TARGETING TARGETED TREATMENT FOR IMMUNE AND NON-IMMUNE KIDNEY DISEASES

Abstract

We have found that calcium calmodulin kinase IV is increased in T cells, podocytes, and mesangial cells from patients with systemic lupus erythematosus, as well as in lupus-prone mice, podocytes of patients with focal segmental glomerulosclerosis, and in mice injected with doxorubicin. We showed that this accounts for aberrant T cell function and glomerular damage. Using nanoparticles (nlg) loaded with a small drug inhibitor of calcium calmodulin kinase IV and tagged with antibodies directed to CD4 we have been able to show inhibition of autoimmunity and lupus nephritis. Also, using nlg tagged with antibodies to nephrin, we showed suppression of nephritis in lupus-prone mice and of glomerular damage in mice exposed to doxorubicin. We propose the development of approaches to deliver drugs to cells in a targeted and precise manner.

CALCIUM/CALMODULIN-DEPENDENT PROTEIN KINASES

Calcium (Ca2+) is a ubiquitous intracellular second messenger involved in the control of crucial cellular processes (1). Engagement of the T cell receptor (TCR) in T cells from patients with systemic lupus erythematosus (SLE) results in increased and earlier free intracytoplasmic Ca²⁺ response (2) followed by increased protein tyrosine phosphorylation of a number of cytosolic proteins (3). We have reported that the CD3 complex in T cells from patients with SLE is rewired to produce an aberrantly enhanced TCR signal (4). In normal T cells TCR stimulation signals through immune receptor tyrosine-based motifs containing CD3ζ and CD3ζ associates with ZAP70 to propagate the signal. In contrast, T cells from patients with SLE have reduced CD3ζ and this leads to the recruitment of the fragment crystallizable (Fc) receptor common γ (FcRγ) chain in the TCR complex. Instead of associating with ZAP70, FcRγ associates with spleen tyrosine kinase (5). The FcRγ–spleen tyrosine kinase complex transmits a stronger signal than the CD3ζ-ZAP70 complex and this leads to enhanced intracellular Ca²⁺ flux (2,6).

Transient receptor potential canonical (TRPC) channels are receptor-operated, nonselective, cationic channels that control the influx of Ca²⁺ through the cell membrane. In the kidney, TRPC channels came to the forefront when gain-of-function TRPC6 mutations were linked to familial focal segmental glomerulosclerosis (FSGS) and TRPC6 was shown to be associated with the slit diaphragm. It appears that TRPC function is downstream of pathways known to cause podocyte injury (7).

Ca²⁺ forms a complex with calmodulin (CaM), a 148–amino acid key protein that transduces signals in response to elevation of intracellular Ca²⁺ (8). The Ca⁺²-CaM complex binds to target molecules to regulate their activity. CaM kinases (CaMKs), as indicated by their name, acquire kinase activity upon binding to Ca2+/CaM which removes the auto-inhibitory domain and exposes the catalytic pocket enabling substrate access (8,9). CaMK4 is localized in the brain, T-lymphocytes, and post-meiotic germ cells (10-12). Full activation of CaMK4 requires phosphorylation on a threonine residue in the activation loop which is accomplished by the upstream Ca2+/CaM-dependent kinase kinases (13).

Following cell activation, CaMK4 translocates to the nucleus where it regulates the activity of several transcription-related components, including cyclic adenosine monophosphate (cAMP) response element binding protein, cAMP response element modulator α (CREMα), histone deacetylase 4, monocyte enhancer factor 2A, and retinoid orphan receptor (ROR) (8,14-20). These molecules are important in the development and function of the immune system, including the regulation of T cell differentiation, cytokine secretion, and cell signaling (17,21-23).

CaMK4 SUPPRESSES INTERLEUKIN 2 PRODUCTION IN SLE

T cells from both patients with SLE and lupus-prone mice produce decreased amounts of interleukin 2 (IL-2), a cytokine vital to the function of the immune system (24,25). One of the main repercussions of the decreased production of IL-2 in SLE is the decreased numbers and function of T regulatory (Treg) cells because they depend highly on IL-2 (26). Also, decreased production of IL-2 accounts for the decreased cytotoxic activity among T cells in SLE and this, among other factors, contributes to the increased rates of infections, the main cause of morbidity and mortality (27).

In SLE T cells, engagement of the CD3/TCR complex by the abundantly circulating autoantigen and/or circulating CD3 autoantibodies cause translocation of CaMK4 to the nucleus where it forces the binding of the CREMα to the promoter and other noncoding sequences of the IL-2 locus and represses its transcription (18). Mechanistically, CREMα recruits DNA methyltransferase 3a and histone deacetylase 1 at the promoter and across the IL-2 locus to accomplish closure of the locus (28). Inhibition of CaMK4 with a small drug inhibitor or silencing CaMK4 results in the restoration of IL-2 production. In the MRL/lpr lupus-prone mouse, systemic administration of a CaMK4 small drug inhibitor or genetic depletion of CaMK4 restores serum levels of IL-2 as well as Treg cell numbers and function (20).

