D conditions for the induction of various degrees of renal injury, these are only directive and may substantially differ from conditions applied by others using other strains or slightly different procedures [43,87]. Therefore, before applying conditions and procedures reported by others, it is recommended to, at first instance, reproduce their validated findings and observations. Depending on the research focus, such observations can either be biochemical (creatinine, BUN, proteinuria), molecular (fibrosis-related gene or protein expression) or histological (e.g. percent positivity on collagen I immunostaining or the number of Ki67 positive nuclei per 400x field). If Chaetocin site results (including mortality rate) diverge substantially (>20 ) from the selected reference data, additional fine-tuning is warranted. Since our data indicate that ischemia time, rather than core body temperature during ischemia, influences the extent of the fibrotic outcome (Figs 1?), fine-tuning is preferentially done by adjusting the duration of ischemia, e.g. in 5 minute steps at first. It needs to be stressed however, that this does not rule out the importance of tight temperature control during the ischemia-reperfusion procedure, as this is still an important source of variation. For accurate results we recommend to use a heating pad with L-660711 sodium salt web feedback system through a rectal temperature probe along with a heat lamp. To further increase temperature control, we found additional benefit in keeping the mice in a neonate incubator during ischemia. In view of the above, it should be mentioned that applying reported ischemia-reperfusion conditions or procedures remains a challenge. This is mainly due to the overall lack of consensus on which information regarding the conditions of the IRI-model, e.g. body temperature during, ischemia time, method of temperature control, strain, gender and age of the animals, etc., should minimally be reported. This complicates the interpretation of results from different laboratories and often does not allow reproduction or unbiased comparison of data. To this end we provide some recommendations to obviate this important issue. First, it is important to mention whether ischemia-reperfusion is conducted bilaterally or unilaterally, and in case of unilateral IRI, whether or not a contralateral nephrectomy is performed, and, if so, at which time following ischemia. Second, when referring to previous reported methods, authors have to ensure that j.jebo.2013.04.005 these references cover the required information that allows a good insight in the details of the model. Third, authors often do not report the temperature at which ischemia-PLOS ONE | DOI:10.1371/journal.pone.0152153 March 23,17 /An Ischemic Mouse Model for AKI to CKDreperfusion is conducted, or only mention the temperature of the operating mat, which differs from the body temperature of the animal. Fourth, the strain and number of animals included per group, as well as mortality rate should be mentioned, as this is an additional indicator of severity of the insult. Last, experimental end-points (e.g. time-point of euthanasia, criteria for early euthanasia) should be mentioned in the methods-section. In conclusion, we demonstrate that UIRI without nephrectomy is a very robust model for induction of long-term tubulo-interstitial fibrosis. In addition, we demonstrate that varying the two main determinants of IRI induced AKI, i.e. body temperature during and duration of ischemia, in the unilateral IRI without.D conditions for the induction of various degrees of renal injury, these are only directive and may substantially differ from conditions applied by others using other strains or slightly different procedures [43,87]. Therefore, before applying conditions and procedures reported by others, it is recommended to, at first instance, reproduce their validated findings and observations. Depending on the research focus, such observations can either be biochemical (creatinine, BUN, proteinuria), molecular (fibrosis-related gene or protein expression) or histological (e.g. percent positivity on collagen I immunostaining or the number of Ki67 positive nuclei per 400x field). If results (including mortality rate) diverge substantially (>20 ) from the selected reference data, additional fine-tuning is warranted. Since our data indicate that ischemia time, rather than core body temperature during ischemia, influences the extent of the fibrotic outcome (Figs 1?), fine-tuning is preferentially done by adjusting the duration of ischemia, e.g. in 5 minute steps at first. It needs to be stressed however, that this does not rule out the importance of tight temperature control during the ischemia-reperfusion procedure, as this is still an important source of variation. For accurate results we recommend to use a heating pad with feedback system through a rectal temperature probe along with a heat lamp. To further increase temperature control, we found additional benefit in keeping the mice in a neonate incubator during ischemia. In view of the above, it should be mentioned that applying reported ischemia-reperfusion conditions or procedures remains a challenge. This is mainly due to the overall lack of consensus on which information regarding the conditions of the IRI-model, e.g. body temperature during, ischemia time, method of temperature control, strain, gender and age of the animals, etc., should minimally be reported. This complicates the interpretation of results from different laboratories and often does not allow reproduction or unbiased comparison of data. To this end we provide some recommendations to obviate this important issue. First, it is important to mention whether ischemia-reperfusion is conducted bilaterally or unilaterally, and in case of unilateral IRI, whether or not a contralateral nephrectomy is performed, and, if so, at which time following ischemia. Second, when referring to previous reported methods, authors have to ensure that j.jebo.2013.04.005 these references cover the required information that allows a good insight in the details of the model. Third, authors often do not report the temperature at which ischemia-PLOS ONE | DOI:10.1371/journal.pone.0152153 March 23,17 /An Ischemic Mouse Model for AKI to CKDreperfusion is conducted, or only mention the temperature of the operating mat, which differs from the body temperature of the animal. Fourth, the strain and number of animals included per group, as well as mortality rate should be mentioned, as this is an additional indicator of severity of the insult. Last, experimental end-points (e.g. time-point of euthanasia, criteria for early euthanasia) should be mentioned in the methods-section. In conclusion, we demonstrate that UIRI without nephrectomy is a very robust model for induction of long-term tubulo-interstitial fibrosis. In addition, we demonstrate that varying the two main determinants of IRI induced AKI, i.e. body temperature during and duration of ischemia, in the unilateral IRI without.