HHS Public Access

Author manuscript
Hypertension. Author manuscript; available in PMC 2019 November 01.
Author Manuscript

Published in final edited form as:

Hypertension. 2018 November ; 72(5): 1172–1179. doi:10.1161/HYPERTENSIONAHA.118.11694.

Renal medullary interstitial COX-2 is essential in preventing salt-

sensitve hypertension and maintaining renal inner medulla/
papilla structural integrity
Ming-Zhi Zhang1,2, Suwan Wang1,2, Yinqiu Wang1,2, Yahua Zhang1, Chuan Ming Hao3, and
Raymond C Harris1,2,4
1Divisionof Nephrology and Hypertension, Department of Medicine, Vanderbilt University School
Author Manuscript

of Medicine, Nashville, Tennessee, U.S.A.

2Vanderbilt
Center for Kidney Disease, Vanderbilt University School of Medicine, Nashville,
Tennessee, U.S.A.
3Division of Nephrology, Huashan Hospital, Fudan University, Shanghai, China
4Department of Veterans Affairs, Nashville, Tennessee, U.S.A.

Abstract
Cyclooxygenase (COX)-derived prostaglandins regulate renal hemodynamics and salt and water
homeostasis. Inhibition of COX activity causes blood pressure elevation. In addition, chronic
analgesic abuse can induce renal injury, including papillary necrosis. COX-2 is highly expressed in
Author Manuscript

the kidney papilla in renal medullary interstitial cell (RMICs). However, its role in blood pressure
and papillary integrity in vivo has not been definitively studied. In mice with selective, inducible
RMIC COX-2 deletion, a high salt diet led to an increase in blood pressure that peaked at 4–5
weeks and was associated with increased papillary expression of aquaporin 2 and epithelial
sodium channel and decreased expression of cystic fibrosis transmembrane conductance regulator.
With continued high salt feeding, the mice with RMIC COX-2 deletion had progressive decreases
in blood pressure from its peak. After return to a normal salt diet for 3 weeks, blood pressure
remained low and was associated with a persistent urinary concentrating defect. Within two weeks
of institution of a high salt diet, increased apoptotic RMICs and collecting duct cells could be
detected in papillae with RMIC deletion of COX-2, and by 9 weeks of high salt, there was a
striking loss of the papillae. Therefore, renal medullary interstitial cell COX-2 expression plays a
crucial role in renal handling water and sodium homeostasis, preventing salt-sensitive
Author Manuscript

hypertension, and maintaining structural integrity of papilla.

Keywords
kidney; salt and water homeostasis; prostaglandin; chronic salt intake

Dr. Raymond C. Harris, C-3321 Medical Center North, Department of Medicine, Vanderbilt University School of Medicine, Nashville,
TN 37232Tel#: 615-322-2150. Fax #:615-343-2675. ray.harris@vanderbilt.edu. Dr. Ming-Zhi Zhang, S3223 Medical Center North
Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN 37232, ming-zhi.zhang@vanderbilt.edu.
Disclosures
None.
 Zhang et al. Page 2

Introduction
Author Manuscript

In the mammalian kidney, prostaglandins are important mediators of vascular tone and salt
and water balance and are involved in the mediation and/or modulation of hormonal action,
such as renin release and antagonism of vasopressin. Prostaglandins arise from enzymatic
metabolism of free arachidonic acid, which is cleaved from membrane phospholipids by
phospholipase A2 activity. Cyclooxygenase (prostaglandin synthase G2/H2, COX) is the
rate-limiting enzyme in metabolizing arachidonic acid to prostaglandin G2 and subsequently
to prostaglandin H2, which serves as the precursor for subsequent metabolism by
prostaglandin and thromboxane synthases. Two isoforms of COX exist in mammals,
“constitutive” COX-1 and inflammatory-mediated and glucocorticoid-sensitive COX-2.
COX-1 is expressed in mammalian kidney in vasculature, glomerular mesangial cells and
collecting duct. The highest expression of COX-2 in mammalian kidney is in the macula
densa in the cortex and in lipid-rich interstitial cells in the medulla 1, although other cell
Author Manuscript

types have also been shown to express detectable levels of the enzyme under some
conditions and in some species, including afferent arterioles, podocytes, and intercalated
collecting duct cells 2, 3.

Essential hypertension is a major source of morbidity and mortality in the general

population, and a significant percentage of hypertensive patients manifest salt-sensitive
hypertension. In addition, at least a quarter of normotensive individuals also show salt
sensitivity 4. The etiology of salt-sensitive hypertension is undoubtedly multifactorial, but
both experimental and epidemiologic evidence link abnormalities in the COX-2/
prostaglandin system to its pathogenesis 5. COX-2 inhibitors, as well as non-selective non-
steroidal anti-inflammatory drugs (NSAIDs), are among the most commonly prescribed and
over-the-counter medications, and are known to elevate blood pressure and antagonize the
Author Manuscript

blood pressure-lowering effect of antihypertensive medication to an extent that may

potentially increase hypertension-related morbidity in many users 6–8.

