Am_J_Physiol_Renal_Physiol-2009-Kelly-ajprenal.00205.2009
Articles in PresS. Am J Physiol Renal Physiol (August 5, 2009). doi:10.1152/ajprenal.00205.2009 THE POST-ISCHEMIC INFLAMMATORY SYNDROME: A CRITICAL
1
MECHANISM OF PROGRESSION IN DIABETIC NEPHROPATHY
2
3
4
5
Running title: Ischemia, Inflammation and Cell Death in the Diabetic Kidney
6
Subject: Pathophysiology of Renal Disease
7
8
9
K. J. Kelly MD, MSc
10
11
BS
James
L.
Burford,
MD
12
Dominguez,
Jesus
H.
13
14
15
Address Correspondence to: K.J. Kelly, MD, MSc
16
Medicine
University
of
Indiana
School
17
Nephrology
Division
of
18
RII-202 950
West
Walnut
Street,
19
IN
46202
Indianapolis,
20
317-274-7453
Telephone:
21
FAX:
317-274-8575
22
kajkelly@https://www.wendangku.net/doc/dc5946727.html,
e-mail:
23
24
ABSTRACT
25
26
Diabetes is a major epidemic, and diabetic nephropathy is currently the most 27
common cause of end-stage renal disease. Two critical components of diabetic 28
nephropathy are persistent inflammation and chronic renal ischemia from widespread 29
vasculopathy. Moreover, acute ischemic renal injury is common in diabetes, potentially 30
causing chronic kidney disease or end-stage renal disease. Accordingly, we tested the 31
hypothesis that acute renal ischemia accelerates nephropathy in diabetes by activating pro-inflammatory pathways. Lean and obese-diabetic ZS rats (F1 hybrids of
32
33
spontaneously hypertensive heart failure and Zucker fatty diabetic rats) were subjected to bilateral renal ischemia or sham surgery prior to the onset of proteinuria. The post-
34
35
ischemia state in rats with obesity-diabetes was characterized by progressive chronic 36
renal failure, increased proteinuria and renal expression of pro-inflammatory mediators.
37
Leukocyte number in obese-diabetic rat kidney was markedly increased for months after 38
ischemia. Intrarenal blood flow velocity was decreased postischemia in lean control and 39
obese-diabetic rats, although it recovered in lean rats. Two months postischemia, blood flow velocity decreased further in both sham surgery and post-ischemia obese-diabetic
40
41
rats, so that erythrocyte flow velocity was only 39% of control in the obese-diabetic/post-42
ischemia rats. In addition, microvascular density remained depressed at 2 months in 43
obese-diabetic/post-ischemia kidneys. Abnormal microvascular permeability and 44
increases in interstitial fibrosis and apoptotic renal cell death were also more 45
pronounced after ischemia in obese-diabetic rats. These data support the hypothesis 46
that acute renal ischemia in obesity-diabetes severely aggravates chronic inflammation 47
and vasculopathy, creating a self-perpetuating post-ischemia inflammatory syndrome, 48
which accelerates renal failure.
INTRODUCTION
49
50
Metabolic syndrome (diabetes, dyslipidemia, obesity and hypertension) (15)
51
afflicts an ever-expanding proportion of the world’s population, frequently leading to
52
diabetic nephropathy and end-stage renal disease (ESRD) (43). An enlarging body of
53
data supports the novel hypothesis that anomalous immunological responses, triggered
54
by metabolic derangements, are critical at every stage of diabetic nephropathy.
