. Effect of colostrum and milk on small intestine expression of AQP4 and AQP5 in
newborn buffalo calves. Res. Vet. Sci. 2015, 103, 149–155.
• Jay R Thiagarajah, Dan Zhao, A S Verkman. Impaired enterocyte proliferation in aquaporin-3
deficiency in mouse models of colitis . Gastroenterology 2015;126:511–9.
• Pelagalli, A.; Squillacioti, C.; De Luca, A.; Pero, M.E.; Vassalotti, G.; Lombardi, P.; Avallone,
L.;Mirabella, N. Expression and localization of aquaporin 4 and aquaporin 5 along the large
intestine of colostrum-suckling buffalo calves. Anat. Histol. Embryol. 2015.
• Yamamoto, T.; Kuramoto, H.; Kadowaki, M. Downregulation in aquaporin 4 and aquaporin 8
expression of the colon associated with the induction of allergic diarrhea in a mouse model of food
allergy. Life Sci. 2007, 81, 115–120.

40. AQP8
• AQP8 was cloned from different rat ,mouse and human tissues and shows species differences
with respect to transport selectivity; only mouse AQP8 is capable of urea transport .
• In the kidney, AQP8 is distributed in intracellular vesicles throughout the cytoplasm in proximal
tubule and collecting duct cells .
• AQP8 is speculated to play a role in osmoequilibration between the cytoplasmic and vesicular
compartments , but functional studies regarding renal AQP8 expression have not been
published.
• Although precise subcellular localization of AQP8 remains to be identified by immunostaining,
it may be feasible to speculate a significant involvement of AQP8 in the enormous water
movement in jejunum and colon.
Elkjaer et al., 2001