Prostaglandins inhibit medullary sodium and water excretion by integrated actions to inhibit
thick ascending limb NaCl reabsorption and to inhibit collecting duct sodium and water
reabsorption. COX-2 expression increases in renal medullary interstitial cells (RMICs) in
response to a high salt diet 9. However, it remains uncertain whether these cells are the major
source of the prostaglandins that mediate the natriuretic and diuretic effects. The goal of the
present studies was to determine whether selective deletion of RMIC COX-2 alters the
kidney’s ability to regulate salt and water homeostasis using inducible Tenascin-C-
CreER2:COX-2f/f mice (RMIC COX-2−/− mice).
Author Manuscript

Materials and Methods

The data will not be available to other researchers. We will make the mice available to other
researchers.

Animal studies.
All animal experiments were performed in accordance with the guidelines and with the
approval of the Institutional Animal Care and Use Committee of Vanderbilt University.

Hypertension. Author manuscript; available in PMC 2019 November 01.

 Zhang et al. Page 3

Generation of mice with COX-2 deletion in renal medullary interstitial cells.

Author Manuscript

COX-2f/f mice in which exons 6, 7, and 8 of Cox-2 gene are flanked by Lox P sites were
originally generated in Dr. Fitzgerald’s laboratory 10. The COX-2f/f mice were crossed with
inducible Tenascin-C-CreER2 mice, in which Cre recombinase activity is induced in adult
mice by tamoxifen administration 11. Tenascin-C is an effective marker of medullary
interstitial cells 12. Both mouse strains were on the C57BL/6 background, which is normally
resistant to COX-2 deficiency-mediated hypertension 13. At 2 months of age, both COX-2f/f
(wild type) and Tenascin-C-CreER2; COX-2f/f (RMIC COX-2−/−) mice were treated with
tamoxifen (Sigma, T6648, dissolved in core oil) at a dose of 160 mg/kg by intraperitoneal
injection for 5 consecutive days. Two weeks after the last injection, the mice were used for
experiments.

Genotyping.
Author Manuscript

DNA was isolated from tail snips. Cre and Cox-2 allele genotypes were determined as
previously described 10, 11. All PCR reactions were carried out using an MJ Research
thermal cycler.

TUNEL staining.
Apoptotic cells were detected by TACS®2 TdT-DAB in situ apoptosis detection kit
(Trevigen, Catalog#: 4810–30-K) according to manufacturer’s instruction.

RNA isolation and quantitative RT-PCR.

Total RNAs from inner medullae/papillae were isolated using TRIzol reagents (Invitrogen).
Quantitative RT-PCR was performed using TaqMan real time PCR (7900HT, Applied
Biosystems). The Master Mix and all gene probes were also purchased from Applied
Author Manuscript

Biosystems. The probes used in the experiments included mouse S18 (Mm02601778),
COX-2 (Mm00478374), cystic fibrosis transmembrane conductance regulator (CFTR)
(Mm00445197), aquaporin 2 (AQP2) (Mm00437575), Atp1a1 (Mm00523255), Atp1b1
(Mm00437612), epithelial sodium channel subunit α (ENaC-α) (Mm00803386), ENaC-β
(Mm00441215), and ENaC-γ (Mm00441228).

Antibodies.
Rabbit anti-AQP2 antibody was purchased from Abcam (ab15116, Cambridge, MA), rabbit
anti-COX-2 antibodies were from LifeSpan BioSciences (LS-C210609) as well as Thermo
Fisher (MA5–14568), and monoclonal 4-Hydroxynonenal (4-HNE) antibody was from
R&D systems (MAB3249).
Author Manuscript

Immunohistochemistry staining.
The animals were anesthetized with Nembutal (70 mg/kg, i.p.) and given heparin (1,000
units/kg, i.p.) to minimize coagulation. One kidney was removed for qRT-PCR, and the
animal was perfused with FPAS (3.7% formaldehyde, 10 mM sodium m-periodate, 40 mM
phosphate buffer, and 1% acetic acid) through the aortic trunk cannulated by means of the
left ventricle. The fixed kidneys were dehydrated through a graded series of ethanols,

Hypertension. Author manuscript; available in PMC 2019 November 01.

 Zhang et al. Page 4

embedded in paraffin, sectioned (4 μm), and mounted on glass slides. Immunostaining was
carried out as in previous reports 14.
Author Manuscript

Blood pressure measurement using tail-cuff and carotid catheterization.

For tail-cuff blood pressure measurements, mice were trained for 3 consecutive days at room
temperature before systolic blood pressure was recorded using a tail-cuff monitor (BP-2000
BP Analysis System, Visitech System). Systolic blood pressures recorded in 2 days were
averaged and used as values from one mouse. For catheterization blood pressure
measurements, blood pressure was recorded every minute for one hour, and the average of
all recorded blood pressure was used as blood pressure from one mouse. Blood pressure
measurement using carotid catheterization was performed through the Vanderbilt MMPC.
Mice were anesthetized with 80 mg/kg of ketamine (Ft Dodge Laboratories) and 8 mg/kg of
inactin (BYK) by intraperitoneal injection and were placed on a temperature-controlled pad.
Author Manuscript

After tracheostomy, phycoerythrin 10 tubing was inserted into the right carotid artery. The
catheter was tunneled under the skin, exteriorized, secured at the back of the neck, filled
with heparinized saline, and sealed. The catheterized mouse was housed individually and 24
h later, data were collected with a Blood Pressure Analyzer (Micro-Med) 15. Systolic blood
pressure (SBP), diastolic blood pressure (DBP), mean arterial pressure (MAP), and heart
rate were recorded every minute for 60 minutes. The average of the recorded data from each
individual awake mouse was used.