Diabetics with renal disease have increased levels of inflammatory markers including C-
55
56
reactive protein, interleukin (IL)-6 and tumor necrosis factor-α (TNF) (9, 10) as well as 57
markedly abnormal leukocyte function (48). Serum TNF levels are correlated with
urinary protein excretion in diabetics without or with overt nephropathy (39), and specific 58
59
cytokine genotypes are associated with diabetic nephropathy (33). Renal infiltrates of
60
inflammatory cells with concurrent renal upregulation of leukocyte adhesion receptors,
61
including intercellular adhesion molecule-1 (ICAM-1), are found in human diabetic
62
nephropathy (4). Moreover, the number of interstitial macrophages is strongly
63
correlated with renal dysfunction, proteinuria and fibrosis in renal biopsy specimens
from diabetics (40). In addition, immune suppression is protective in models of diabetic
64
65
nephropathy (11, 53), and conversely, macrophages, in adoptive transfer studies,
66
induce proteinuria and mesangial expansion in rat kidneys (21).
67
In humans, acute kidney injury (AKI) from renal ischemia is often superimposed
68
on diabetic injury (20, 24). Furthermore, inflammation may contribute to AKI in humans,
69
as elevated urinary IL-6 and IL-8 in renal allograft recipients can predict AKI (32). In
70
diabetic animals, greater vulnerability to renal ischemia has been shown (17, 56), and
71
emerging data reveal that inflammation is a likely aggravating factor. For example,
72
leukocyte-binding molecules ICAM-1 and LOX-1 (28, 30) are increased after renal
ischemia in rats, and increases in renal leukocytes, TNF and IL-1 are all seen after renal
73
74
ischemia in mice (28).Thus, we tested the hypothesis that in obesity-diabetes, acute 75
renal ischemia would further activate renal pro-inflammatory pathways and accelerate 76
the progression of renal injury. We used the ZS model of the metabolic syndrome: lean 77
and obese F1 hybrid rats derived from the Zucker diabetic (ZDF) and the spontaneously 78
hypertensive heart failure (SHHF) rat. Obese ZS rats are known to develop albuminuria, 79
glomerulosclerosis, interstitial fibrosis and renal failure (13, 29). The lean ZS litter mates 80
served as normal controls. Acute renal ischemia in obese-diabetic rats evoked severe, 81
progressive renal inflammation that persisted well after the acute ischemic insult. The inflammation occurred in conjunction with far more severe renal vasculopathy, fibrosis,
82
83
apoptotic cell death and organ failure. These findings indicate that a single episode of acute renal ischemia has long-term and self-sustained adverse renal consequences in
84
85
obesity-diabetes.
86
87
MATERIALS and METHODS
88
89
Animal Protocols. All experiments were conducted in conformity with the "Guiding
90
Principles for Research Involving Animals and Human Beings." The investigations were
91
approved by the Institutional Animal Care and Use Committee of Indiana University
92
School of Medicine. Lean controls and obese-diabetic male ZSF1 rats (ZS, Jackson
93
Labs, Bar Harbor, ME), 8-32 weeks old, were fed Purina diet # 5008 with 27% protein,
27% animal fat and 56% carbohydrate. Body weights were recorded and sera plus urine 94
95
samples were collected at biweekly intervals. The sera were analyzed for glucose,
96
creatinine and urea, and the urine for protein and creatinine on a Beckman CX4CE
system (11). The rats were anesthetized with intraperitoneal (i.p.) pentobarbital (50
97
98
mg/kg) and placed on a homeothermic table to maintain core body temperature at 99
~37o C. After insuring adequate anesthesia, renal ischemia was induced by occluding
100
both renal pedicles for 25 minutes with microaneurysm clamps as described (27). This
101
results in a relatively mild acute functional insult. Mean BUN levels were 27 ± 1 mg/dl in
102
the lean control rats 24 hours postischemia (vs 12 ± 0.5 mg/dl in sham surgery
controls). Sham surgery was an identical surgical procedure with exposure of both 103
104
kidneys, but renal ischemia was not induced. For intravital imaging (below), a small
105
flank incision was made to expose the left kidney. Systolic blood pressure was
106
measured by tail cuff or femoral artery catheter prior to imaging.
