Proximal tubular injury and rapid formation of atubular glomeruli in mice with unilateral ureteral obstruction: a new look at an old model

Abstract

Unilateral ureteral obstruction (UUO), employed extensively as a model of progressive renal interstitial fibrosis, results in rapid parenchymal deterioration. Atubular glomeruli are formed in many renal disorders, but their identification has been limited by labor-intensive available techniques. The formation of atubular glomeruli was therefore investigated in adult male mice subjected to complete UUO under general anesthesia. In this species, the urinary pole of Bowman's capsule is normally lined by tall parietal epithelial cells similar to those of the proximal tubule, and both avidly bind Lotus tetragonolobus lectin. Following UUO, these cells became flattened, lost their affinity for Lotus lectin, and no longer generated superoxide (revealed by nitroblue tetrazolium infusion). Based on Lotus lectin staining, stereological measurements, and serial section analysis, over 80% of glomeruli underwent marked transformation after 14 days of UUO. The glomerulotubular junction became stenotic and atrophic due to cell death by apoptosis and autophagy, with concomitant remodeling of Bowman's capsule to form atubular glomeruli. In this degenerative process, transformed epithelial cells sealing the urinary pole expressed α-smooth muscle actin, vimentin, and nestin. Although atubular glomeruli remained perfused, renin immunostaining was markedly increased along afferent arterioles, and associated maculae densae disappeared. Numerous progressive kidney disorders, including diabetic nephropathy, are characterized by the formation of atubular glomeruli. The rapidity with which glomerulotubular junctions degenerate, coupled with Lotus lectin as a marker of glomerular integrity, points to new investigative uses for the model of murine UUO focusing on mechanisms of epithelial cell injury and remodeling in addition to fibrogenesis.

Fig. 1.

Mouse kidney structure compared in 0.25-μm “semithin” plastic sections (a, d, g), 4-μm paraffin sections with Lotus tetragonolobus immunostaining (b, e, h, i), and 10-μm sections of nitroblue tetrazolium (NBT)-perfused material (c, f, j). a: Sagittal section through glomerulus which joins with its proximal tubule (PT) at the glomerulotubular junction (GTJ). The PT is composed of tall epithelial cells, continuous with cells (arrows), similar in appearance, which form a substantial portion of Bowman's capsule. The remainder of the capsule is composed of flattened parietal epithelial cells (PECs). b: Similarly oriented glomerulus showing that Lotus lectin stains the tall epithelial cells of both PT and Bowman's capsule, but leaves the flat PECs unstained. c: In this nephron from an unobstructed (contralateral) kidney, NBT staining for the presence of superoxide produces a distinct outlining (arrowheads) concentrated at the basal surfaces of the tall epithelial cells of Bowman's capsule and the adjacent PT. d: Ureteral obstruction leads to rapid and progressive degeneration of nephrons, characterized by shift in the morphology of capsule cells from tall to flattened (arrows) and atrophy of the GTJ and contiguous PT. e: Epithelial cells of the capsule, GTJ, and initial PT segment lose their affinity for Lotus lectin. f: NBT reaction product disappears from the glomerulus and becomes localized to the luminal side of epithelial cells of the downstream PT. g-j: Atubular glomeruli, traced in serial consecutive sections to confirm the lack of attached PTs. In g, capsules now consist entirely of flattened PECs, but the internal glomerular components (podocytes, mesangium, and capillaries) exhibit normal morphology. Two tubule profiles (*) were followed and found to be disconnected, isolated segments that had lost their lumina and were composed of epithelial cells containing numerous autophagic bodies (also see Fig. 3, g and h). h and i: Loss of the tall cell configuration of PECs results in glomerular capsules devoid of Lotus staining. j: Although nearby fragments of PTs are heavily stained by NBT perfusion, the atubular glomerulus shows no evidence of superoxide production. Scale bar in j = 50 μm and applies to all panels.

Fig. 2.