Acute volume expansion

Mice (on a high salt diet for 2 week to increase RMIC COX-2) were intraperitoneally
injected with isotonic saline (10% of their body weight), then immediately placed in a 96
well chamber as shown in supplemental Figure S1, and 4-hour urine was collected. Urinary
Author Manuscript

sodium was determined using a flame photometer.

Urinary osmolality.
Mice were water deprived for 12 h (8:00 PM to 8:00 AM), and then urine was collected for
measurement of urine osmolality using a microosmometer (μOsmette, Precision
Systems,Natick, MA).

Statistics.
All values are presented as means, with error bars representing ± SEM. P values were
calculated by Student’s t test.

Results
Author Manuscript

In order to delete COX-2 expression selectively in RMICs, we crossed COX-2f/f mice with
inducible Tenascin-C-CreER2 transgenic mice (Tenascin-C-CreER2:COX-2f/f mice, RMIC
COX-2−/−), in which Cre recombinase activity is induced in adult mice by tamoxifen
administration 16. Tenascin C is an effective RMIC marker 12. Inducible deletion of COX-2
expression in RMIC in adult mice avoided any potential developmental abnormality of the
inner medulla/papilla. As indicated in Figure 1A, COX-2 mRNA expression was markedly
decreased in papillae after tamoxifen administration. To confirm further the effectiveness of

Hypertension. Author manuscript; available in PMC 2019 November 01.

 Zhang et al. Page 5

COX-2 deletion in renal medullary interstitial cells, COX-2 immunostaining was performed
Author Manuscript

in kidneys after high salt diet for 2 weeks, because high salt intake increases interstitial
COX-2 expression 17, 18. As indicated in Figure 1B, strong COX-2 immunoreactivity was
apparent in many interstitial cells (arrows) in wild type (COX-2f/f) mice, but only a few
interstitial cells were COX-2 positive in kidneys of RMIC COX-2−/− mice. Therefore,
COX-2 expression was effectively deleted in medullary interstitial cells in RMIC COX-2−/−
mice.

To investigate the role of RMIC COX-2 in regulation of salt and water excretion (natriuresis
and diuresis) in response to acute volume expansion, mice with or without a high salt diet
for 2 weeks in order to increase RMIC COX-2 expression were intraperitoneally
administrated 10% body weight of warm isotonic saline. At baseline or after high salt diet
for 2 weeks, RMIC COX-2−/− mice had impaired ability to rapidly excrete salt and water,
compared to littermate COX-2f/f mice (Figure 2). Therefore, RMIC COX-2 plays an
Author Manuscript

important role to facilitate sodium and water excretion efficiently in response to acute
volume expansion.

To investigate the potential mechanisms underlying renal medullary interstitial cell COX-2
mediated-natriuresis and diuresis, we determined the expression of transporters expressed in
the inner medulla. Expression and activation of the water channel, aquaporin 2 (AQP2) is
known to be down-regulated by medullary prostaglandins 19–22. As indicated in Figure 3A,
AQP2 mRNA levels were markedly higher in high salt-treated RMIC COX-2−/− mice than
high salt-treated COX-2f/f mice. In addition, immunostaining indicated that the density of
AQP2 expression in renal medullary collecting duct epithelial cells (particularly, inner
medulla) was more intense in RMIC COX-2−/− mice than in COX-2f/f mice. Furthermore,
more AQP2 immunoreactivity was observed in the apical membrane of collecting duct
Author Manuscript

epithelial cells from RMIC COX-2−/− mice, consistent with higher water channel activity.

The cystic fibrosis transmembrane conductance regulator (CFTR) is expressed along all the
nephron segments and facilitates chloride secretion in the medullary collecting duct 23, 24.
We found that CFTR mRNA levels were markedly lower in RMIC COX-2−/− mice than in
COX-2f/f mice (Figrue 3B), suggesting that COX-2-mediated prostanoids from renal
medullary interstitial cells may increase chloride secretion through upregulation of CFTR in
the medulla. In addition, deletion of RMIC COX-2 led to increases in renal medullary
mRNA levels of α and γ subunits of ENaC and β subunit of Na+/K+ ATPase (Figure 3C).

Global COX-2 deletion did not affect blood pressure in C57/BL6 mice on a normal salt diet
13,and high salt alone did not markedly increase blood pressure in wild type mice or rats
while high salt plus selective COX-2 inhibition increased blood pressure in both 25, 26. In
Author Manuscript

preliminary studies, we also found that RMIC COX-2−/− did not affect blood pressure when
fed a normal salt diet. However, when mice were administered a high salt diet and blood
pressure was continuously measured by tail cuff plethysmography, COX-2f/f mice had
minimal increases in systolic blood pressure, but RMIC COX-2−/− mice developed
progressively increased systolic blood pressure for the first 3–5 weeks of treatment (Figure
4A). Similarly, increased SBP, DBP, and MAP were seen when blood pressure was
measured with indwelling carotid catheterization in awake mice on a high salt diet for 4

Hypertension. Author manuscript; available in PMC 2019 November 01.