107
Intravital multi-photon fluorescence microscopy (14). Intravital imaging was performed 108
109
with a Bio-Rad MRC-1024MP confocal/multiphoton microscope (Hercules, CA)
equipped with a titanium-sapphire laser (Spectraphysics, Mountain View, CA). Imaging 110
111
was performed at 12, 20 and 32 weeks of age (2, 8 and 12 weeks after renal ischemia
or sham surgery). The rats were placed on the heated (37oC) microscope stage and 112
113
covered with a temperature controlled pad. General anesthesia was accomplished with
114
pentobarbital (50 mg/kg) or thiobarbital (80 mg/kg) given intraperitoneally. The left
115
kidney was surgically exposed, placed in a cell culture dish with a glass bottom (Warner
116
Instruments, Hamden, CT), and bathed in warm 0.9% NaCl (14). Hoeschst 33342
117
(250μg in 0.5 ml 0.9% NaCl; Molecular Probes, Eugene, OR) was injected intravenously
immediately prior to imaging to identify nuclei and the focal plane. Renal microvascular 118
119
flow was visualized using fluorescein isothiocyanate (FITC) conjugated 100,000 Dalton
120
(large) dextran (400 μg in 0.5ml in 0.9% NaCl; Molecular Probes, Eugene, OR), injected
intravenously (IV) immediately prior to imaging. A smaller (20,000 Dalton) Texas Red-121
122
conjugated dextran (2 mg in 0.5 ml 0.9% NaCl injected IV) was used to assess
123
microvascular permeability. Excitation wavelength (800nm), laser output (approximately
124
30%) and photomultiplier settings were chosen on the basis of prior studies [14], so that
125
the fluorescence intensity of nuclei was approximately 50% of maximum across different
126
animals observed at different times.
127
128
Immunohistochemistry(22). Tissue was fixed in 3.8% paraformaldehyde and preserved 129
in 30% sucrose before 10 μm frozen sections were obtained. Sections were incubated 130
with rabbit anti-rat LOX-1 (12) and mouse anti-rat ICAM-1 (28) followed by a Texas
131
Red-conjugated donkey anti-rabbit IgG and FITC-conjugated donkey anti-mouse IgG
132
(Jackson ImmunoResearch, West Grove, PA) and the nuclear dye Dapi (Molecular
133
Probes, Eugene, OR). Images were collected with a Zeiss LSM 510 confocal
134
microscope and analyzed with Zeiss LSM software and MetaMorph (Universal Imaging
135
Corporation, Downingtown, PA). Renal collagen was rendered visible with second
harmonic imaging microscopy, which was performed directly on noncentrosymmetric 136
137
collagen with the two-photon microscope (7), without exogenous fluorphores. Standard 138
trichrome and chloracetate esterase (Leder) staining were also performed (12) to confirm collagen and leukocyte infiltration, respectively.
139
140
141
Microvascular flow/permeability/density. Renal capillary red blood cell velocity (RBCV) 142
was measured to estimate renal capillary blood flow rates. We used Metamorph 143
software (Molecular Devices, Downingtown, PA) to determine the displacement of 144
intracapillary erythrocytes on sequential images with correction for microvascular angle.
Vascular leak was quantified by analyzing initial intravital images in 4 x 4 grids, with 145
146
each grid scored for the presence or absence of small and large dextran extravasations, 147
expressed as fractions of total grid segments (47). Renal capillary density was 148
quantified as the fractional area in each section representing intravascular large 149
molecular weight dextran in the initial images obtained after dextran injection.
150
151
Image Scoring. All quantification was performed on coded images. Intravascular 152
leukocytes were identified as nucleated cells within the vasculature in intravital images 153
and classified as free flowing (non-adherent to vessel wall) or adherent (adherent to 154
vessel wall for >10 seconds) leukocytes. Erythrocyte aggregates were identified as 155
shadows of stacked red blood cells moving in unison, and were recorded as either 156
present or absent in each quadrant of the coded images. Fluorescence corresponding 157
to immunoreactive LOX-1 and ICAM-1 was quantified via Metamorph software. Fibrosis, 158
capillary area and fraction of abnormal tubules were also quantified using Metamorph. 159
Fibrosis is expressed as the fraction of the tissue area imaged as collagen. Tubules with
areas of denudation, shrunken cells or intraluminal casts were classified as abnormal. 160
161
Apoptotic cells were identified as those with condensed, fragmented nuclei and 162
expressed as the fraction of total nuclei in the image.
