Nephrotoxicity of Calcineurin Inhibitors in kidney epithelial cells is independent of Calcineurin and NFAT signaling

Background Calcineurin inhibitors (CNI) such as Cyclosporine A (CsA) and Tacrolimus (FK506) are commonly used after renal transplantation in order to suppress the immune system. In lymphoid cells, CsA acts via the Calcineurin-NFAT axis, whereas in non-lymphoid cells, such as kidney epithelial cells, CsA induces Calcineurin inhibitor toxicity (CNT). Up to date, it is unknown via which off-targets CsA induces CNT in kidney epithelial cells. Methods In vitro experiments using a surrogate marker to measure CNT induction as well as in vivo studies with acute CNT, were used in order to elucidate the underlying molecular mechanism. Results Inhibition of the NFAT axis does not show any nephrotoxicity. However, inhibition of p38 and PI3K/Akt Kinases showed induction of nephrotoxicity. Conclusions These findings show that CsA acts NFAT independent on kidney epithelial cells. Moreover, inhibition of certain protein kinases mimic CsA activity on kidney epithelial cells indicating that p38 and PI3K/Akt kinase pathways might be involved in CNT progression on kidney epithelial cells.


Introduction
Calcineurin Inhibitors (CNI), e.g., Cyclosporin A (CsA), Tacrolimus (FK506) are pivotal drugs for the prevention of rejection after solid organ transplantation. CNI show a highly interindividual pharmacokinetic profile and have a narrow therapeutic range. Even mild overdosing may lead to substantial acute and chronic side effects, including tumour development, infections, metabolic disturbances, and kidney failure. Calcineurin inhibitors induce nephrotoxicity through (reversible) vasoconstriction in the vas afferent with consecutive relative hypoxia and progressive athero-and arteriolohyalinosis, tubular atrophy and interstitial fibrosis 1 . Recent register studies have demonstrated that virtually all kidney transplanted patients develop signs of chronic CNT within 10 years after kidney transplantation 2,3 .
In lymphocytes, one major activity of CsA is the inhibition of the Calcineurin-NFAT axis leading to repression of transcriptional programs important for activation, proliferation, and cytokine production. This inhibition primarily contributes to the immune suppressive effect of CsA 4,5 .
Members of the TNF superfamily have a non-redundant role in the pathogenesis of tissue regeneration and wound healing but are also critically involved in chronic inflammatory and fibrosis [6][7][8] . We have recently shown that the TNF superfamily member TWEAK (TNFSF12) is indispensable for the development of CNT in mice 9 . CsA induces the expression of TWEAK's receptor, Fn14, in kidney epithelial cells, which facilitates inflammatory and fibrotic signals critical for progressive nephrotoxicity. Deficiency for TWEAK or treatment with TWEAKneutralizing reagents indeed protected animals from acute CNT lesions [9][10][11][12] . Furthermore, administration of recombinant TWEAK (rTWEAK) induced similar disease as when animals are treated with CsA alone. Interestingly the combination of rTWEAK and CsA induced severe tubulopathy and interstitial inflammation 9 . The receptor Fn14 is low ubiquitously expressed on epithelial cells and can be induced upon stress and injury stimuli 13 . Fn14 is the only known receptor to the cytokine TWEAK 14 . TWEAK is produced by infiltrating and tissue-resident immune cells, largely monocytes and neutrophils [15][16][17] .
Currently, it is unclear, how CNI (CsA) interfere with kidney epithelial cells and induce nephrotoxicity. The presented work aims investigating the molecular mechanism in CNT. We hypothesize that CsA elicits its inflammatory and fibrotic activities in renal epithelial cells independent of NFAT, but via inhibition kinase pathways.

Cell Sort -Aria
Mouse kidney samples were digested and processed as described above. Cells were stained with CD45-PerCP/Cy5.5 and CD326/ EpCAM-PE and DAPI. Cells were sorted according to live cells (DAPI negative). All CD45 positive cells were excluded and only CD326 positive cells were collected and used for RNAseq.

