P2X7 activation is altered in UCH-L1 KO mice

The antibody applied detects both the proform and activated mature ADAM17.

ADAM17 activation is P2X7-co-mediated.

Analyzing the blotting pattern and densitometry of pro-ADAM17 in wildtype and P2X7 KO mice, more intense bands and significantly (p<0,05) higher density of pro-ADAM17 were observed in wildtype PI compared to wildtype APN and P2X7 KO PI to P2X7 KO APN lysates respectively. This finding may account for an enhanced ADAM17 activation in glomerulonephritic kidneys compared to healthy tissue. In UCH-L1 KO kidney lysates, pro-ADAM17 bands do not show any significant difference in intensity or density when comparing PI and APN samples. This observation might be indicative of an impaired ADAM17 activation due to altered P2X7 function in mice lacking UCH-L1.

Bands of mature ADAM17 showed similar features as those of pro-ADAM17. While in wildtype and P2X7 KO kidney lysates, protein amounts were diminished in APN compared to PI preparations both band-wise as density-wise, UCH-L1 KO samples were inert concerning ADAM17 band intensity and density between PI and APN state. P2X7 KO animals showed most intense ADAM17 bands and highest density compared to wildtype and UCH-L1 KO mice. In P2X7 KO APN kidney lysates, mature ADAM17 content was significantly (p<0,05) higher than in wildtype APN and UCH-L1 KO APN preparations respectively.

4.4.1.2. ADAM17 mRNA expression

In contrast to ADAM17 protein content, which showed a decline in tissue lysates prepared from kidneys of animals that had been issued anti-podocyte serum (Figure 41), ADAM17 mRNA expression was upregulated in anti-podocyte serum treated compared to preimme-serum treated wildtype and P2X7 KO mouse kidneys. While basal ADAM17 expression was low in wildtype preimmune-serum mouse kidneys, a significant, 7-fold increase could comparatively be denoted in anti-podocyte-serum treated tissues. The same tendency, yet without significance, could be observed in P2X7 KO anti-podocyte serum treated compared

Figure 42: Relative renal expression of ADAM17 mRNA. Expression levels measured via qPCR in kidney tissues of wildtype, P2X7 KO and UCH-L1 KO mice in PI- and APN-serum treated groups. Values are expressed as mean +/- SEM. Statistical significance was calculated using the Mann-Whitney-U-test. A p-value <0.05 was accepted statistically significant. Figures in bars = n of samples measured per group.

to P2X7 KO preimmune-serum treated littermates (3-fold increased ADAM17 expression).

Relative ADAM17 expression coincided with ADAM17 protein levels among the UCH-L1 KO groups. There was no significant upregulation in ADAM17 expression between preimmune- and anti-podocyte serum treated animals, but basal expression in UCH-L1 KO preimmune-serum kidney lysates was already as high as wildtype and P2X7 KO anti-podocyte serum values. UCH-L1 KO anti-podocyte serum tissue preparations showed similar ADAM17 expression levels. These findings argue for a proper ADAM17 regulation in wildtype animals, whereas mice lacking UCH-L1 seem unable to adjust expression and function of the sheddase. This observation might be indicative of an altered P2X7 activation or function in UCH-L1 KO animals.

4.4.2. EGFR

4.4.2.1. EGFR Western Blot

ADAM17 activation results in proinflammatory signals in most systems through shedding of TNFα.

However, ADAM17 has also been described to function antiinflammatorily via the activation of the notch pathway and EGFR. EGFR protein content was hence considered an indicator of ADAM17 - and, in consequence, P2X7 – function in wildtype, P2X7 KO and UCH-L1 KO mouse kidneys.

Evaluation of protein bands and densitometry after EGFR antibody detection (Figure 43) showed a significant (p<0,001) increase of EGFR protein amounts in lysates prepared from wildtype and P2X7 KO anti-podocyte serum-treated mouse kidneys compared to kidney preparations obtained from their respective preimmune-serum-treated littermates. This finding coincided with the upregulated ADAM17 mRNA

Figure 43: EGFR Western blot in P2X7 KO and UCH-L1 KO kidney lysates. Representative Western blot from kidney lysates of wildtype, P2X7 KO and UCH-L1 KO mice;

EGFR detection. Densitometry of n=2 Western Blots per mouse group.

Values are expressed as mean +/- SEM. Statistical significance was calculated using the Mann-Whitney-U-test. A p-value <0.05 was accepted statistically significant.

Figures in bars = n of samples measured per group.

podocyte serum described above. P2X7 KO anti-podocyte serum treated mouse kidney lysates contained the highest amounts of EGFR protein (p<0,001 to WT APN).