163
164
Statistics. Data are expressed as means ± 1 standard error. Analysis of variance was 165
used to determine if differences among mean values reached statistical significance. 166
Tukey’s test was used to correct for multiple comparisons. Correlations were 167
determined using non-parametric (Spearman’s) correlation coefficients. The null 168
hypothesis was rejected at p<0.05.
169
170
RESULTS
171
172
Metabolic and Renal Functional Parameters
173
ZS rats were randomly divided into four study groups: lean sham controls (LS, N 174
=4) only had their kidneys surgically exposed. Lean ischemic rats (LI, N = 4) had their 175
kidneys exposed and both renal pedicles clamped for 25 minutes. Obese-diabetic sham 176
surgery rats (OS, N = 3) had their kidneys exposed, whereas the surgically exposed kidneys in the obese-diabetic ischemic group (OI, N = 7) were also clamped for 25 177
178
minutes. The initial values for serum creatinine were similar in LS control rats and OS 179
rats at eight weeks of age, the point of entry to the study (two weeks prior to surgery).
However, even at this early age obese-diabetic rats were significantly heavier and their 180
181
blood glucose levels were higher than in their lean litter mates, figure 1. These 182
differences persisted throughout the study. Mean serum creatinine progressively 183
increased in OI from 16 to 24 weeks of age, well after the acute ischemic insult. In 184
contrast, serum creatinine remained unchanged in the other three groups, figure 1. 185
Urinary protein excretion was elevated in OS and OI, but it increased to significantly higher levels in the OI group. Proteinuria at 12 weeks was highly correlated with 186
187
fibrosis at study termination (r=0.98). Systolic blood pressure was also higher in OS and 188
OI rats and it increased to higher levels in OI, figure 1. There were 2 fatalities in the OI 189
group within two days of periodic intravital imaging at 12 and 32 weeks of age. This 190
outcome was unique to OI, with all the other rats recovering from imaging and finishing 191
the study successfully (p<0.05). Accordingly, direct comparisons among the four 192
groups were only made at those time points when the OI group numbered 4 rats or 193
more. An additional 3 OI rats expired during the course of the study, while all LI rats 194
completed the study.
195
196
Effect of Renal Ischemia and Time on Microvascular Blood Velocity in the Diabetic 197
Kidney
198
Renal capillary blood flow and leukocyte dynamics were visualized in living rats 199
using intravital, multiphoton fluorescence microscopy, figure 2. Peritubular capillary 200
plasma flow was determined from direct measurements of red blood cell (RBC) velocity 201
(RBCV). The initial set of RBCV values was obtained in 12-week-old rats, 2 weeks after 202
surgery; the second and third sets of measurements were collected in the same rats 203
when they had reached 20 and 32 weeks of age. The initial RBCV values, at 12 weeks of age, were similar in LS rats and OS rats, and were depressed in both lean and 204
205
obese-diabetic rats postischemia (LI controls, 211 ± 58, and OI diabetic 209 ± 39). In 206
20 and 32 week-old rats mean RBCV was significantly lower in OS diabetic than in 207
same age LS control rats. More severely depressed RBCV was seen in the LI group at 208
20 weeks, the time when serum creatinine begins to increase. RBCV decreased further 209
with time in LI.