Kinase Inhibitor Library
MCT were treated with various kinase inhibitors (Enzo, BML-2832) at 10µM. After, cells were dissociated and stained with APC-Fn14. Flow cytometry was used to analyse Fn14 expression on the cell surface.

Protein Isolation and Analysis
Cells were lysed in Laemmli buffer and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane (ThermoFisher, 88518). Membranes were blocked with 5% milk in TBST for 1h. Incubation with the primary antibody, aFn14 (#4403, Cell Signaling), aGAPDH (Merck, cB1001), was conducted overnight at 4°C. The horseradish peroxidase-conjugated secondary antibody was added to the membrane for 1h at RT. Antibodies were diluted according to manufactures recommendation.

RNA isolation, reverse transcription and qRT-PCR
Total RNA was isolated from samples using TRIzol reagent (Invitrogen, 15596026). Nanodrop 1000 spectrophotometer was used to measure RNA concentration and evaluate its quality.
PrimeScript RT Reagent Kit (Takara, RR037A) was used to generate cDNA. Real-time polymerase chain reaction was performed by using the TaqMan method. For this, probes, which are UPL labelled (Roche) and gene-specific primers were mixed with TaqMan Fast Universal PCR Master Mix (Appliued Biosystems, 4352042). Gene Expression was calculated with the dCt relative to the housekeeping gene. A RT 2 Profiler PCR Array for mouse nephrotoxicity (PAMM-094Z) was used to determine expression of nephrotoxic genes.

RNAseq
Total RNA was isolated by using TRIzol reagent according to manufactures protocol. RNA sequencing was performed on illumine NovaSeq 600.

In vivo experiments: Acute toxicity model
As previously described 9 , acute CNT lesions were induced by injecting (ip) animals with 100mg/kg CsA (Sandimmun Neoral®), 10mg/kg p38 Inhibitor (SB203580, SB202190) and 10mg 11R-VIVIT on 3 consecutive days twice daily. The fourth day, animals were euthanized, and kidney and blood were collected for subsequent analysis.

Statistical analysis
Analysis was performed using Prism 5 software (GraphPad, San Diego, CA). Results are the mean ± the standard deviation (SD). Groups were analysed by Student's t-test (unpaired) or one-way ANOVA. P-values below 0.05 were considered as significant.

Surface Fn14 is induced upon CsA exposure in kidney epithelial cells, but not in response to cell-permeable NFAT inhibitors.
We have previously demonstrated that the calcineurin inhibitor CsA induces Fn14 surface expression on kidney epithelial cells in vitro and in vivo 9 . To further support these findings, we  (Fig. 1C, D). Also, CsA induced further upregulation of Fn14 on cell surfaces even NFAT axis was inhibited (plasmid mediated) (Fig. 1D). It has been shown that CsA sensitizes cells to the cytokine TWEAK 9 . We show in our experiments how cells express significantly higher ICAM (adhesion molecule) after TWEAK treatment when pre-sensitized with CsA. This pre-sensitization is no longer observed when NFAT is inhibited in these cells, comparable to control (Fig. 1E). These experiments indicate, that CsA's activity in kidney epithelial cells on Fn14 expression cannot be mimicked by direct NFAT inhibitors. Notably, two p38 kinase inhibitors (SB203580 and SB202190) showed potential to upregulate Fn14 on cell surfaces. Titration of both inhibitors revealed similar potential to induce Fn14 (Fig.   2C). Protein from SB203580 treated cells showed similar upregulation of Fn14 as cells treated with CsA (Fig. 2D). Cells treated simultaneously with a p38 kinase inhibitor and CsA showed an increased expression of Fn14 compared to cells only treated with CsA or one of the p38 kinase inhibitor (Fig. 2E). To corroborate these findings, we generated cells expressing a constitutive active p38 kinase (pCMV-p38-CA-EGFP). Basal Fn14 expression (MFI) was moderate in all untreated mock-transfected (pCMV-eGFP-N1) cells and p38-CA-expressing cells. Mock-transfected control (GFP) showed a highly significant upregulation of Fn14 upon SB203580 or CsA stimulation. Meanwhile, for the p38-CA-expressing cells, the p38 inhibitor or CsA induced significantly lower Fn14 expression (Fig. 2F). Furthermore, a nephrotoxic gene array revealed that CsA upregulates numerous genes involved in toxicity in the kidney. What is more, that SB203580 also shows a high induction potential of nephrotoxic genes (Fig. 2G).

