Prabal K. Chatterjee

Cytokine-Stimulated Nitric Oxide Production in the Human Renal Proximal Tubule and Its Modulation by Natriuretic Peptides: A Novel Immunomodulatory Mechanism?

Although the importance of the human kidney in a variety of disease states is well recognised, the exact mechanisms involved remain unclear. Animal disease models suggest that while high local concentrations of nitric oxide (NO) may play a key role in the initiation and progression of renal disease, low levels may also be essential for normal renal function and cell protection, possibly explaining the variable reports of both beneficial and detrimental responses of renal disease models following NO inhibition. NO has both physiological and pathological roles and clearly a balance between these two primary roles is likely to prevail leading to the conclusion that partial rather than total inhibition of NO production may be beneficial. Despite increasing evidence for the role of NO from animal disease models, little is known of the role of NO and potential modulators within the human kidney. In this review we describe three series of studies during which we examined the ability of primary cultures of human proximal tubular cells to produce NO in response to inflammatory cytokines and the possible role of potential modulators such as the natriuretic peptides. Following challenge with the combination of inflammatory cytokines IL-1β, TNF-α, and IFN-γ, such cultures exhibit a time-dependent increase in inducible NO synthetase induction and corresponding NO production, an effect which was inhibited by L-NMMA. In the second series of studies we demonstrated that increasing concentrations of atrial natriuretic factor (ANF) or C(4–23)ANF could stimulate a time- and concentration-dependent increase in nitric oxide production which was again abolished by L-NMMA. These results suggested that ANF acting at the natriuretic peptide receptor C could stimulate nitric oxide production in human proximal tubular cells. In the final series of studies we demonstrated that pro-inflammatory cytokine-induced nitric oxide production could be inhibited by ANF, brain natriuretic peptide, C-type natriuretic peptide or C(4–23)ANF. The actions of the natriuretic peptides and C(4–23)ANF was to return pro-inflammatory nitric oxide production to those observed when human proximal tubular cells were incubated with ANF alone indicating that this inhibition was mediated via the natriuretic peptide receptor C. The function of NO in the kidney is unclear but undoubtedly it has both beneficial and detrimental actions which in health remain in balance. However, when the kidney is subjected to an immune challenge, high cytotoxic levels of NO are produced locally and appear to be responsible for local damage, unfortunately total inhibition of NO production during such disease states does not always result in benefit. Clearly total abolition of an NO response removes important integral protective actions such as vasodilation. In the ideal situation, treatment of disease processes related to NO excess would involve the inhibition of these high local levels while still protecting vital dependent mechanisms. We believe that the NO natriuretic peptide interaction, which we have reported in this review, places ANF in a unique position of being able to maintain the essential or protective actions of NO while inhibiting potentially cytotoxic or detrimental effects.

Atrial natriuretic factor modulates nitric oxide production : an ANF-C receptor-mediated effect

Objectives: To investigate the possible immunomodulatory and regulatory functions of atrial natriuretic factor (ANF) and the natriuretic peptide C (NPR-C) receptor in the control of cytokine-stimulated nitric oxide production in primary cultures of human proximal tubular cells. Methods: Freshly prepared human proximal tubular cells were seeded on plastic plates and allowed to reach confluence. The confluent cells were then incubated with ANF or cycliC(4_23)ANF (c(4_23)ANF) alone, or preincubated with ANF or C(4_23)ANF before incubation with the nitric oxide-stimulating combination of cytokines interleukin-1|3 (10u/ml), tumour necrosis factor-cc (10ng/ml) and interferon-y (100 u/ml). Results: In the present series of experiments we have found that incubation of primary cultures of human proximal tubular cells with ANF or C(4_23)ANF stimulates nitric oxide production dose-dependently. Paradoxically, ANF acting via the NPR-C receptor also inhibits cytokine activation of the enzyme-inducible nitric oxide synthase via a cyclic GMP-independent mechanism. Both of these effects were reproduced by the NPR-C receptor-specific ligand cIn the present series of experiments we have found that incubatio4_23)ANF. Conclusions: These findings represent novel actions of ANF mediated via the NPR-C receptor. The results also provide a simple model system in which to study the subcellular mechanisms of NPR-C receptor activation.

Evidence for Atrial Natriuretic Factor Induced Natriuretic Peptide Receptor Subtype Switching in Rat Proximal Tubular Cells during Culture

Culture and natriuretic peptide dependent changes in the expression of the natriuretic peptides atrial natriuretic factor (ANF), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) and the natriuretic peptide receptors A, B, and C in primary cultures of rat proximal tubular cells were demonstrated using polymerase chain reaction analysis and cyclic guanosine monophosphate response to ANF and CNP. Freshly isolated cells expressed mRNA coding for the natriuretic peptide receptor C only, with no expression of the natriuretic peptides or the natriuretic peptide receptors A or B. At confluence natriuretic peptide receptor C expression was lost, while mRNA transcripts for both ANF and BNP and the A and B receptors became apparent. The appearance of mRNA transcripts for the natriuretic peptide receptors A and B during cell growth correspond with a significant increase in the cyclic guanosine monophosphate respone to both ANF and CNP, confirming the presence of functionally active guanylate cyclase linked A and B natriuretic peptide receptors. The observed changes in peptide receptor expression during culture were preceded by changes in natriuretic peptide mRNA expression, suggesting the possibility that natriuretic peptide receptor subtype switching may be under the control of endogenous peptide release. Incubation of freshly isolated proximal tubular cells with ANF, BNP, or CNP for 3 h induced similar changes in receptor expression. Incubation with ANF induced expression of the natriuretic peptide receptor B and CNP while inhibiting natriuretic peptide receptor C. Incubation with BNP induced expression of the natriuretic peptide receptor B and CNP. Incubation with CNP induced expression of the natriuretic peptide receptors A and B and CNP. These results suggest that primary cultures of rat proximal tubular cells may experience natriuretic peptide and natriuretic peptide receptor subtype switching as they approach confluence under the control of endogenously expressed natriuretic peptides.