210
211
Effect of Renal Ischemia on Microvascular Density
212
The impaired renal capillary blood velocity in obese-diabetic rats was accompanied by 213
renal microvascular attenuation (51). Accordingly, collected intravital two-photon renal 214
images were used to measure the effect of ischemia on the extent of the renal 215
microvascular network (figures 2 and 3). Specific intravascular fluorescence was 216
quantified as the number of intravascular pixels per image, and then expressed as a 217
fraction of the total number of pixels in the same image, or fractional intravascular 218
fluorescence. Renal ischemia caused an early attenuation of the microvasculature
measured two weeks postischemia in both control lean and obese-diabetic rats which 219
220
worsened with time. The percent of total tissue area attributable to intravascular
221
fluorescence in kidneys of 32 week old rats was 10.4 ± 0.3% in LS and 8.5 ± 0.2% in OI,
222
p<0.01), figure 3. The significant pruning of the renal peritubular vasculature was
223
associated with extensive renal capillary leak as shown in the following section.
224
225
Effect of Renal Ischemia on Microvascular Integrity (Permeability) in Diabetes
Representative two-photon intravital images of LS, LI, OS and OI kidneys are 226
227
shown in figures 2 and 4 and the data summary in figure 3. In preparation for imaging,
228
rats were injected intravenously with three fluorescent dyes: Hoechst 33342, a blue
229
fluorescing dye to label nuclei, a 20,000 Dalton (small) Texas Red conjugated dextran, a
230
red fluorescing dye, and a 100,000 Dalton (large) FITC labeled dextran, a green
231
fluorescing dye to label intravascular spaces. Sequential images were obtained
beginning 20 minutes after injection of Hoechst 33342, and 2 minutes after injection of 232
233
dextrans. Specific fluorescence intensities for both small and large dextrans in the
234
interstitial space were analyzed with MetaMorph software. The fraction of grids in each
235
image with evidence of interstitial leaked dextran is shown in figure 3. Two weeks post-
236
ischemia, interstitial fluorescence indicating leak of the smaller Texas Red-dextran was
237
indistinguishable from background in LS, and slightly elevated in LI rats (0.019 ± 0.01
and 0.11 ± 0.05, respectively). The dye leak values in LS and LI remained relatively 238
239
constant though-out the study. Initial and subsequent dye leakage was high in OS and
240
even more abnormal in OI (0.24 ±0.03 and 0.63 ± 0.06, respectively), figures 3 and 4.
The larger dextran leakage was similarly distributed, albeit at a lower rate. Leaked 241
242
interstitial fluorescence of larger FITC-dextran was indistinguishable from background in
LS and in LI rats and remained relatively constant throughout the study. Microvascular 243
244
permeability to the large dextran was elevated in OS and more markedly increased in
245
OI (0.13 ± 0.03 and 0.40 ± 0.06, respectively) at 2 weeks postischemia. Hence, these
data show that generalized renal capillary leakage in obesity-diabetes was further 246
247
aggravated by ischemia.
248
Effect of Renal Ischemia on Leukocyte Dynamics in the Diabetic Kidney
249
250
Renal intravascular leukocytes were identified and quantified by intravital microscopy
251
and confirmed in post-mortem renal sections. In vivo, total, free flowing and adherent
leukocytes were quantified in intravital images, and expressed as numbers of 252
253
intravascular leukocytes in microscopic fields magnified 60 times. Initially, few
254
intravascular renal leukocytes/field were seen in LS, LI, and OS kidneys. Leukocyte
255
number was much higher in OI rats (13.7 ± 1.5; p<0.0001 vs OS), figure 5.
256
Subsequently, capillary leukocyte number increased further in OI and to a lesser extent
257
in OS and LI, figure 5. Furthermore, those leukocytes that adhered to vascular
endothelium were identified and counted in vivo. Initially, there were far more 258
259
adherent renal leukocytes in OI rats than in OS rats (OI 47 ± 5%, OS 3 ± 3%, p<0.0001
260
vs OI), or LI (3 ± 3%), or LS (0 ± 0%, ANOVA p<0.0001). Subsequently, leukocyte
number remained higher in OI than in the two lean groups, figure 5. Neutrophils were 261
262
counted in post-mortem renal sections stained with Leder’s stain and identified by their
263
typical multilobed nuclei. Neutrophil numbers were higher in OS than in LS (OS, 17.7
± 1.0 vs. LS, 0.2 ± 0.2, p<0.0001) and post-ischemia, renal neutrophils in diabetic OI 264
265
rats were higher than in corresponding LI controls (OI, 25.8 ± 3.0 vs. LI, 1.2 ±0.4,
p<0.01). Total intravascular leukocyte number was highly correlated with serum 266
267
creatinine (r=0.83).
