CaMKIV: A Potential Target for a Treatment for Lupus Nephritis

Systemic lupus erythematosus (SLE) is an autoimmune disorder that currently has no cure (PubMed Health, 2011). SLE may result in abnormal deposits in kidney cells, which leads to complications in the kidneys. This results in the condition lupus nephritis, in which extensive damage to the kidney occurs and ultimately results in kidney failure (PubMed Health, 2011). There has been much speculation that Calcium/calmodulin-dependent protein kinases (CaMKs) is a factor in the progression of SLE, for it has previously been demonstrated that CaMKIV contributes to decreased IL-2 production in SLE T cells (Karin, 1995). CaMKIV may also play a role in the production of IL-6, for it has been demonstrated that just before the age when lupus nephritis is developed, spleen cells of mice contain a subpopulation of Th cells that selectively induced their B cells in vitro to produce highly cationic IgG autoantibodies to both single-stranded DNA and double-stranded DNA (Datta, 1987). This may be of critical importance, for it has also been shown that IL-6 is a feed forward process in which macrophages respond to IgG to support further B-cell production of IgG (Maeda, 2010).

It has previously been demonstrated that inhibiting CaMKIV in lupus-prone MRL/lpr mice mitigates disease pathology (Karin, 1995). Ichinose et al. have recently elaborated on the cellular abnormalities that may be controlled by CaMKIV (2011). These results have helped to clarify the cellular processes that result in the damage of kidneys in lupus nephritis by producing large amounts of autoimmune mesangial cells and increasing production of IL-6 and have shown that CaMKIV may be a critical target in developing treatments for lupus nephritis.

Ichinose et al. have recently proposed a critical functioning of CaMKIV in the progression of SLE and lupus nephritis (2011). To first test this hypothesis, Camkiv null locus was transferred into lupus-prone mouse strain MRL/lpr. However, it was found that there was no difference in number of splenocytes, peripheral lymph node cells, and major B and T cell compartments amounts between MRL/lpr and mice lacking the CaMKIV gene, MRL/lpr.Camkv^-/- mice.

However, after 16 weeks of development, the kidney was considerably less damaged in MRL/lpr.Camkiv^-/- mice compared to MRL/lpr mice. Even though immune cell levels remained comparable, mice without functional CaMKIV developed significantly less glomerular, tubulointerstitial, and pervascular lesions as well as decreased proteinuria and serum titers of anti-dsDNA antibodies. From these results, it was concretely determined that the presence of CaMKIV was responsible for kidney damage, but not from the functioning of the major immune cells.

KN-93 is a known inhibitor of CaMKIV, therefore treatment with KN-93 will diminish the function of CaMKIV. To test the function of CaMKIV on the number of mesangial cells, MRL/MPJ and MRL/lpr mice were pretreated with KN-93 to assess the differences in mice when CaMKIV is blcoked. Treatment with KN-93 was found to suppress levels of mesangial cells. Therefore, inhibition of CaMKIV in lupus-prone MRL/lpr mice results in decreased mesangial cell proliferation.

Cell division was monitored in order to determine the rates of cell prliferation. Cells undergoing division were synchronized by serum starvation for 24 hours and labeled with propidium iodide in order to observe their proliferation capabilities by flow cytometry. It was found that the percentage of cells actively commencing the cell cycle in order to divide and multiply was much higher in lupus prone MRL/lpr mice compared with the control MRL/MPJ mice. This indicates that mesangial cells in MRL/lpr mice actively divide, even in the absence of exogenous stimuli. However, upon treatment with KN-93, cells were arrested in the cell cycle and the percentage of mesangial cells dividing and proliferating was clearly diminished.

Mice were then genetically depleted of CaMKIV in MRL/lpr.Camkiv^-/- mice in order to determine the impact of the absense of CaMKIV. Mesangial cells in mice without functional CaMKIV were then measured. It was found that large percentages of mesangial cells from MRL/lpr.Camkiv^-/- mice were arrested in the cell cycle and not actively dividing compared with control MRL/lpr and MRL/MPJ mice. PDGF-BB, which acts as a stimulus to promote cell proliferation, was not able to overcome the block imposed by the genetic deletion of CaMKIV, demonstrating that CaMKIV plays an important role in mesangial proliferation, with an intrinsic ability to proliferate without exogenous stimulation.