 Zhang et al. Page 6

weeks (DBP: 101.1 ± 0.9 vs. 88.8 ± 2.4 mmHg, P < 0.005; SBP: 131.6 ± 1.5 vs. 114.3 ± 3.0
mmHg, P < 0.001; MAP: 117.2 ± 4.0 vs. 101.8 ± 1.8 mmHg, P < 0.005. N = 8 in COX-2f/f
Author Manuscript

group and n = 6 in RMIC COX-2−/− group) (Figure 4B). Heart rate difference was minimal
between two groups (524 ± 13 vs. 473 ± 17 of COX-2f/f, P > 0.05).

When the mice were treated with high salt for an extended period of time (up to 9 weeks),
blood pressure in the RMIC COX-2−/− mice progressively decreased from its peak, and
eventually fell to levels lower than in the COX-2f/f mice (Figure 4A). Even after return to a
normal salt diet for 3 weeks after 9-weeks of high salt diet, RMIC COX-2−/− mice still had
persistently lower blood pressures (Figure 4C), and a persistent urinary concentrating defect
compared to COX-2f/f mice (Figure 4D). However, baseline urinary concentrating ability
was comparable between COX-2f/f mice and RMIC COX-2−/− mice (mOsm/kg H2O: 3300
± 95 vs. 3129 ± 113 of COX-2f/f, P > 0.05. N = 8 in COX-2f/f group and n = 9 in COX-2−/−
group).
Author Manuscript

Morphologic examination either by MRI or direct examination indicated striking loss of

papillae in the RMIC COX-2−/− mice in response to chronic high salt exposure (Figure 5),
which was not seen in RMIC COX-2−/− mice maintained on the normal salt diet (not
shown). Of note, COX-2f/f mice fed the chronic high salt diet exhibited normal papillae.

To investigate whether RMIC COX-2-derived prostanoids exerted cytoprotective effects on

structures in the inner medulla, we determined that there was increased TUNEL staining in
papillae of RMIC COX-2−/− mice as early as two weeks after institution of a high salt diet
(Figure 6A) and increased oxidative stress, as indicated by increased papillary 4-HNE
immunostaining (Figure 6B).
Author Manuscript

Discussion
With salt loading, PGE2 production in the medulla increases and inhibits water reabsorption
by antagonizing vasopressin action and decreasing AQP2 expression 22 and stimulates salt
excretion by inhibiting sodium reabsorption by decreasing ENaC expression and stimulating
chloride excretion through activation of cystic fibrosis transmembrane conductance regulator
(CFTR) 24. The current studies utilized inducible COX-2 deletion in renal medullary
interstitial cells to investigate the potential role of RMIC COX-2 expression in renal
handling of sodium and water homeostasis and maintenance of the structural integrity of the
papilla. RMIC COX-2−/− mice exhibited impaired renal sodium and water excretion in
response to acute volume expansion; inner medullary AQP2 mRNA and protein levels were
higher in RMIC COX-2−/− mice than in COX-2f/f mice in response to a high salt intake, and
mRNA expression of other relevant sodium transporters, including α and γ subunits of
Author Manuscript

eNaC and β subunit of Na+/K+-ATPase were also increased in RMIC COX-2−/− mice while
expression of CFTR was decreased. The other striking finding was that RMIC COX-2−/−
mice initially developed salt-sensitive hypertension, but long-term high salt intake led to
papillary loss in RMIC COX-2−/− mice, in association with lower blood pressure and
impaired urinary concentrating ability, at least in part due to increased apoptosis and
oxidative stress in both RMIC and surrounding collecting duct cells. Of note, the persistent
low blood pressure and the impaired urinary concentrating ability seen in RMIC COX-2−/−

Hypertension. Author manuscript; available in PMC 2019 November 01.

 Zhang et al. Page 7

mice after long-term high salt diet is due to papillary damage or loss, rather than a
Author Manuscript

physiological effect.

The adult mammalian kidney is a particularly rich source of prostaglandin as well as an

important biological target of these prostaglandins 27. In the medulla, COX-2 is primarily
expressed in medullary interstitial cells, particularly in the papilla tip, and a high salt diet has
been shown to up-regulate COX-2 expression in renal medullary interstitial cells 1, 28.
COX-2 inhibitors reduce urinary sodium excretion during an initial period of salt loading
25, 29–32. In rats with uninephrectomy plus high salt diet, medullary infusion of a selective

COX-2 inhibitor resulted in salt-sensitive hypertension 33. However, these studies did not
determine the source of COX-2 derived prostanoids. It has been suggested that NSAID/
COX-2 inhibitor-induced increases in blood pressure might be due in part to inhibition of
endothelial COX-2-derived prostacyclin/PGI2 34, 35 or myeloid COX-2-derived PGE2 26.
Our current study provides clear proof of the importance of COX-2 expression in RMIC in
Author Manuscript

mediating renal salt and water regulation and demonstrate that inhibition of renal medullary
interstitial cell COX-2 contributes to salt-sensitive hypertension.

In addition to their role in regulating salt and water homeostasis, there is evidence that inner
medullary prostanoids provide an important survival function for inner medullary structures
since papillary necrosis or apoptosis has been reported with both non-selective and COX-2-
selective inhibitors 36, 37. In mouse renal inner medullary collecting duct cells, inhibition of
PGE2 production did not affect cell proliferation or apoptosis 38, indicating that collecting
duct was not the source of cytoprotective prostanoids. Although cultured medullary
fibroblasts have been shown to undergo apoptosis in response to hypertonic stress following
COX-2 inhibition 39, the role of COX-2 derived prostaglandins from RMICs in maintenance
of papillary integrity in vivo was previously undetermined. The present studies clearly
Author Manuscript

demonstrate that COX-2 in RMICs plays an important role in maintaining structural

integrity of all papillary structures subjected to the stress of a high salt diet.