In contrast, UCH-L1 KO anti-podocyte serum kidney lysates contained less EGFR protein than lysates prepared from preimmune-serum treated mouse tissues. This observed decrease was significant (p<0,001) and corresponded to the impaired ADAM17 regulation found via ADAM17 Western blot and qPCR.

4.4.2.2. EGFR expression/qPCR

Among wildtype mice injected either with preimmune(PI)- or anti-podocyte(APN) serum, results obtained from qPCR for EGFR (Figure 44) corresponded with EGFR protein contents evaluated via WB (Figure 43). Kidney lysates prepared from anti-podocyte serum-treated wildtype mice showed a significantly higher (5-fold) EGFR mRNA expression than preimmune-serum-treated littermates (p<0,05). In contrast, results differed from WB findings in the P2X7 KO mouse group. EGFR mRNA expression was significantly higher than in in WT anti-podocyte serum treated mouse kidneys (p<0,05), but did not alter between P2X7 KO preimmune- and anti-podocyte serum treated kidneys.

P2X7 KO animals treated with preimmune serum and those that obtained anti-podocyte serum expressed the same amounts of EGFR mRNA.

Among UCH-L1 KO animals, Western blotting observations were approved by qPCR.

UCH-L1 KO preimmune-serum treated animals expressed 2,8-fold higher levels of EGFR mRNA than anti-podocyte-serum-treated littermates. Interestingly, UCH-L1 KO PI kidneys exhibited the highest EGFR expression levels of all mice investigated (p<0,05 to WT APN).

Figure 44: Relative renal expression of EGFR mRNA is highest in UCH-L1 KO mice injected with preimmune serum, 14d past serum injection. Expression levels measured via qPCR in kidney tissues of wildtype, P2X7 KO and UCH-L1 KO mice in PI- and APN-serum treated groups. Values are expressed as mean +/- SEM. Statistical significance was calculated using the Mann-Whitney-U-test. A p-value <0.05 was accepted statistically significant. Figures in bars = n of samples measured per group.

WT PI WT APN

P2X7 KO PI

P2X7 KO APN

UCHL1 KO PI UCHL1 KO APN 0

5 10 15 20 25 30 35 40 45

*

*

*

*

* **

7 13

12 17

8

**

*

**

**

EGF-R relative expression

4.4.3. HeyL

HeyL is a member of the Hes and Hey families of basic helix-loop-helix transcription factors, that are regarded as Notch target genes and mediate EGFR expression.

HeyL is among the last links of the intracellular Notch1-involving reaction chain activated by ADAM17. HeyL expression was hence considered an indicator of ADAM17 activity.

HeyL mRNA expression levels among wildtype, P2X7 KO and UCH-L1 KO mice injected with either preimmune- or anti-podocyte serum coincided with ADAM17 mRNA expression patterns among the different mouse groups (Figure 45).

HeyL mRNA expression was upregulated in anti-podocyte serum treated mice compared to wildtype and P2X7 KO mouse kidneys of animals that received preimmune serum. While basal HeyL expression was low in wildtype preimmune-serum mouse kidneys, a significant (p<0,05 to WT PI), 8-fold increase could comparatively be denoted in anti-podocyte-serum treated tissues. The same tendency, yet without significance, could be observed in P2X7 KO mice injected with anti-podocyte serum compared to preimmune-serum-treated P2X7 KO littermates (3-fold increased HeyL expression).

Among the two UCH-L1 KO group, there was no significant upregulation in HeyL expression between preimmune- and anti-podocyte-serum treated animals, but basal expression in UCH-L1 KO preimmune-serum kidney lysates was already as high as in wildtype and P2X7 KO podocyte serum conspecifics. UCH-L1 KO anti-podocyte serum tissue preparations showed similar HeyL expression levels. These findings again argue for a proper ADAM17 regulation in wildtype animals, whereas mice deprived of UCH-L1 seem incapable to adjust expression and function of the enzyme.

Figure 45: Relative renal expression of HeyL mRNA is significantly higher in wildtype and P2X7-deficient mice injected with anti-podocyte serum than in wildtype and P2X7 KO animals treated with preimmune serum. Expression levels measured via qPCR in kidney tissues of wildtype, P2X7 KO and UCH-L1 KO mice in PI- and APN-serum treated groups. Values are expressed as mean +/- SEM. Statistical significance was calculated using the Mann-Whitney-U-test. A p-value <0.05 was accepted statistically significant.

Figures in bars = n of samples measured per group.

4.5. Therapeutic antagonization of the P2X7 receptor attenuates the