CsA, but not VIVIT induces nephrotoxicity in vivo.
Exposure of mice with high dose of CsA induces nephrotoxicity, reflected in induction of acute phase, fibrosis and inflammatory genes, e.g. Angptl4, CD24 and TNFRSF12a. Furthermore, mice treated with p38 inhibitors SB203580 and SB202190 show similar upregulation of genes involved in nephrotoxicity (Fig. 3A). Meanwhile, animals treated with VIVIT showed no such signatures. Moreover, immune cell infiltration (CD45+) into the kidney is upregulated in animals treated with CsA or p38 kinase inhibitors (Fig. 3B). Not only, could we detect such changes in kidney infiltrated cells but also immune cell compartment, notably CD45+ and CD11b+ cells, seem to be increased upon CNI or p38 kinase treatment (Fig. 3C).

Discussions
CNI play a crucial role in effective immunosuppression after kidney transplantation. However, long term treatment lead to chronic nephrotoxicity. In lymphoid cells, CsA and FK506 interfere with the cytosolic phosphatase Calcineurin and thereby prevent dephosphorylation of nuclear factor of activated T cells (NFAT) and consequently gene transcription. To date, it is unclear, if biological effects of CNI in non-lymphoid cells result similarly from disrupted Calcineurin-NFAT signalling. This work aimed to investigate the molecular mechanism of CNT in epithelial cells. We present experimental evidence, that CNT lesions are induced in kidney epithelial cells in vivo and in vitro independent of canonical Calcineurin -NFAT signalling, yet induced by kinase inhibitors, including inhibitors of the p38, IKK and PI3K pathway.
Firstly, we showed that Fn14 upregulation is achieved by treatment of kidney epithelial cells by CNI (CsA and FK506) whereas other compounds fail to upregulate Fn14. Furthermore, we show that also an effective inhibitor (11R-VIVIT) of the NFAT axis fails to induce Fn14 in epithelial cells. These results confirm previous studies 9 that Fn14 is specifically upregulated in kidney epithelial cells when treated with a nephrotoxic agent and that Fn14 is a good surrogate marker to measure nephrotoxicity and pro-fibrotic signals. Furthermore, did our result show that nephrotoxic CNI signalling in kidney epithelial cells is not mediated via the canonical

Calcineurin-NFAT pathway in vitro and in vivo.
Likely, CNT is the consequence of off-target effects of CsA in kidney epithelial cells. It has been hypothesized that CsA targets disparate signalling pathways in lymphoid and nonlymphoid cells 19 . As we showed that CsA's fibrotic effect on kidney cells is stable and easy to measure (Fn14) and that the nephrotoxic effect is NFAT-independent, we show here synthetic compounds and their capacity to induce Fn14. Our screen reveals that protein inhibitors of the CNT shows a highly variable clinical presentation, and some patients develop early and progressive lesions within months of SOT, while others tolerate similar CNT doses/through levels over many years or decades without significant graft or kidney function impairment and absence of CNT lesions in biopsy specimen 3,28,29 . The reason for this highly inter-individual predisposition is unclear, but likely lies in single nucleotide polymorphisms (from the donor in a setting of kidney transplantation, and the recipient). To date, no GWAS study has been performed to decipher risk genes for rapid and progressive CNT after SOT.
In conclusion, these results suggest that CsA acts NFAT-independently on kidney cells yet requires p38 kinase and PI3K/Akt kinase pathways. Furthermore, could we show in vitro and in vivo that inhibition of proposed pathways (p38 and PI3K/Akt) mimics the toxicity of CsA on epithelial cells. However, we cannot exclude that CNT is rather not a highly specific interaction with a single molecular pathway.