Molecular Pathogenesis of Post-Transplant Acute Kidney Injury: Assessment of Whole-Genome mRNA and MiRNA Profiles.

Acute kidney injury (AKI) affects roughly 25% of all recipients of deceased donor organs. The prevention of post-transplant AKI is still an unmet clinical need. We prospectively collected zero-hour, indication as well as protocol kidney biopsies from 166 allografts between 2011 and 2013. In this cohort eight cases with AKI and ten matched allografts without pathology serving as control group were identified with a follow-up biopsy within the first twelve days after engraftment. For this set the zero-hour and follow-up biopsies were subjected to genome wide microRNA and mRNA profiling and analysis, followed by validation in independent expression profiles of 42 AKI and 21 protocol biopsies for strictly controlling the false discovery rate. Follow-up biopsies of AKI allografts compared to time-matched protocol biopsies, further baseline adjustment for zero-hour biopsy expression level and validation in independent datasets, revealed a molecular AKI signature holding 20 mRNAs and two miRNAs (miR-182-5p and miR-21-3p). Next to several established biomarkers such as lipocalin-2 also novel candidates of interest were identified in the signature. In further experimental evaluation the elevated transcript expression level of the secretory leukocyte peptidase inhibitor (SLPI) in AKI allografts was confirmed in plasma and urine on the protein level (p<0.001 and p = 0.003, respectively). miR-182-5p was identified as a molecular regulator of post-transplant AKI, strongly correlated with global gene expression changes during AKI. In summary, we identified an AKI-specific molecular signature providing the ground for novel biomarkers and target candidates such as SLPI and miR-182-5p in addressing AKI

Estrogen- and Satiety State-Dependent Metabolic Lateralization in the Hypothalamus of Female Rats

<div><p>Hypothalamus is the highest center and the main crossroad of numerous homeostatic regulatory pathways including reproduction and energy metabolism. Previous reports indicate that some of these functions may be driven by the synchronized but distinct functioning of the left and right hypothalamic sides. However, the nature of interplay between the hemispheres with regard to distinct hypothalamic functions is still unclear. Here we investigated the metabolic asymmetry between the left and right hypothalamic sides of ovariectomized female rats by measuring mitochondrial respiration rates, a parameter that reflects the intensity of cell and tissue metabolism. Ovariectomized (saline injected) and ovariectomized+estrogen injected animals were fed <i>ad libitum</i> or fasted to determine 1) the contribution of estrogen to metabolic asymmetry of hypothalamus; and 2) whether the hypothalamic asymmetry is modulated by the satiety state. Results show that estrogen-priming significantly increased both the proportion of animals with detected hypothalamic lateralization and the degree of metabolic difference between the hypothalamic sides causing a right-sided dominance during state 3 mitochondrial respiration (St3) in <i>ad libitum</i> fed animals. After 24 hours of fasting, lateralization in St3 values was clearly maintained; however, instead of the observed right-sided dominance that was detected in <i>ad libitum</i> fed animals here appeared in form of either right- or left-sidedness. In conclusion, our results revealed estrogen- and satiety state-dependent metabolic differences between the two hypothalamic hemispheres in female rats showing that the hypothalamic hemispheres drive the reproductive and satiety state related functions in an asymmetric manner.</p></div

Correlation coefficients (Spearman's rho) of (A) miR-21-3p and (B) miR-182-5p and mRNAs plotted against the corresponding raw <i>p</i>-values of baseline adjusted mRNA levels comparing the AKI and PGF group.

<p>Correlation coefficients (Spearman's rho) of (A) miR-21-3p and (B) miR-182-5p and mRNAs plotted against the corresponding raw <i>p</i>-values of baseline adjusted mRNA levels comparing the AKI and PGF group.</p

SLPI protein concentration (ng/ml) measured by sandwich ELISA in (A) EDTA-plasma and (B) urine of AKI and PGF patients.

<p>Three AKI patients were anuric at the time of post-TX biopsy. (C) Relative gene expression levels of SLPI in post-TX biopsies. Individual data points as well as median, 1^st and 3^rd quartile are provided.</p

Percentage of animals with hypothalamic asymmetry in state 3 mitochondrial respiration rate (<i>mrr</i>).

<p>E2 treatment, regardless of satiety states, caused significantly higher proportion of sided animals (Fisher’s exact test, *: p<0.05). In absence of E2, 24 hours food restriction further reduced the extent of hypothalamic asymmetry. Experimental groups: E2+<i>ad lib</i>: estrogen treated, <i>ad libitum</i> fed animals (n = 5); E2+fasted: estrogen treated, fasted animals (n = 6); S+<i>ad lib</i>: vehicle injected, <i>ad libitum</i> fed animals (n = 5); S+fasted: vehicle injected, fasted animals (n = 4). Sidedness was considered if the difference between the left and right hypothalamic sides of the individual was 20% or higher.</p

Kidney donor and recipient characteristics comparing AKI and control groups in the Basic and independent validation datasets.

<p>Continuous data are provided as median and 1^st and 3^rd quartile; categorical data are shown as counts.</p><p>na … not applicable,</p>a<p>Fisher's exact test,</p>b<p>mean (range),</p>c<p>median (range),</p><p>n. st. … not stated.</p><p>PGF … primary graft function, AKI … acute kidney injury.</p

Raw <i>p</i>-values and area under the curve (AUC) of the investigated mRNAs and miRNAs comparing post-TX AKI and PGF allografts.

<p>(A) full p-value range of mRNAs (B) mRNAs with <i>p</i>-value<0.08 on the x-axis for specific visualization of features with a raw <i>p</i>-value<0.05 (C) full p-value range of microRNAs (D) microRNAs with raw <i>p</i>-value<0.08 on the x-axis; red: features verified in an independent dataset (20 mRNAs and two microRNAs), green: significant features after baseline adjustment (39 mRNAs and 29 miRNAs), blue: significant features identified by SAM (245 mRNAs and 49 microRNAs), black: remaining features (11,788 mRNAs and 393 microRNAs).</p

AKI mRNA signature as verified in the independent evaluation dataset.

<p><b>bold</b>......molecular features discussed as biomarker candidates of acute kidney injury.</p><p>Raw p-values and fold changes of verified differentially regulated genes are provided.</p

AKI miRNA signature as verified in the evaluation dataset.

<p>Raw p-values and fold changes of verified differentially regulated miRNAs are provided.</p