268
Effect of Renal Ischemia on Erythrocyte Aggregation in the Diabetic Kidney
269
270
Renal capillary blood flow in obesity-diabetes was distorted by increased numbers of 271
circulating red blood cell (RBC) aggregates. The adherent RBCs formed free flowing 272
microclusters, easily identified by intravital microscopy. RBC aggregates were virtually 273
non-existent in LS, 0.06 ± 0.06 micro-aggregates per 60 X microscopic field, and 0.23 ± 274
0.07 in OS diabetic rats, p=0.07. Initially, the effect of ischemia on the number of RBC
micro-aggregates was striking in LI and OI: 0.65 ± 0.16 and 1.09 ± 0.15, respectively, p 275
276
<0.05. RBC aggregation resolved in LI but it subsequently increased further in OS and 277
OI, figure 5.
278
279
Effect of Renal Ischemia on the Expression of the Pro-inflammatory Receptors ICAM-1 280
and LOX-1 in the Diabetic Kidney
The renal pro-inflammatory state in obesity-diabetes is characterized not only by 281
282
invading inflammatory cells, but also by induction of adhesion receptor molecules that 283
anchor and retain leukocytes (11, 28, 35, 45). Two of these critical recognition 284
molecules are LOX-1 and ICAM-1, which have restricted expression in lean rats, but are 285
strongly expressed in renal tubules of obese-diabetic rats (29). Accordingly, these two 286
recognition molecules were sought and identified in post-mortem renal sections 287
obtained at 32 weeks, figure 6. Immunoreactive ICAM-1 and LOX-1 were barely 288
detected in LS controls, but they increased markedly after ischemia (LI, 2.01± 0.32 and 289
1.58 ± 0.14 fold respectively, p <0.05). LOX1 and ICAM-1 were also strongly expressed
in diabetes-obesity (OS, 1.94 ± 0.38 and 4.53 ± 1.63-fold, respectively) consistent with 290
291
previous results (29). Ischemia further enhanced their expression (LOX-1 7.92 ± 0.95 292
and ICAM-1 8.98 ± 3.02-fold in OI, both p<0.01). Both ICAM-1 and LOX-1 expression were correlated with serum creatinine, proteinuria and fibrosis (via trichrome stain; table 293
294
1).
295
Effect of Renal Ischemia on Fibrosis in the Diabetic Kidney
296
297
Renal fibrosis is a decisive morbid outcome in nephropathy, and it was followed in vivo 298
by second harmonic two-photon imaging microscopy of renal noncentrosymmetric collagen. In addition, the final extent of renal fibrosis was measured in post-mortem 299
300
renal sections stained with Mason’s trichrome. In vivo, renal fibrosis was first 301
noticeable in sham operated 20 week-old obese-diabetic rats (OS), and more extensive 302
in the same-age ischemic group of obese-diabetic rats (OI): 24.5 ± 1.7 and 29.8 ± 1.4% 303
of tissue area, respectively, p < 0.05. In contrast, early renal fibrosis in corresponding 304
lean controls (LS), and lean post-ischemic rats (LI) was not detectable, 13.5 ± 2.1 and 305
15.2 ± 1.6%, although later on fibrosis increased slightly in the latter group, figure 7. 306
307
Effect of Renal Ischemia on Cell Death in the Diabetic Kidney
308
Apoptotic cell death has been proposed as a cause of diabetic nephropathy important in 309
tubular atrophy and tubulointerstitial fibrosis (31, 46, 55). Tubular apoptosis has been 310
demonstrated in renal biopsies of patients with early and advanced diabetic 311
nephropathy and correlated with subsequent loss of function as well as low density 312
lipoprotein levels and duration of diabetes (55). Multiple abnormalities in the metabolic 313
syndrome, including hyperglycemia, inflammation, reactive oxygen species and
dyslipidemia result in apoptosis in cultured cells (1, 42, 50, 54) . The loss of cells in the 314
315
kidney via apoptosis may be a significant contributor to loss of function and interstitial
316
fibrosis. Therefore, we quantified apoptosis in sham and obese-diabetic rat kidneys after
renal ischemia or sham surgery. Renal apoptosis was markedly increased in sham-317
318
operated obese-diabetic rats as indicated by intravital microscopy (figure 8). Renal cell
319
death was increased further after ischemia, and escalated progressively in OI with time.