Progressive loss of glomerulotubular integrity resulting from unilateral ureteral obstruction (UUO) as indicated by Lotus staining. a: 14-day obstructed kidney. b: 14-day contralateral kidney. Staining in Bowman's capsules or the connected PTs (arrowheads) is severely diminished in the obstructed kidney, in which the majority of glomeruli appear as lectin-negative profiles (*). Scale bar in b = 250 μm and applies to a and b. c: Fractional distribution of Lotus-positive glomeruli expressed as a percentage of total glomeruli counted in a single sagittal kidney section of obstructed and contralateral kidneys of each animal (n = 7 in each category). The 37% reduction in Lotus-positive glomerular staining from 7 to 14 days of UUO reflects the rapidity with which glomerulotubular integrity is lost in this model of renal injury (filled bars = obstructed kidney; open bars = contralateral kidney; means ± SE; *P < 0.05 vs. contralateral kidney; #P < 0.05 vs. 14-day UUO kidney).

Fig. 3.

TdT-mediated dUTP nick end labeling positivity identifies cell death in 1-day (a), 3-day (b and c), and 7-day (d, e) obstructed kidney. a: Apoptosis (arrows) is initiated in the flat PECs at the vascular pole of Bowman's capsule, but it shifts to tall PECs at the urinary pole by 3 days (b). c: Details in a semithin plastic section of an apoptotic tall PEC (arrow). d and e: Apoptotic figures continue to appear in both PECs and PT epithelial cells at 7 days. Autophagy is also present in PTs (f, g, h). f: Epithelial cells of a collapsed PT (between arrows) stain strongly for the autophagy-related protein LC3B. Inset: inclusions in a PT stain positively for beclin, another protein involved in autophagy (arrowheads). g: Transmission electron micrograph of a collapsed PT in cross section, demonstrating the lack of a lumen and the presence of autolysosomes (arrows). Nuclei (N) in these cells have a normal appearance, and the cytoplasm is unremarkable except for the numerous large inclusions and a paucity of mitochondria. h: Semithin plastic section of a glomerulus in which the GTJ and initial segment of the PT have transformed into a solid cord of dark cells that joins (at *) with a tubule segment that, although collapsed, still consists of large epithelial cells containing autophagic inclusions (arrows). Scale bar in e = 50 μm and applies to a-e. Scale bars in f = 50 μm, in g = 10 μm, and in h = 50 μm.

Fig. 4.

Immunostaining for vimentin (a, b), nestin (c), and α-smooth muscle actin (SMA; d) in nephrons from kidneys from animals with 14 days of UUO. a: In the contralateral (unobstructed) kidney, vimentin is present primarily in podocytes (POD) and blood vessels (BV). b: In the obstructed kidney, vimentin is expressed not only in POD and the afferent arteriole (AA) but also in the atrophied GTJ and detaching PT. c: Glomeruli in the obstructed kidney show strong nestin immunostaining both in POD and epithelial cells of the capsule (arrowheads) and the attached atrophic PT (*) d: Serial 2-μm section of the nephron in c, showing α-SMA positivity in capsule cells, particularly in a “shelf” of cells extending through the GTJ (between arrows). Scale bar in b = 50 μm and applies to a and b; scale bar in d = 50 μm and applies to c and d.

Fig. 5.

a–d: Renin staining for juxtaglomerular cells. a and b: After 7 days of UUO, renin expression is similar between the obstructed and contralateral kidneys. By 14 days (c, d), renin has become expressed along the AA of the obstructed kidney, but it is absent or only weakly expressed in the contralateral kidney. e and f: Neuronal nitric oxide synthase (nNOS) staining for macula densa (MD). Each of the 2 glomeruli in e was traced in serial sections to its connection both with an atrophic PT (example shown by PT) and a collapsed MD that retained its specific staining for nNOS. Atubular glomeruli lacked the MD, however. Typical morphology of the MD is seen in the contralateral kidney (f). g and h: Modified Masson trichrome staining to emphasize erythrocytes. Glomeruli in both the obstructed (g) and contralateral kidneys (h) display open capillaries and few erythrocytes (arrowheads) following fixation by vascular perfusion. Scale bar in h = 50 μm and applies to all panels.