An intriguing recent study examining the role of COX-2 vs. COX-1 found that although
COX-1 generates orders of magnitude more prostanoids than COX-2, it cannot compensate
for loss of COX-2 in renal development, suggesting that COX-2 may serve other functions in
addition to, or instead of prostanoid function 40. Further studies will be necessary to
determine the mechanisms by which RMIC COX-2 provides papillary cytoprotection.

In summary, renal medullary interstitial cell COX-2 expression plays an important role in
renal handling water and sodium homeostasis, preventing salt-sensitive hypertension, and
maintaining structural integrity of papilla.
Author Manuscript

Perspectives
Our data clearly demostarte that COX-2 is essential to promote sodium and water excretion
in response to acute salt loading as well as chronic salt loading. Specific inhibition of
cyclooxygenase 2 in interstitial cells in the kidney medulla is an important mediator of
NSAID-induced hypertension and analgesic nephropathy.

Hypertension. Author manuscript; available in PMC 2019 November 01.

 Zhang et al. Page 8

Supplementary Material
Author Manuscript

Refer to Web version on PubMed Central for supplementary material.

Acknowledgments
Sources of Funding

This work was supported by NIH grants DK-95785, DK-51265, and DK-114809 (to MZZ and RCH); DK-62794
and DK-103067 (to RCH); and funds from the Department of Veterans Affairs (to RCH). Carotid catheterization
was performed through the Vanderbilt Mouse Metabolic Phenotyping Center (MMPC, DK059637).

References
1. Harris RC. Cyclooxygenase-2 in rat nephron development. Am J Physiol. 1997;273:F994–1002.
[PubMed: 9435689]
Author Manuscript

2. Harris RC. Cox-2 and the kidney. J Cardiovasc Pharmacol. 2006;47 Suppl 1:S37–42. [PubMed:
16785827]
3. Stegbauer J, Chen D, Herrera M, Sparks MA, Yang T, Konigshausen E, Gurley SB, Coffman TM.
Resistance to hypertension mediated by intercalated cells of the collecting duct. JCI Insight.
2017;2:e92720. [PubMed: 28405625]
4. Kanbay M, Chen Y, Solak Y, Sanders PW. Mechanisms and consequences of salt sensitivity and
dietary salt intake. Curr Opin Nephrol Hypertens. 2011;20:37–43. [PubMed: 21088577]
5. Kohsaka S, Volcik KA, Folsom AR, Wu KK, Ballantyne CM, Willerson JT, Boerwinkle E.
Increased risk of incident stroke associated with the cyclooxygenase 2 (cox-2) g-765c
polymorphism in african-americans: The atherosclerosis risk in communities study. Atherosclerosis.
2008;196:926–930. [PubMed: 17350020]
6. Johnson AG, Nguyen TV, Day RO. Do nonsteroidal anti-inflammatory drugs affect blood pressure?
A meta-analysis. Ann Intern Med. 1994;121:289–300. [PubMed: 8037411]
7. Cheng Y, Wang M, Yu Y, Lawson J, Funk CD, Fitzgerald GA. Cyclooxygenases, microsomal
prostaglandin e synthase-1, and cardiovascular function. J Clin Invest. 2006;116:1391–1399.
Author Manuscript

[PubMed: 16614756]
8. Elliott WJ. Do the blood pressure effects of nonsteroidal antiinflammatory drugs influence
cardiovascular morbidity and mortality? Curr Hypertens Rep. 2010;12:258–266. [PubMed:
20524091]
9. Abou-Issa HM, Alshafie GA, Seibert K, Koki AT, Masferrer JL, Harris RE. Dose-response effects of
the cox-2 inhibitor, celecoxib, on the chemoprevention of mammary carcinogenesis. Anticancer
Res. 2001;21:3425–3432. [PubMed: 11848504]
10. Wang D, Patel VV, Ricciotti E, Zhou R, Levin MD, Gao E, Yu Z, Ferrari VA, Lu MM, Xu J, Zhang
H, Hui Y, Cheng Y, Petrenko N, Yu Y, FitzGerald GA. Cardiomyocyte cyclooxygenase-2
influences cardiac rhythm and function. Proc Natl Acad Sci U S A. 2009;106:7548–7552.
[PubMed: 19376970]
11. He W, Xie Q, Wang Y, Chen J, Zhao M, Davis LS, Breyer MD, Gu G, Hao CM. Generation of a
tenascin-c-creer2 knockin mouse line for conditional DNA recombination in renal medullary
interstitial cells. PLoS One. 2013;8:e79839. [PubMed: 24244568]
Author Manuscript

12. Neelisetty S, Alford C, Reynolds K, Woodbury L, Nlandu-Khodo S, Yang H, Fogo AB, Hao CM,
Harris RC, Zent R, Gewin L. Renal fibrosis is not reduced by blocking transforming growth factor-
beta signaling in matrix-producing interstitial cells. Kidney Int. 2015;88:503–514. [PubMed:
25760325]
13. Yang T, Huang YG, Ye W, Hansen P, Schnermann JB, Briggs JP. Influence of genetic background
and gender on hypertension and renal failure in cox-2-deficient mice. Am J Physiol Renal Physiol.
2005;288:F1125–1132. [PubMed: 15613621]

Hypertension. Author manuscript; available in PMC 2019 November 01.