320
This sustained increase in the rate of renal cell death induced by ischemia was still
321
evident 22 weeks following the single episode of ischemia, figure 8.
322
323
DISCUSSION
324
325
Diabetic patients and those with chronic kidney disease are predisposed to acute 326
ischemic renal injury (20). Thus, we tested the hypothesis that ischemia would exacerbate injury in the diabetic kidney. We investigated the effect of a single episode 327
328
of renal ischemia on function, structural abnormalities, inflammation, microvascular 329
dysfunction and cell death in an animal model of obesity-diabetes. Lean and obese-330
diabetic ten week-old rats were subjected to either sham surgery or bilateral renal 331
ischemia and followed for 22 weeks. Months after renal ischemia, renal function as well 332
as fibrosis, inflammation and apoptosis were accelerated in the obese-diabetic (OI) rats.
Long term, serum creatinine levels remained unchanged in sham operated lean rats 333
334
(LS), post-ischemic lean rats (LI), and in obese-diabetic sham operated rats (OS). In 335
contrast, after an initial recovery, serum creatinine increased progressively in post-ischemic obese-diabetic rats (OI), reaching levels consistent with advanced renal failure 336
337
(13). This supports the hypothesis that AKI exacerbates chronic renal injury. 338
Conversely, proteinuria was progressive postischemia, consistent with impaired 339
recovery from an acute insult in chronic kidney disease (CKD). Urine protein excretion 340
was elevated in OI rats compared to LS, LI and OS litter mates.
341
Three critical determinants of nephropathy--renal vasculopathy, inflammation, and fibrosis--were monitored by direct intravital multiphoton fluorescence microscopy in 342
343
the four groups of rats. The goal was to examine the modifying role of ischemia on 344
these key effectors of nephropathy. Progressive inflammation, fibrosis and microvascular dysfunction, as well as apoptotic cell death in the diabetic kidney were 345
346
exacerbated by ischemia.
Nephropathy in obese-diabetic rats is characterized by protracted inflammation 347
348
and subsequent fibrosis (11), the end-point consequence to metabolic abnormalities
349
and regional vascular hypoperfusion (12, 29). Hence, we investigated the potential
350
magnifying role of inflammation on the progression of nephropathy in post-ischemic
351
obese-diabetic rats. Although all models have limitations, chronic kidney disease is the
352
hallmark of human diabetic nephropathy and, as the Animal Models of Diabetic
Complications Consortium has pointed out, “the major deficiency in [prior] animal 353
354
models of diabetic nephropathy is the absence of kidney failure” (5, 6, 34). Although the
355
pathognomonic change in early diabetic kidney disease is glomerular basement
membrane thickening, inflammation and apoptosis markers in urine (58) and serum (41) 356
357
early in diabetes and can predict declines in renal function in longitudinal studies.
358
Tubulointerstitial inflammation is found in human diabetic nephropathy specimens (4).
