Seymour Rosen

Nitric oxide and prostanoids protect the renal outer medulla from radiocontrast toxicity in the rat.

Human radiocontrast nephrotoxicity is predicted by the presence of multiple risk factors, often associated with compromised renal circulation. To produce a simple model of radiocontrast nephropathy, rats were pretreated with indomethacin and N omega-nitro-L-arginine methyl ester (L-NAME, to inhibit nitric oxide synthesis) before the administration of iothalamate. Acute renal failure consistently developed, with a decline in creatinine clearance from 1.05 +/- 0.10 to 0.27 +/- 0.05 ml/min (P < 0.001) associated with selective necrosis of 49 +/- 9% of medullary thick ascending limbs. Hemodynamic studies using laser-Doppler probes revealed that when injected alone, iothalamate increased outer medullary blood flow to 196 +/- 25% of baseline (P < 0.001). Pretreatment by L-NAME or indomethacin both reduced basal medullary blood flow and transformed the medullary vasodilator response to radiocontrast into vasoconstriction, with a prolonged reduction of medullary blood flow to less then half of baseline. Combined administration of indomethacin, L-NAME, and iothalamate lowered medullary blood flow to 12 +/- 4% of baseline. We conclude that prostanoids and nitric oxide have an important protective role in the renal response to radiocontrast material. Reduced synthesis of these vasoactive substances in renal/vascular diseases may predispose patients to radiocontrast nephropathy.

Role of nitric oxide in renal medullary oxygenation. Studies in isolated and intact rat kidneys.

We investigated the role of the endothelial-derived relaxing factor nitric oxide (NO) in the homeostasis of O2 supply to the renal medulla, a region normally operating on the verge of hypoxia. Sensitive Clark-type O2 microelectrodes were inserted into renal cortex and medulla of anesthetized rats. The inhibitor of NO formation, L-NG-monomethylarginine (LNMMA), while increasing blood pressure and reducing renal blood flow, decreased medullary pO2 from 23 +/- 3 mmHg to 12 +/- 3 (P less than 0.001), with no change in the cortex. These responses were promptly reversed by L-arginine, which bypasses the LNMMA blockade. In isolated rat kidneys, LNMMA reduced perfusion flow without altering glomerular filtration rate, and augmented deep medullary hypoxic injury to thick ascending limbs from 68 to 90% of the tubules (P less than 0.02). These changes were prevented by L-arginine. Nitroprusside had a protective effect upon thick limb injury. Finally, in a previously reported model of radiocontrast nephropathy (1988. J. Clin. Invest. 82:401), LNMMA increased the severity of renal failure (final plasma creatinine from 2.3 +/- 2 mg% to 3.4 +/- 3, P less than 0.005) and the proportion of damaged thick limbs (from 24 +/- 6% to 53 +/- 9, P less than 0.01). Nitrovasodilatation may participate in the balance of renal medullary oxygenation and play an important role in the prevention of medullary hypoxic injury.

Acute renal failure with selective medullary injury in the rat.

Since human acute renal failure (ARF) is frequently the result of multiple rather than single insults, we used a combination of treatments to induce ARF in rats. Uninephrectomized, salt-depleted rats injected with indomethacin developed ARF after administration of radiocontrast. After 24 h, the plasma creatine rose from 103 +/- 3 to 211 +/- 22 mumol/liter (mean +/- SE) and the creatinine clearance dropped from 0.7 +/- 0.1 to 0.2 +/- 0.04 ml/min (P less than 0.001). Severe injury was confined to the outer medulla and comprised necrosis of medullary thick ascending limbs (mTALs), tubular collapse, and casts. Other nephron segments were free of damage except for the proximal convoluted tubules which showed vacuole formation originating from lateral limiting membranes that resembled changes reported in human contrast nephropathy. Cell damage to mTALs included mitochondrial swelling, nuclear pyknosis, and cytoplasmic disruption with superimposed calcification; these changes were most severe in the deepest areas of the outer medulla, away from vasa recta in zones remote from oxygen supply. The fraction of mTALs with severe damage was 30 +/- 7% (range 2-68) and the extent of injury was correlated with a rise in plasma creatinine (r = 0.8, P less than 0.001). Thus, the nature of mTAL injury was similar to the selective lesions observed in isolated kidneys perfused with cell-free medium and was shown to derive from an imbalance between high oxygen demand by actively transporting mTALs and the meager oxygen supply to the renal medulla. Combined multiple renal insults in the rat produce ARF that resembles the clinical syndrome of contrast nephropathy and is characterized by selective mTAL injury conditioned by medullary hypoxia.