IL-6, a product of mesangial cells, was then measured to determine what the influence of mesangial cells have on the progression of kidney failure. It was found that cells with genetic deletions of CaMKIV had lower levels of IL-6. IL-6mRNA expression in activated splenocytes was significantly decreased in MRL/lpr.Camkiv^-/- mice. Furthermore, mice treated with KN-93 also had suppressed levels of IL-6 in MRL/lpr mesangial cells at mRNA and protein levels. These results indicate that either the genetic deletion or inhibition of CaMKIV will not only decrease the proliferation of mesangial cells, but will also decrease the production of IL-6.

Ichinose et al. further delved into the transcriptional mechanisms underlying IL-6 expression. It was found that IL6 promoter activity in MRL/lpr.Camkiv^-/- mesangial cells decreased compared with MRL/lpr mesangial cells. Mutating AP-1 resulted in limited promoter activity, demonstrating that AP-1 is involved in IL-6 gene transcription. AP-1 member c-jun was also found in increased levels in MRL/lpr.Camkiv^-/- mesangial cells compared with MRL/lpr mesangial cells. This was further confirmed using DNA binding assays, demonstrating that the AP-1 motif showed an increased binding of nucleoprotein from MRL/lpr mesangial cells stimulated with PDGF-BB, which was diminished when nucleoprotein lysates from MRL/lpr.Camkiv^-/- mesangial cells were used.

This study elaborated on the mechanisms that potentially influence the progression of lupus nephritis. Ichinose et al. provide concrete evidence that mesangial cells from MRL/lpr mice are able to proliferate in vitro in the absence of exogenous stimuli, along with increasing IL-6 production. Ichinose et al. were able to convincingly link these cellular abnormalities to CaMKIV, for the genetic absence or the blocking of function of CaMKIV resulted in the suppression of mesangial cell proliferation. CaMKIV has been identified as an important regulator of cellular abnormalities that are observed in lupus nephritis, suggesting it as a potential target in the mitigation of autoimmunity and suppression of kidney inflammation. These results should be elaborated on by assessing the effects of CaMKIV on the functioning of macrophages and the production of IgG to further understand the effects of IL-6 on the immune system and progression of lupus nephritis. Further research may also be required to assess the practicality of blocking CaMKIV in lupus nephritis treatment. Even though inhibiting the function of CaMKIV may help regulate cell abnormalities in lupus nephritis, CaMKIV has been shown to have neuroprotective effects, and its inhibition may potentially prove to be disastrous in other body functions (Sée et al., 2001). Ichinose et al. have made much progress on targeting a potential controller of lupus nephritis. This lead should be followed in order to advance the understanding of SLE and potentially propose the first successful treatment for this autoimmune disease.

Works Cited:

Datta, S. K., Patel H., Berry D. Induction of a cationic shift in IgG anti-DNA autoantibodies. Role of T helper cells with classical and novel phenotypes in three murine models of lupus nephritis. (1987). J. Exp. Med. 165, 1252-1268.

Ichinose K., Rauen T., Juang Y., Kis-Toth K., Mizui M., Koga T., Tsokos G. C. Cutting Edgue: Calcium/Calmodulin-Dependent Protein Kinase Type IV is Essential for Mesangial Cell Proliferation and Lupus Nephritis. J. Immunol. 1102357.

Karin M. 1995. The regulation of AP-1 activity by mitogen-activated protein kinases. J. Biol. Chem. 270: 16483-16486.

Maeda K., Mehta H., Drevets D. A., Coggeshall K. M. IL-6 increases B-cell IgG production in a feed-forward proinflammatory mechanism to skew hematopoiesis and elevate myeloid production. (2010). Blood 115: 4699-4706.

PubMed Health, A.D.A.M. Medical Encyclopedia. Systemic lupus erythematosus. (2011). National Center for Biotechnology Information.

Sée V., Boutillier A. L., Bito H., Loeffler J. P. Calcium/calmodulin-dependent protein kinase type IV (CaMKIV) inhibits apoptosis induced by potassium deprivation in cerebellar granule neurons. FASEB J. 2001 Jan;15(1):134-144. PubMed PMID: 11149901.