ABERRANT CaMK4 ACTIVATION DISRUPTS THE BALANCE OF TREG AND TH17 CELLS IN SLE

At the single-cell level, the differentiation into either Treg or Th17 cell lineage appears reciprocal in nature (29). For example, upon TCR stimulation, the addition of transforming growth factor β drives naïve T cells to express FoxP3 and differentiate into Treg cells. However, the addition of IL-6 to tumor growth factor β promotes RORγt expression, steers cells towards Th17 differentiation, and inhibits FoxP3 and Treg cell differentiation. The reciprocal nature of Treg and Th17 cells is also apparent in SLE where numbers and activity of anti-inflammatory Treg cells are reduced while proinflammatory Th17 cells are increased (30).

Previously, in collaboration with Klaus Tenbrock (31), we had generated a mouse overexpressing CREMα in T cells only. That mouse produced decreased amounts of IL-2, as expected, but it produced increased amounts of the proinflammatory cytokine IL-17 (31). This set off a series of studies to determine how CREMα controls the production of IL-17 which can be summarized as follows. CREMα binds to the promoter of IL-17 gene and at various other sites across the IL-17 locus. Yet, upon binding to the IL-17 locus instead of closing it, it keeps it open (32). CREMα binding to the IL-17 promoter facilitates the transcription of IL-17 in T cells from SLE patients (28).

An additional pathway whereby CaMK4 promotes Th17 differentiation is through the activation of RORγt. RORγt is a key transcription factor for Th17 differentiation and IL-17 production (17). In T cells from MRL/lpr mice, CaMK4 binds and activates AKT and the AKT/mechanistic target of rapamycin (mTOR)/SP6 pathway (19) is known to activate RORγt. Thus, CaMK4 promotes the differentiation of Th17 cells indirectly by inhibiting IL-2 transcription and directly by promoting IL-17 transcription through CREMα and by activating RORγt through the AKT/mTOR/SP6 pathway. In normal T cells, overexpression of CaMK4 increases differentiation of Th17 cells in vitro and genetic depletion of CaMK4 disrupts Th17 cell differentiation in T cells derived from normal or autoimmune prone MRL/lpr mice (19). Moreover, mice lacking CaMK4 or mice subjected to pharmacological inhibition of CaMK4 are resistant to experimental autoimmune encephalomyelitis, which is a well-established Th17 cell-dependent process (19,33).

In T cells from autoimmune-prone MRL/lpr mice, CaMK4 is mostly induced after stimulating naïve T cells under Th17 but not Th1, Th2, or Treg polarizing conditions (19). Importantly, depletion of CaMK4 restores IL-2 production (20) and improves Treg number and function in MRL/lpr mice (34). At the same time, depletion of CaMK4 inhibits Th17 development in SLE T cells (19) and prevents infiltration of IL-17 producing cells in the kidney (33).

It appears that activation of CaMK4 directs T cells towards Th17 differentiation and away from Treg cells differentiation. Inhibition of CaMK4 restores the Treg/Th17 imbalance, limits lymphocyte proliferation and activation, suppresses nephritis and skin disease, and improves survival in lupus-prone mice (20,33,35).

CaMK4 IN RESIDENT KIDNEY CELLS

Lupus nephritis is a major manifestation of SLE occurring in more than 60% of SLE patients and is characterized by glomerular immune complex deposition and cell proliferation (36). Resident kidney cells including mesangial cells and podocytes have been implicated in the expression of nephritis in patients with SLE. CaMK4 also plays a role in resident kidney cells. Specifically, it contributes to the pathogenesis of lupus nephritis by promoting mesangial cell proliferation through IL-6 production. Mesangial cells in the glomerulus are known to produce IL-6 when exposed to double-stranded DNA antibodies (37), and in an autocrine fashion, IL-6 stimulates mesangial cell proliferation (38,39). This is thought to contribute to the pathogenesis of lupus nephritis because blockade of IL-6 or the IL-6 receptor ameliorates kidney disease in lupus-prone mice (40-42). In MRL/lpr mice, IL-6 production by mesangial cells is increased, especially upon stimulation with platelet-derived growth factor. This increased production is reversed when mice are treated with a CaMK4 inhibitor or genetic depletion of CaMK4. Moreover, global depletion of CaMK4 reduces mesangial cell proliferation, and greatly reduces kidney damage (43).

