Distinct effects on long-term function of injured and contralateral kidneys following unilateral renal ischemia-reperfusion

Abstract

Salt-sensitive hypertension and chronic kidney disease (CKD) following recovery from acute kidney injury (AKI) may occur secondary to incomplete repair, or by activation of circulating factors stimulated by injury. We created two types of renal injury induced by unilateral ischemia-reperfusion (I/R); in a direct/ipsilateral AKI group, rats were subjected to unilateral I/R and the untouched contralateral kidney was removed by unilateral nephrectomy after 5 wk to isolate effects on the injured kidney. In the remote/contralateral AKI group, the injured kidney was removed after 5 wk to isolate effects on the untouched kidney. When these animals were subsequently challenged with elevated dietary sodium for an additional 4 wk (0.4 to 4%), both remote/contralateral and direct/ipsilateral AKI rats manifested a significant increase in blood pressure relative to sham-operated controls. Similarly, in acute studies, both ipsilateral and contralateral kidneys had impaired pressure natriuresis and hemodynamic responses. Reductions in vascular density were observed following direct/ipsilateral injury, but were not observed in the remote/contralateral kidney. However, both remote/contralateral and direct/ipsilateral kidneys contained interstitial cells, some of which were identified as activated (low CD62L/CD4+) T lymphocytes. In contrast, only the direct/ipsilateral AKI group demonstrated significant CKD following exposure to elevated salt. This was characterized by a significant reduction in creatinine clearance, an increase in albuminuria, and a dramatic expansion of interstitial inflammation. Taken together, these data suggest that the salt-sensitive features of AKI on hypertension and CKD are segregable such that effects on hemodynamics and hypertension occur independent of direct renal damage. However, prior direct injury to the kidney is required to elicit the full manifestation of CKD induced by elevated sodium intake.

Fig. 1.

Experimental schema of studies carried out to distinguish the effects of unilateral injury on chronic function in both direct/ipsilateral and remote/contralateral injured kidneys. A: illustrates groups used in study I to distinguish between direct and remote effects of ischemia-reperfusion (I/R) injury on salt sensitivity. The time line is similar to that used in previous studies (25). All rats are maintained on standard 0.4% NaCl diet during recovery for 5 wk of unilateral I/R (red circle) or sham I/R (black circle). The “direct/contralateral” injury group is studied by subjecting rats to unilateral nephrectomy (UNx; depicted by X) of the uninjured, contralateral kidney at 33 days, such that only the injured kidney remains. The “remote/contralateral” injury group is prepared by removal of the injured kidney at 33 days, such that contralateral kidney remains. UNx is also carried out in the sham group as a control for both direct and remote injury groups. At 35 days, rats are exposed to an elevated NaCl diet (4.0%) for the remaining 4 wk of the study. At 8 wk, telemetric transducers are implanted to evaluate the effects of treatments on mean arterial blood pressure, which was evaluated on the final 3 consecutive days of the study. B: illustrates the time line for study II, in which the effects of “direct/ipsilateral” and “remote/contralateral” injury to the kidney are subjected to acute renal function studies. In these studies, animals recover for 1 wk following UNx (from week 5–6) and are maintained for the entire study period on standard 0.4% NaCl diet. At 6 wk of recovery, the effects of these injuries on renal autoregulation and pressure natriuresis were studied.

Fig. 2.

Effect of direct and remote injury on serum creatinine and creatinine clearance. A: serum creatinine values are shown for sham, remote/contralateral, and direct/ipsilateral groups at 24 h, 7 days, 33 days, and 56 days post-I/R or sham. B: creatinine clearance values are shown for sham, remote/contralateral, and direct/ipsilateral groups based on 24-h urine collections at the indicated times; n = 7, 11, and 7 for sham, remote/contralateral, and direct/ipsilateral injury groups, respectively. Data in A and B are means ± SE. *P < 0.05 vs. sham-operated control by ANOVA and Student-Newman-Keuls post hoc test. NS, not significant.

Fig. 3.

Renal histology of direct and remote I/R injury at 33 days of recovery. Representative renal histology of ipsilateral and contralateral kidneys following unilateral I/R, obtained at the time of UNx, before exposure to elevated dietary salt. Shown are representative periodic acid Schiff (PAS)-stained sections of a kidney from a sham-operated animal at day 33 (A), a contralateral injured kidney (B), and an ipsilateral injured kidney (C). Note renal tubular morphology is largely intact in both direct and remote injury. Evidence of increased interstitial cells is present in both the contralateral and ipsilateral kidneys (black arrows). Magnification is shown.