 Zhang et al. Page 9

14. Cheng H, Wang S, Jo YI, Hao CM, Zhang M, Fan X, Kennedy C, Breyer MD, Moeckel GW,
Harris RC. Overexpression of cyclooxygenase-2 predisposes to podocyte injury. J Am Soc
Author Manuscript

Nephrol. 2007;18:551–559. [PubMed: 17202413]

15. Yao B, Harris RC, Zhang MZ. Intrarenal dopamine attenuates deoxycorticosterone acetate/high
salt-induced blood pressure elevation in part through activation of a medullary cyclooxygenase 2
pathway. Hypertension. 2009;54:1077–1083. [PubMed: 19770404]
16. Abu El-Asrar AM, Mohammad G, Nawaz MI, Siddiquei MM, Kangave D, Opdenakker G.
Expression of lysophosphatidic acid, autotaxin and acylglycerol kinase as biomarkers in diabetic
retinopathy. Acta Diabetol. 2013;50:363–371. [PubMed: 22864860]
17. Harris RC, McKanna JA, Akai Y, Jacobson HR, Dubois RN, Breyer MD. Cyclooxygenase-2 is
associated with the macula densa of rat kidney and increases with salt restriction. J Clin Invest.
1994;94:2504–2510. [PubMed: 7989609]
18. Chen CL, Yu X, James IO, Zhang HY, Yang J, Radulescu A, Zhou Y, Besner GE. Heparin-binding
egf-like growth factor protects intestinal stem cells from injury in a rat model of necrotizing
enterocolitis. Lab Invest. 2012;92:331–344. [PubMed: 22157721]
19. Jia Z, Wang H, Yang T. Mice lacking mpges-1 are resistant to lithium-induced polyuria. Am J
Author Manuscript

Physiol Renal Physiol. 2009;297:F1689–1696. [PubMed: 19692487]

20. Soodvilai S, Jia Z, Wang MH, Dong Z, Yang T. Mpges-1 deletion impairs diuretic response to
acute water loading. Am J Physiol Renal Physiol. 2009;296:F1129–1135. [PubMed: 19225050]
21. Kim GH, Choi NW, Jung JY, Song JH, Lee CH, Kang CM, Knepper MA. Treating lithium-induced
nephrogenic diabetes insipidus with a cox-2 inhibitor improves polyuria via upregulation of aqp2
and nkcc2. Am J Physiol Renal Physiol. 2008;294:F702–709. [PubMed: 18216147]
22. Cheng X, Zhang H, Lee HL, Park JM. Cyclooxygenase-2 inhibitor preserves medullary
aquaporin-2 expression and prevents polyuria after ureteral obstruction. J Urol. 2004;172:2387–
2390. [PubMed: 15538275]
23. Souza-Menezes J, da Silva Feltran G, Morales MM. Cftr and tnr-cftr expression and function in the
kidney. Biophys Rev. 2014;6:227–236. [PubMed: 28510183]
24. Rajagopal M, Thomas SV, Kathpalia PP, Chen Y, Pao AC. Prostaglandin e2 induces chloride
secretion through crosstalk between camp and calcium signaling in mouse inner medullary
collecting duct cells. Am J Physiol Cell Physiol. 2014;306:C263–278. [PubMed: 24284792]
Author Manuscript

25. Yao B, Harris RC, Zhang MZ. Interactions between 11beta-hydroxysteroid dehydrogenase and
cox-2 in kidney. Am J Physiol Regul Integr Comp Physiol. 2005;288:R1767–1773. [PubMed:
15718388]
26. Zhang MZ, Yao B, Wang Y, Yang S, Wang S, Fan X, Harris RC. Inhibition of cyclooxygenase-2 in
hematopoietic cells results in salt-sensitive hypertension. J Clin Invest. 2015;125:4281–4294.
[PubMed: 26485285]
27. Zhang MZ, Wang JL, Cheng HF, Harris RC, McKanna JA. Cyclooxygenase-2 in rat nephron
development. Am J Physiol. 1997;273:F994–1002. [PubMed: 9435689]
28. Schnermann J, Traynor T, Yang T, Arend L, Huang YG, Smart A, Briggs JP. Tubuloglomerular
feedback: New concepts and developments. Kidney Int Suppl. 1998;67:S40–45. [PubMed:
9736251]
29. Catella-Lawson F, McAdam B, Morrison BW, Kapoor S, Kujubu D, Antes L, Lasseter KC, Quan
H, Gertz BJ, FitzGerald GA. Effects of specific inhibition of cyclooxygenase-2 on sodium balance,
hemodynamics, and vasoactive eicosanoids. J Pharmacol Exp Ther. 1999;289:735–741. [PubMed:
10215647]
Author Manuscript

30. Rodriguez F, Llinas MT, Gonzalez JD, Rivera J, Salazar FJ. Renal changes induced by a
cyclooxygenase-2 inhibitor during normal and low sodium intake. Hypertension. 2000;36:276–
281. [PubMed: 10948090]
31. Qi Z, Hao CM, Langenbach RI, Breyer RM, Redha R, Morrow JD, Breyer MD. Opposite effects of
cyclooxygenase-1 and −2 activity on the pressor response to angiotensin ii. J Clin Invest.
2002;110:61–69. [PubMed: 12093889]
32. Zewde T, Mattson DL. Inhibition of cyclooxygenase-2 in the rat renal medulla leads to sodium-
sensitive hypertension. Hypertension. 2004;44:424–428. [PubMed: 15314032]

Hypertension. Author manuscript; available in PMC 2019 November 01.