CaMK4 also appears to contribute to podocyte dysfunction in autoimmune kidney disease. Podocytes from lupus nephritis patients exhibit elevated levels of CaMK4. Although the exact mechanism responsible for CaMK4 upregulation is unknown, autoantibodies likely play a role because podocytes exposed to immunoglobulin G (IgG) from patients with lupus nephritis display increased CaMK4 and reduced expression of proteins known to be important for the structure and function of podocytes including podocin and nephrin (44). Also, exposure of podocytes to IgG from patients with SLE causes an increase in the expression of the costimulatory molecules CD80 and CD86 on the surface membrane (44,45). Global genetic ablation of CaMK4 in MRL.lpr mice greatly reduces proteinuria (43).

Podocytes from patients with FSGS also express increased levels of CaMK4 suggesting that this kinase may represent a shared molecule in the expression of immune and non-immune podocytopathies. At the biochemical level, increased levels of CaMK4 disrupt the maintenance of the slit diaphragm by phosphorylating the adaptor molecule 14-3-3β. 14-3-3β stabilizes synaptopodin, an actin-binding molecule that is critical for the maintenance of normal actin fiber dynamics. Therefore, phosphorylation of 14-3-3β by CaMK4 causes the release and degradation of synaptopodin leading to destabilization of the actin fiber network (46).

TARGETING CaMK4 IN LUPUS

To address potential off-target concerns of systematic delivery of CaMK4 inhibitors, our lab has explored nlg-based delivery of the CAMK4 inhibitor KN-93 to target CaMK4 specifically in T cells or podocytes. Use of KN-93 packaged nlg coated with an antibody recognizing CD4 targeted KN-93 specifically to CD4 T cells without increasing cell death. In vitro studies have shown the cell-specific delivery of the drugs, whereas color-leaded nlg tagged with a CD4 antibody, but not a control antibody, were detected in the spleen. Subsequently, we injected KN-93–loaded nlg coated with a CD4 antibody targeted to lupus-prone MRL/lpr mice. Such treatment resulted in increased IL-2 levels in the serum, reduced IL-17 producing infiltrating cells in the kidneys, limited glomerular and interstitial lupus pathology, and improved kidney function as measured by proteinuria. Importantly, the effective dose of KN-93 delivered by nlg was 10% of the dose necessary to warrant an effect from systemically delivered KN-93 (33). This treatment resulted in decreased numbers of T cells lacking CD4 and CD8 (double-negative T cells), but the titers of autoantibodies were not affected presenting an example of dissociation between the contribution of T cells and autoantibodies in the expression of lupus-related pathology. This implies that targeted administration of this drug should have fewer if any side effects. As noted above, CaMK4 is expressed by neurons and germ cells.

Having established that CaMK4 is increased in mesangial cells and podocytes in MRL.lpr mice, we considered delivering the CaMK4 inhibitor to either of these cells. Because mesangial cells express surface molecules shared by many other cells and only podocytes express surface molecules almost exclusive of other cells, we proceeded with the latter.

Using the same delivery approach, KN-93 was loaded in nlg and targeted to podocytes tagged with nephrin or podocin (podocin is expressed on the surface of injured podocytes in vitro) of MRL.lpr mice at the beginning of their clinical disease and, surprisingly, they never developed proteinuria and immune complexes never deposited, despite the fact that humoral and cellular elements of autoimmunity were rampant in these mice. This observation suggests that immune complexes do not deposit if the function of the podocytes is kept intact. The treated mice did not develop crescents (46) which have been claimed to originate from podocytes (47). Pharmacologic inhibition or silencing of CaMK4 in cultured podocytes inhibited their motility, which increases when the actin network is destabilized because when they were subjected to the scratch-wound assay they did not move to fill up the empty space. To the extent crescents originate from podocytes, this inability of CaMK4-deficient podocytes to fill up the scratch-wounded area explains why targeted delivery of a CaMK4 inhibitor to podocytes in lupus-prone mice mitigated the formation of crescents (46). While targeted delivery of the CaMK4 inhibitor to podocytes had a profound effect on glomerular pathology, systemic autoimmunity including autoantibody titers and cellular abnormalities in the spleen and the lymph nodes remained absolutely unchanged.

TARGETING CaMK4 IN FSGS

FSGS is the most common primary glomerular disease that results in end-stage renal disease. It is a heterogeneous clinical entity characterized by a characteristic histologic pattern. The origin of FSGS is diverse and genetic, metabolic, infectious, and unknown factors (idiopathic forms) have been claimed to be involved in its expression. Proteinuria is the typical clinical finding of FSGS (48). It accounts for the majority of the people who require dialysis and organ transplantation and the health-care cost is enormous. There is no available treatment to prevent evolution of the pathologic process and current efforts (and clinical trials) are limited to renoprotective drugs (https://nephcure.org). The podocyte is the target cell for injury in FSGS and a growing list of disease-causing gene mutations encoding proteins that regulate podocyte survival and homeostasis has been identified in FSGS patients (49).

