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Updated: Aug 25, 2026
Optimizing Isolation and Purification of Murine Glomerular Mesangial Cells
Published on: March 7, 2025
Characterization of the PGI2/IP system in cultured rat mesangial cells
Rania Nasrallah1, Anne Landry, James W Scholey
1Department of Cellular and Molecular Medicine, Kidney Research Centre, Faculty of Medicine, University of Ottawa, 451 Smyth Road, Room 1337, Ottawa, ON, Canada K1H 8M5.
Abstract:
Mesangial cells play an important role in glomerular function. They are an important source of cyclooxygenase (COX)-derived arachidonic acid metabolites, including prostaglandin E(2) and prostacyclin. Prostacyclin receptor (IP) mRNA was amplified from cultured mesangial cell total RNA by RT-PCR. While the prostaglandin E(2) receptor subtype EP(2) was not detected, EP(1,3,4) mRNA was amplified. Also, IP protein was noted in mesangial cells, proximal tubules, inner medullary collecting ducts, and the inner and outer medulla. But no protein was detected in whole cortex preparations. Prostacyclin analogues: cicaprost and iloprost, increased cAMP levels in mesangial cells. On the other hand, arginine-vasopressin and angiotensin II increased intracellular calcium in mesangial cells, but cicaprost, iloprost and prostaglandin E(2) had no effect. Moreover, a 50% inhibition of cicaprost- and iloprost-cAMP stimulation was observed upon mesangial cell exposure to 25 and 35 mM glucose for 5 days. But no change in IP mRNA was observed at any glucose concentration or time exposure. Although 25 mM glucose had no effect on COX-1 protein levels, COX-2 was increased up to 50%. In contrast, PGIS levels were reduced by 50%. Thus, we conclude that the prostacyclin/IP system is present in cultured rat mesangial cells, coupling to a cAMP stimulatory pathway. High glucose altered both enzymes in the PGI(2) synthesis pathway, increasing COX-2 but reducing PGIS. In addition, glucose diminished the cAMP response to prostacyclin analogues. Therefore, glucose attenuates the PGI(2)/IP system in cultured rat mesangial cells.
Insights
High glucose levels impair the prostacyclin/IP system in rat mesangial cells, reducing cAMP signaling and altering key enzymes in prostaglandin synthesis.
Area of Science:
- Renal physiology
- Cellular signaling
- Molecular biology
Background:
- Mesangial cells are crucial for glomerular function and produce cyclooxygenase (COX)-derived metabolites like prostaglandin E2 and prostacyclin.
- Prostacyclin signaling, mediated by the prostacyclin receptor (IP), plays a role in kidney function.
Purpose of the Study:
- To investigate the presence and function of the prostacyclin/IP system in cultured rat mesangial cells.
- To determine the effects of high glucose on this system and its associated signaling pathways.
Main Methods:
- Reverse transcription-polymerase chain reaction (RT-PCR) to detect mRNA for IP and prostaglandin E2 receptors (EP).
- Western blotting or immunohistochemistry to detect IP protein expression.
- Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels in response to prostacyclin analogues and other stimuli.
- Assessment of COX-1, COX-2, and prostacyclin synthase (PGIS) protein levels under high glucose conditions.
Main Results:
- IP mRNA and protein were detected in cultured mesangial cells, along with EP(1,3,4) receptor mRNA.
- Prostacyclin analogues (cicaprost, iloprost) increased cAMP levels in mesangial cells.
- High glucose exposure (25-35 mM for 5 days) significantly reduced the cAMP response to prostacyclin analogues.
- High glucose increased COX-2 expression and decreased PGIS levels, while COX-1 remained unchanged.
- IP mRNA levels were not affected by glucose concentration or exposure time.
Conclusions:
- The prostacyclin/IP system is functionally present in cultured rat mesangial cells, linked to a cAMP stimulatory pathway.
- High glucose conditions attenuate the prostacyclin/IP system by altering prostaglandin synthesis enzymes (increasing COX-2, decreasing PGIS) and diminishing the cAMP response.
- These findings suggest a potential mechanism by which hyperglycemia may negatively impact glomerular function.