 Zhang et al. Page 10

33. Zewde T, Mattson DL. Inhibition of cyclooxygenase-2 in the rat renal medulla leads to sodium-
sensitive hypertension. Hypertension. 2004;44:424–428. [PubMed: 15314032]
Author Manuscript

34. Francois H, Athirakul K, Howell D, Dash R, Mao L, Kim H-S, Rockman HA, FitzGerald GA,
Koller BH, Coffman TM. Prostacyclin protects against elevated blood pressure and cardiac
fibrosis. Cell Metabolism. 2005;2:201–207. [PubMed: 16154102]
35. Yu Y, Ricciotti E, Scalia R, Tang SY, Grant G, Yu Z, Landesberg G, Crichton I, Wu W, Pure E,
Funk CD, FitzGerald GA. Vascular cox-2 modulates blood pressure and thrombosis in mice. Sci
Transl Med. 2012;4:132ra154.
36. Segasothy M, Samad SA, Zulfigar A, Bennett WM. Chronic renal disease and papillary necrosis
associated with the long-term use of nonsteroidal anti-inflammatory drugs as the sole or
predominant analgesic. Am J Kidney Dis. 1994;24:17–24. [PubMed: 8023820]
37. Akhund L, Quinet RJ, Ishaq S. Celecoxib-related renal papillary necrosis. Arch Intern Med.
2003;163:114–115. [PubMed: 12523925]
38. Rocha GM, Michea LF, Peters EM, Kirby M, Xu Y, Ferguson DR, Burg MB. Direct toxicity of
nonsteroidal antiinflammatory drugs for renal medullary cells. Proc Natl Acad Sci U S A.
2001;98:5317–5322. [PubMed: 11320259]
Author Manuscript

39. Lopez-Rodriguez C, Antos CL, Shelton JM, Richardson JA, Lin F, Novobrantseva TI, Bronson RT,
Igarashi P, Rao A, Olson EN. Loss of nfat5 results in renal atrophy and lack of tonicity-responsive
gene expression. Proc Natl Acad Sci U S A. 2004;101:2392–2397. [PubMed: 14983020]
40. Li X, Mazaleuskaya LL, Ballantyne LL, Meng H, FitzGerald GA, Funk CD. Differential
compensation of two cyclooxygenases in renal homeostasis is independent of prostaglandin-
synthetic capacity under basal conditions. FASEB J. 2018:fj201800252R.
Author Manuscript
Author Manuscript

Hypertension. Author manuscript; available in PMC 2019 November 01.

 Zhang et al. Page 11
Author Manuscript

Novelty and Significance

What is New?
Using an inducible COX-2 knockout mdoel, we investigated the potential role of renal
medullary interstitial cell COX-2 in renal handling of sodium and water homeostasis and
maintenance of the structural integrity of the papilla. We definitively determined that
renal medullary interstitial cell COX-2 is essential to excrete excessive sodium in
response to acute salt loading and to prevent salt-sensitive hypertension and to protect
against papillary damage in response to chronic high salt intake.

What is Relevant?
These studies demonstrate that specific inhibition of cyclooxygenase 2 in interstitial cells
in the kidney medulla is an important mediator of NSAID-induced hypertension and
Author Manuscript

analgesic nephropathy.

Summary
Renal medullary interstitial cell COX-2-mediated prostaglandin plays an important role
in handing water and sodium homeostasis, preventing salt-sensitive hypertension, and
maitaining structural integrity of papilla.
Author Manuscript
Author Manuscript

Hypertension. Author manuscript; available in PMC 2019 November 01.

 Zhang et al. Page 12
Author Manuscript
Author Manuscript

Figure 1. Renal medullary interstitial cell COX-2 was effectively deleted in Tenascin-C-CreER2:
COX-2f/f (RMIC COX-2f/f) mice.
A: After tamoxifen administration for 2 weeks, renal inner medullae/papillae were dissected
and COX-2 mRNA was quantitated by qPCR. COX-2 mRNA levels in renal medulla/papilla
were significantly reduced in RMIC COX-2−/− mice. ***P < 0.001, n = 6 in COX-2f/f mice
Author Manuscript

and n = 5 in RMIC COX-2−/− mice. All values are means ± SEM. P value was calculated by
Student’s t test.
B: After administration of a high salt diet for 2 weeks, COX-2 positive cells were found in
many inner medullary interstitial cells in COX-2f/f mice (arrows), but only a few inner
medullary interstitial cells were COX-2 positive in RMIC COX-2−/− mice. Scale bar: 50 μM.
Author Manuscript

Hypertension. Author manuscript; available in PMC 2019 November 01.