Doxorubicin has been used extensively to study several aspects of FSGS (50). We found that injection of doxorubicin into mice increases the expression of CaMK4 in podocytes. When we delivered nlg loaded with KN-93 and tagged with nephrin or podocin antibodies at the time of the injection of doxorubicin, the mice did not develop proteinuria, the glomeruli did not display any damage, and the podocyte processes were not effaced when examined by electron microscopy. Interestingly, and more relevant to the treatment of patients with FSGS, delivery of the CaMK4 inhibitor 7 days later reversed all damage (46). This evidence strongly urges the consideration of novel approaches to limit FSGS which, through the invariable need of kidney dialysis and transplantation, is responsible for major taxation of health system expenses.

OTHER TARGETING DELIVERY APPROACHES

Prominent among the known cellular abnormalities in patients with SLE are the activation and expansion of pathogenic Th1, Th17, and TCR-αβ ⁺CD4^–CD8^–double-negative (DN) T cells and the failure of regulatory CD4/CD8 T lymphocytes (30). The differentiation and function of each T cell subset is controlled by the cooperative action of different transcription factors able to bind to open DNA loci. DNA methylation represents a core epigenetic control mechanism that directs T cell activation and differentiation. Generalized DNA hypomethylation has been linked to autoimmunity, presumably by favoring the expression of proinflammatory genes, but such a claim is qualified by the multidirectional abnormal expression of genes in T cells from patients with SLE (51). Both hypomethylated and hypermethylated cytosine-guanine sites have been found in T cells from patients with SLE compared with normal controls in several genome-wide DNA methylation studies.

We loaded nlg with the most widely used epigenetic modulator, 5-azacytidine (5-Aza), a chemical analog of cytidine that inhibits DNA methylation, and, after tagging them with specific antibodies for delivery to T cell subsets, we found unexpectedly that 5-Aza promoted Treg expansion when delivered to CD4 T cells and inhibited the generation of DN cells (52-54) when delivered to CD8 T cells. Both targeted deliveries, unlike the systemic delivery of 5-Aza, resulted in disease suppression. Our studies have revealed an unexpected therapeutic effect of DNA demethylation exercised precisely on either CD4 or CD8 T cells and its importance in reversing established disease (55).

CONCLUSIONS

CaMK4 is a central molecule that regulates multiple processes that significantly contribute to the pathology of SLE by controlling both T cells and kidney resident T cells. In T cells it accounts for the aberrant production of IL-2 and IL-17 and in the kidneys for the proliferation of mesangial cells and the loss of function of podocytes. Unexpectedly, we have found that CaMK4 is also upregulated in podocytes from patients with FSGS. In both immune and non-immune podocytopathies, CaMK4 compromises the structure and function of podocytes. Although in lupus nephritis it appears that IgG which enters podocytes elicits an increase in the expression of CaMK4, the involved mechanisms are still at large. Similarly, we have no information regarding the mechanism that leads to increased expression of CaMK4 in patients with FSGS, although the known increased Ca⁺² flux certainly contributes (7). In lupus-prone mice, targeted delivery of a CaMK4 inhibitor to T cells suppresses both autoimmunity and the development of nephritis. Yet, targeted delivery to podocytes averts the deposition of immune complexes without affecting autoimmunity. This observation suggests that immune complexes may deposit after podocytes have been injured and changes the approach we should take to prevent kidney damage. It appears that delivery of a CaMK4 inhibitor to podocytes holds high therapeutic promise for both immune (lupus nephritis) and non-immune (FSGS) podocytopathies (56).

DISCUSSION

Reeves, San Antonio: Really interesting, but why are immune complexes not forming?

Tsokos, Boston: They're formed, but they're not deposited. They're formed in the circulation.

Reeves, San Antonio: Why are they not deposited?

Tsokos, Boston: Our position right now is that if you preserve the structure and the function of the podocytes they cannot deposit there. All of us form immune complexes, right? Every time we sneeze we have immune complexes. If we cannot raise CaMK4 in our podocytes, immune complexes are not deposited and indeed most of us don't develop glomerulonephritis. People who have increased levels of CaMK4 or who increase CaMK4 in podocytes are likely to have immune complexes deposited there because the structure and function of the podocytes have been compromised. We think about this a lot and would like to know the physical chemistry which may underlie the process of deposition, but we're not there yet.