Intrarenal Renin-Angiotensin-System Dysregulation after Kidney Transplantation

Figures

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  Figure 1. Plasma equilibrium Ang levels. Plasma equilibrium Ang II (A), Ang I (B) and Ang-(1–7) (C) levels were measured in patients without RAS blockade and with ACEi therapy (n = 6 per group). Additionally, ACE activity (D), Ang II/Ang I-Ratio (E) and renin concentration (F) were analyzed in the respective patient groups. Horizontal bars represent median values. Lower limit of quantification: LLOQ. (1 pg/mL (Ang II), 1.3 pg/mL (Ang I), 2 pg/mL (Ang-(1–7)), 5 U/L ACE-acitvity, 1µU/mL (renin)). Time after transplantation (no RAS blockde median 3.0 [0.5–12.6] vs ACEi therapy 7.0 [1.9–13.3]). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.005.

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  Figure 2. Intrarenal Ang II and Ang-(1–7) forming RAS enzymes. Representative IHC stainings of RAS enzyme expressions of ACE, chymase, NEP, PEP, ACE2 and PCP in kidney biopsies of early biopsy groups with no RAS blockade and with ACEi therapy. (A) No differences in immunohistologic expression patterns were observed between different time periods after transplantation except higher incidence of chymase bearing mast cells. Black arrowheads indicate the respective enzymes or cells containing specific enzymes (chymase in mast cells) of representative biopsies. Scale bar equals 50 µm; 400× magnification. Simplified scheme of enzymatic Ang II and Ang-(1–7) synthesis from Ang I and Ang II, respectively (B).

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  Figure 3. Ang II and Ang-(1–7) synthesis by allograft RAS enzymes. Absolute Ang II synthesis rates from Ang I (A) in kidney biopsy homogenates of KTx patients with various graft vintages without RAS blockade (no RASi) or with ACEi therapy were analyzed by mass spectrometry. Similarly, Ang-(1–7) synthesis from Ang I (B) was determined.

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  Figure 4. Ang II and Ang-(1–7) synthesis in the transplanted kidney. Contribution of ACE (A) and chymase (B) activity to form Ang II from Ang I was measured in kidney biopsy homogenates of KTx patients with various graft vintage. After spiking the samples with Ang I as substrate, NEP enzyme activity to form Ang-(1–7) in kidney biopsy homogenates of KTx patient groups by various graft vintage without RAS blockade (no RASi) or with ACEi therapy was analysed by mass spectrometry. (C) Similarly, PEP activity was determined (D).

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  Figure 5. Chymase/NEP-ratio. Calculated Chymase-to-NEP-ratio in kidney biopsies according to graft vintage in patients without RAS blockade (no RASi) or with ACEi therapy.

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  Figure 6. Mast cells in the allograft post KTx. IHC staining of kidney biopsies in both cohorts of analyzed patients revealed accumulation of chymase bearing mast cells (indicated by arrowheads) in allograft biopsies over the years post KTx, independent of ACEi therapy, 100× magnification, scalebar is 50 µm. (A) Cell counts displayed higher mast cell numbers in grafts with a graft vintage of two or more years compared to biopsies early after KTx. (B) Representative mast cell in allograft biopsy, 630× magnification, scalebar is 5 µm (C).

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  Figure 7. RAS dysregulation after kidney transplantation. Early after KTx (<2years), a moderate ACE-mediated Ang II synthesis occurs in the graft. During the first months after transplantation, Ang-(1–7) synthesis is dominantly mediated by NEP, together with ACE -mediated Ang II synthesis from Ang I. In allografts with higher graft vintage (>12 years), a strong increase of local chymase mediated Ang II synthesis and constant NEP mediated Ang-(1–7) synthesis occurs. In patients with allografts with more than a decade of age, ACEi therapy with subsequent negative feedback and systemic renin upregulation and increased Ang I levels might trigger local chymase mediated Ang II synthesis.