 Zhang et al. Page 13
Author Manuscript
Author Manuscript

Figure 2. RMIC COX-2 deficiency led to impaired diuresis and natriuresis in response to acute
volume expansion.
Acute volume expansion was carried out in mice with normal chow or mice on a high salt
diet for 2 weeks. A: RMIC COX-2 deletion led to decreases in urine volume and sodium
excretion at baseline. *P < 0.05, n = 4 in both groups.
B: RMIC COX-2 deficiency decreased urine volume and sodium excretion 2 weeks after
high salt diet. *P < 0.05, n = 5 in COX-2f/f mice and n = 3 in RMIC COX-2−/− mice. All
values are means ± SEM. P values were calculated by Student’s t test.
Author Manuscript
Author Manuscript

Hypertension. Author manuscript; available in PMC 2019 November 01.

 Zhang et al. Page 14
Author Manuscript

Figure 3. RMIC COX-2 deficiency led to increases in expression of sodium transporters and
Author Manuscript

aquaporin 2 but decreases in CFTR.

Mice were fed with high salt diet for 2 weeks. A: RMIC COX-2 deficiency led to increases
in aquaporin 2 (AQP2) mRNA and protein expression in the inner medullae/papillae. In
addition, the immunostaining density in the apical plasma membrane was higher in RMIC
COX-2−/− mice than in COX-2f/f mice (arrows), an indication of increased AQP2 activity.
Original magnification: x 400. ***P < 0.001, n = 6 in the COX-2f/f group and n = 8 in the
RMIC COX-2−/− group. Scale bar: 50 μM.
B: The mRNA levels of CFTR decreased in RMIC COX-2−/− mice. **P < 0.01, n = 6 in the
COX-2f/f group and n = 8 in the RMIC COX-2−/− group.
C: The mRNA levels of ENaCα, EnaCγ, and Atp1b1 were increased in RMIC COX-2−/−
mice. *P < 0.05, **P < 0.01, n = 6 in the COX-2f/f group and n = 8 in the RMIC COX-2−/−
group. All values are means ± SEM. P values were calculated by Student’s t test.
Author Manuscript
Author Manuscript

Hypertension. Author manuscript; available in PMC 2019 November 01.

 Zhang et al. Page 15
Author Manuscript
Author Manuscript

Figure 4. RMIC COX-2 deficiency caused salt-sensitive hypertension and papillary damage in
response to chronic salt loading Both COX-2f/f and RMIC COX-2−/− mice were fed with a high
salt diet (8% NaCl) after tamoxifen administration for 2 weeks.
A: Systolic blood pressure (SBP) was monitored with tail cuff microphonic manometer
following initiation of high salt treatment (8% NaCl). High salt administration had no
apparent effect on blood pressure in COX-2f/f mice throughout experiment period, but
increased blood pressure in RMIC COX-2 deficient mice for the first 5 weeks, and then led
to progressively decreased blood pressure. N= 7 in COX-2f/f mice and n = 6 RMIC
COX-2−/− mice. All values are means ± SEM.
B: Diastolic blood perssure (SBP), systolic blood pressure (SBP) and mean arterial pressure
(MAP) measured by carotid catheterization after a high salt diet for 4–5 weeks was
Author Manuscript

markedly higher in RMIC COX-2−/− mice than in COX-2f/f mice. **P < 0.01; n = 8 in
COX-2f/f mice and n = 6 in RMIC COX-2−/− mice.
C and D: Both COX-2f/f and RMIC COX-2−/− mice were fed with a high salt diet (8%
NaCl) for 9 weeks, and then fed with normal chow for 3 weeks. C: Blood pressure was
markedly lower in RMIC COX-2−/− mice than in COX-2f/f mice. ***P < 0.001; n= 7 in
COX-2f/f mice and n = 6 RMIC COX-2−/− mice.
D: Urine osmolality was markedly lower in RMIC COX-2−/− mice than in COX-2f/f mice
after 16 hours of water deprivation. **P < 0.01; n= 6 in COX-2f/f mice and n = 5 RMIC
COX-2−/− mice.
All values are shown as mean ± SEM. P values were calculated by Student’s t test.
Author Manuscript

Hypertension. Author manuscript; available in PMC 2019 November 01.

 Zhang et al. Page 16
Author Manuscript
Author Manuscript

Figure 5. Chronic high salt intake caused renal papillary loss in RMIC COX-2 deficient mice.
Direct morphologic examination (upper panels) or magnetic resonance imaging (MRI, lower
panels) showed striking papillary loss after long-term high salt exposure (9–10 weeks) in
RMIC COX-2−/− mice, but not in COX-2f/f mice.
Author Manuscript
Author Manuscript

Hypertension. Author manuscript; available in PMC 2019 November 01.

 Zhang et al. Page 17
Author Manuscript
Author Manuscript

Figure 6. RMIC COX-2 deficiency led to apoptosis in the inner medullae/papillae in response to
high salt diet.
A: RMIC COX-2−/− mice had increased papillary apoptotic cells as early as 2 weeks after a
high salt diet, and more apoptotic cells were evident in both interstitial and epithelial cells 4
weeks after a high salt diet. ***P < 0.001 vs. corresponding COX-2f/f mice, †††P < 0.001 vs.
2 weeks of RMIC COX-2−/− mice; n = 6 in each group. Scale bar: 25 μM.
B: Four weeks after high salt intake, there was increased papillary oxidative stress in RMIC
COX-2−/− mice, as indicated by 4-HNE-staining. Original magnification: x 160. All values
are shown as mean ± SEM. P values were calculated by Student’s t test. Scale bar: 160 μM.
Author Manuscript
Author Manuscript

Hypertension. Author manuscript; available in PMC 2019 November 01.