Guanosine supplementation reduces apoptosis and protects renal function in the setting of ischemic injury - PubMed (original) (raw)

Guanosine supplementation reduces apoptosis and protects renal function in the setting of ischemic injury

K J Kelly et al. J Clin Invest. 2001 Nov.

Abstract

Ischemic injury to the kidney is characterized in part by nucleotide depletion and tubular cell death in the form of necrosis or apoptosis. Recently, we linked anoxia-induced apoptosis in renal cell cultures specifically to the depletion of GTP. We therefore hypothesized that enhancing GTP repletion in vivo might protect function by reducing apoptosis in postischemic tubules. Male C57 black mice (the "I" group of animals) underwent bilateral renal artery clamp for 32 minutes to induce ischemia and then received either normal saline ("NS") or guanosine ("G"). After 1 hour of reperfusion, renal GTP levels in NS/I were reduced to nearly half of those in sham operated mice, whereas these levels were nearly unchanged in G/I mice. Morphologic examination of tubular injury revealed no significant differences between the two groups. However, there was a significant reduction in the number of apoptotic tubular cells in the medulla in the G/I group as compared with the NS/I group. At 24 hours, creatinine was significantly elevated in the NS/I group, compared to the G/I group. We conclude that guanosine protects against renal ischemic injury by replenishing GTP stores and preventing tubular apoptosis.

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Figures

Figure 1

Figure 1

Effects of intraperitoneal guanosine on plasma guanosine levels. Animals received 30 mg/kg guanosine intraperitoneally 2 hours before bilateral renal ischemia/reperfusion. Blood was collected at various time points and plasma guanosine levels determined by HPLC as detailed in Methods. Values are means ± SE (n = 3).

Figure 2

Figure 2

Effects of guanosine on renal nucleotide levels during reperfusion. Values are means ± SE. Mice received normal saline (NS, n = 5) or guanosine (G, n = 5) 30 mg/kg intraperitoneally, 2 hours before 32-minute bilateral renal ischemia (I) or sham surgery. Renal tissues were obtained from each kidney 1 hour after surgery and processed for nucleotide determination (*P < 0.02, when GTP in NS/I was compared with GTP in G/I).

Figure 3

Figure 3

Effects of guanosine and enhanced GTP recovery on renal histology at 24 hours. Mice received normal saline (NS) or guanosine (G) 30 mg/kg intraperitoneally 2 hours before renal ischemia (I) or sham surgery. Representative H&E-stained sections from sham, NS/I, and G/I 24 hours after surgery are shown. Compared with the sham group, sections from both NS/I and G/I show patchy tubular dilatation, necrosis, and extensive cast formation. DAPI (blue) and FITC-phalloidin (green) staining show significant loss and/or disruption of the apical actin rim in both NS/I and G/I as compared with sham.

Figure 4

Figure 4

Effects of guanosine and enhanced GTP recovery on renal cell apoptosis at 24 hours. Mice were treated as detailed in legend of Figure 2. Kidney sections were obtained at 24 hours and costained with TUNEL and DAPI. (a) A negative control (NC; TdT omitted) and (b) a DNase-treated positive control (PC) for TUNEL. All TUNEL-positive nuclei had bright green fluorescence. (c) A representative field showing the medulla from NS/I group. Extensive TUNEL-positive staining is seen and is localized primarily to the tubular epithelium (dark green autofluorescence). (d) A representative field from the medulla of the G/I group showing lack of TUNEL-positive staining. (e) A magnification of TUNEL-positive nuclei from NS/I mice, with only the DAPI channel turned on. ×60. Nuclei are condensed, dysmorphic, and show heavy staining. Inset shows a single nucleus. ×120. Typical apoptotic fragmentation of chromatin into four small bodies is seen. (f) A magnification of TUNEL-negative nuclei from G/I mice. ×60. They show lack of condensation and faint DAPI staining.

Figure 5

Figure 5

Morphologic characterization of apoptosis by light and electron microscopy. Mice received normal saline (NS) or guanosine (G) 30 mg/kg intraperitoneally 2 hours before renal ischemia or sham surgery. Representative H&E-stained sections 24 hours after ischemia from the guanosine-treated group are shown (a and b). Nuclei with predominantly necrotic features are observed. They have degraded chromatin without formation of discrete, membrane-bound fragments. (c, d, e, and f) From the NS-treated group. Classic apoptotic bodies with dense condensation and fragmentation of chromatin into discrete fragments are seen (arrows). (g and h) Electron microscopic photomicrographs of sections from the normal saline group show clear apoptotic (g) and necrotic (h) morphologies.

Figure 6

Figure 6

Quantitative evaluation of TUNEL-positive nuclei in cortex and medulla. Values are means ± SE and represent number of TUNEL-positive nuclei expressed as percentage of total number of nuclei. Mice were treated as detailed in the legend of Figure 2. Kidney sections were obtained at 24 hours and processed for TUNEL and DAPI staining as detailed in Methods. Number of fields counted: 20 for G/I, 35 for NS/I, 8 for G/sham, and 8 for NS/sham. (*P < 0.01 when medullary TUNEL-positive nuclei in NS/I were compared to all other groups).

Figure 7

Figure 7

Distribution of TUNEL-positive nuclei among various nephronal segments. (a) A 5-μm thick section from NS/I mice stained with TUNEL and DAPI. The TUNEL positive nuclei were pseudocolored white instead of green to allow clear distinction from the green FITC-phalloidin. (b) A 5-μm section immediately contiguous to the one shown in a is stained with the actin marker FITC-phalloidin (bright green) to identify proximal tubules (P). Distal tubules (D) show minimal or no staining. The section was costained with anti-THP Ab and a Cy5-conjugated secondary Ab (red). This is predominantly a marker for Henle’s loop segments (LH). (c) An overlay of a and b allows the localization of TUNEL-positive nuclei to various tubular segments. (d) A quantitative estimate of the distribution of TUNEL-positive nuclei normalized to total number of DAPI-positive nuclei for each particular tubular segment or lumen. Other refers to interstitial or vascular TUNEL-positive nuclei.

Figure 8

Figure 8

Effects of guanosine and enhanced renal GTP levels on renal function at 24 hours. Values are means ± SE. Creatinine levels at 24 hours are shown after administration of normal saline (NS) or guanosine (G) 2 hours before sham surgery or bilateral renal ischemia. Mean creatinine was significantly higher in the NS/I mice than in the other groups. (*P < 0.01, n = 6 for NS/I and G/I groups).

Figure 9

Figure 9

Effects of guanosine on nucleotide levels and apoptosis after chemical anoxia recovery in LLC-PK1 cells. Values are means ± SE. (a) LLC-PK1 cells were treated for 45 minutes with 0.1 μM antimycin A in depleted media followed by recovery for 2 hours. Nucleotides were measured at 45 minutes of depletion (n = 7) and at 2 hours of recovery (n = 6). (b) Cells were treated identically except for the addition of 200 μM guanosine to the recovery medium (n = 6). (c and d) Representative fields of confocal microscopic images of LLC-PK1 cells at 24 hours after recovery from chemical anoxia. Cells were costained with Hoechst 33342 and propidium iodide as detailed in Methods. In c, cells recovered in regular media. Apoptotic features such as condensation and fragmentation of chromatin is seen in most cells in the field. In d, cells recovered in the presence of 200 μM guanosine. Most nuclei showed normal morphology, and only few had apoptotic features.

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