359
We focused on ischemia-reperfusion renal injury because it drastically depresses
360
capillary renal blood flow in the post-ischemic period in obese-diabetic mice, and it
361
subsequently reduces renal blood flow (44). Renal hemodynamic and inflammatory
changes may interact and worsen renal injury. For example, angiotensin II, critical in 362
363
diabetic nephropathy and arterial hypertension, stimulates the expression of cytokines
364
and growth factors (38) and results in renal infiltration of inflammatory cells in
365
experimental CKD (49). Inflammation can result in synthesis of angiotensin II (57). Both
366
systolic and diastolic blood pressure were decreased in patients treated with
367
immunosuppression for rheumatoid arthritis or psoriasis, and systolic blood pressure
368
was correlated with urine levels of inflammatory mediators (19). Furthermore, acute
369
renal injury (AKI) has synergistic morbid effects on diabetic nephropathy, and
370
conversely, diabetic nephropathy enhances the risk for AKI (20). Accordingly, we
tested if the dynamic interaction of “acute on chronic” renal injury was fueled by renal 371
372
inflammation. We reasoned that the pro-inflammatory role of renal hypoxia has been
373
undeniably demonstrated in the ischemia-reperfusion model of renal injury (22, 27, 28).
374
Moreover, renal hypoperfusion also complicates diabetic nephropathy (36), likely
375
resulting in a pro-inflammatory state (16, 37). However, this earlier work does not fully
376
address the self-sustained chronic nature of the post-ischemic pro-inflammatory state
reported herein. In fact, in otherwise normal rats, an acute episode of ischemic injury 377
378
results in long-lasting vascular effects, including rarefaction of the peritubular capillary
379
network many weeks following the ischemic episode (3). Our results, consistent with
these data, clearly demonstrate that one early episode of ischemia-reperfusion has 380
381
powerful and long-lasting effects in rats with obesity-diabetes. Indeed, the post-
382
ischemic obese-diabetic rats demonstrated an enhanced and sustained renal pro-
383
inflammatory state, which most likely accelerated apoptosis, a critical turning point after
384
reperfusion injury (8, 25, 26), as well as the decay of renal function and structure.
385
Our novel model of “acute on chronic” renal failure reveals that accelerated
persistent renal inflammation is a critical morbid element of progressive renal failure 386
387
complicated by acute injury. We have termed this condition the post-ischemic
388
inflammatory syndrome of diabetic nephropathy. The damaging inflammatory process
389
goes on for months past the acute ischemic insult, and it seems to critically modify the
390
renal outcome. The remarkable features of the long-lasting post-ischemic inflammatory
391
syndrome have received little attention, although its existence was previously described
392
in uninephrectomized rats subjected to ischemia reperfusion consequential to renal
393
auto-transplantation (18). The auto-transplanted rat model also had a sustained
394
inflammatory response, and the main driving stimulus appeared to be ischemia and the
loss of kidney mass (18). In contrast, our rat model has the metabolic syndrome, and 395
396
this morbid entity was likely the driving self-sustaining force, although a significant role 397
of progressive loss of renal mass could not be discounted (13). In either case, our data show that acute kidney injury exacerbates the sustained renal proinflammatory state, 398
399
accelerates apoptosis, renal decay, and caused far more severe renal fibrosis and 400
failure. This critical discovery validates, at least in obese-diabetic rats, the view that 401
acute ischemia can result in chronic renal failure (2). In addition, from our findings we 402
conclude that anti-inflammatory renal rescue therapy, used successfully by us (11) and 403
others (52), can be employed to limit the long-term damage inflicted by ischemia 404
reperfusion injury in diabetes (23).
405
406
407
408
409
ACKNOWLEDGMENTS
410
This work was supported in part by Merit Review funds to JHD and a Clarian Health 411
Partners Values Fund Award and a research grant from DCI Paul Teschan Research 412
and Development Fund to KJK. Imaging was performed at the Indiana Center for 413
Biological Microscopy.
414
Reprint Requests: K.J. Kelly, MD, MSc
415
Indiana University-Nephrology, 950 West Walnut Street-RII 202
416
46202
Indianapolis,
IN
417
418