Fig. 4.

Effect of unilateral I/R on kidney weight of direct/ipsilatereal and remote contralateral kidneys. A: shown are kidney-to-body wt ratios at 33 days, representing weights at the time of UNx. At 33 days, the contralateral kidney is from direct injury group and is significantly hypertrophic relative to the sham. The kidney direct/ipsilateral kidney is from the remote group and is slightly hypotrophic. B: kidney-to-body wt ratios for sham, direct/ipsilateral, and remote/contralateral injury at 63 days. Data in A and B are means ± SE. * And #P < 0.05 vs. sham-operated control by Student's t-test.

Fig. 5.

Effect of elevated NaCl intake on albuminuria in sham, remote/contralateral acute kidney injury (AKI), and direct/ipsilateral AKI groups. Albumin excretion rates were derived from 24-h urine collections at the indicated times. Data for day 63 time points were not obtained due to the need to collect blood pressure measurements. Data are means ± SE. *P < 0.05 vs. sham-operated control by ANOVA and Student-Newman-Keuls post hoc test. (a, b)Significant increases in sham-operated and remote injury groups following exposure to elevated NaCl relative to the final collection under standard salt conditions (i.e., day 33).

Fig. 6.

Effect of direct/ipsilateral injury and remote/contralateral injury on the salt-sensitive changes in blood pressure. Values shown are mean blood pressures derived from telemetry measurements averaged over the 3-day period from days 60–62 of study I. Due to loss of animals at the time of implantation of transducers, n = 5, 6, and 3 for the sham, remote, and direct injury groups. Data are means ± SE. *P < 0.05 vs. sham-operated control by Student's t-test.

Fig. 7.

Representative renal histology from direct/ipsilateral and remote/contralateral injured kidneys following reduction in renal mass by UNx and exposure to elevated dietary salt. Shown are representative PAS-stained sections of a kidney from a sham-operated animal at day 63 (A), a remotely injured kidney (B), and a directly injured kidney (C). Magnification is shown in C. Insets for each micrograph reveal the presence of interstitial infiltrate in remotely injured kidneys and a more severe infiltration in directly injured kidneys.

Fig. 8.

Macrophage deposition following direct/ipsilateral AKI and remote/contralateral AKI. ED-1-positive cells were identified in both remote/contralateral (A) and direct/ipsilateral injured kidneys (B) and are indicated by the black arrows. Magnification is shown.

Fig. 9.

Effect of direct/ipsilateral and remote/contralateral injury on renal function, pressure natriuresis, and medullary blood flow. Rats from study II were analyzed at 6 wk following I/R and renal hemodynamics were measured in response to increases in renal perfusion pressure. A: glomerular filtration rate (GFR) measured by inulin clearance. B: urinary flow rate. C: sodium excretion. D: medullary blood flow by laser Doppler flow. The N for each group is shown. *P < 0.05 by ANOVA and Student-Newman-Keuls post hoc test measuring the high-pressure point in remote and direct groups relative to the sham-operated control group.

Fig. 10.

Effect of unilateral I/R on capillary density in remote/contralateral and direct/ipsilateral kidneys. Quantitative analysis of vessel density scoring based on cablin immunohistochemistry is shown. Data are means ± SE and the N for each group is shown. *P < 0.05 in directly injured vs. sham-operated control by Student's t-test, while remote injury did not influence blood vessel density (N.S.).

Fig. 11.

FACS analysis of renal T cell deposition in sham kidneys, remote/contralateral kidneys, or direct/ipsilateral kidneys 33 days following unilateral I/R. A: forward and side scatter are shown with representative CD4+ and CD8+ characterization corresponding to the indicated population. B: representative histograms of CD62L and CD25 are indicated for the CD4-positive population in spleen, and kidney from sham, direct and remote AKI kidneys. The total numbers of CD4 and CD8 lymphocytes per kidney are shown in C and D, respectively; while the mean fluorescence intensities of CD62L and CD25 of the CD4-positive populations are shown in E and F, respectively. Data are means ± SE and are based on N = 5 per group. *P < 0.05 by Student's t-test relative to sham-operated control. Differences between remote and direct injury groups are indicated above each of these bars in the graphs.