Selective vulnerability of the medullary thick ascending limb to anoxia in the isolated perfused rat kidney.

A specific anatomical lesion sharply localized to the cells of the medullary thick ascending limbs (mTAL) and characterized by mitochondrial swelling progressing to nuclear pyknosis and cell death is elicited reproducibly in isolated rat kidneys perfused for 15 or 90 min with cell-free albumin-Ringers medium gassed with 5% CO2, 95% O2 (O2 content, 1.5 vol/100 ml). The lesion, involving about half of mTALs, appears first in mTALs removed from vascular bundles and near the inner medulla, areas most likely to be anoxic. Hypoxic perfusion (O2 content 0.12 vol/100 ml) exaggerates the lesion, wiping out gradations of damage and extending it to all mTALs. O2-enriched perfusions using rat erythrocytes (O2 content 7.1 vol/100 ml) completely eliminates the lesion (unless gassed with carbon monoxide). Similarly, supplementation of the perfusion medium with a purified hemoglobin (O2 content 5.8 vol/100 ml) prevents mTAL injury. Perfusion with a fluorinated hydrocarbon blood substitute, Oxypherol (O2 content 4.3 vol/100 ml) also attenuates the lesion. These findings suggest that the mTAL is exquisitely susceptible to anoxic damage because of low O2 supply imposed by the medullary vascular system and the high rate of metabolism mandated by active reabsorption of sodium chloride. The vulnerability of the mTAL to anoxic injury could play a key role in the pathogenesis of ischemic renal injury.

Tubulo-Interstitial Nephritis Associated with Polyomavirus (BK Type) Infection

We studied viral injury to the kidney in a six-year-old boy with hyperimmunoglobulin M immunodeficiency who presented with irreversible acute renal failure and eventually died after five months of dialysis. Renal biopsy at the time of his presentation revealed a predominantly tubulo-interstitial process with numerous viral inclusions that were identified as polyomavirus. Urine cultures showed a massive viruria with BK-type, polyomavirus. The kidney disease was end stage, with persistence of BK virus identified by morphologic techniques and by culture. DNA hybridization analysis showed virus in low concentration in the lymph nodes, spleen, and lungs. The marked viruria, the high concentration of BK virus, and the extensive distribution of viral antigen throughout the kidney all suggest that infection with BK virus was the basis of the severe renal parenchymal injury.

Carbonic anhydrase activity in Rana pipiens skin: biochemical and histochemical analysis.

SummaryCarbonic anhydrase activity was demonstrated biochemically and localized histochemically in epidermis of Rana pipiens. The activity observed throughout the lower epidermis was diminished or abolished by periods of fixation which did not affect the activity in upper epidermis. In the latter region, the enzyme was demonstrated only in certain cells with a flask shape known to have numerous mitochondria and a system of convoluted apical membranes. Such findings raise the possibility that these distinctive cells play a role in transport functions of the isolated frog skin suppressed by carbonic anhydrase inhibitors.

Vascular permeability factor (vascular endothelial growth factor) is strongly expressed in the normal male genital tract and is present in substantial quantities in semen

PURPOSE Vascular permeability factor (VPF) is a potent inducer of microvascular hyperpermeability and stimulates endothelial cell growth and angiogenesis. This study examines expression of VPF in the male genital tract. MATERIALS AND METHODS Vascular permeability factor in seminal plasma was quantified by immunoassay. Vascular permeability factor mRNA and protein expression in tissue were studied by situ hybridization and immunohistochemistry. RESULTS All seminal plasmas studied contained high levels of VPF. Prostatic and seminal vesicle epithelium labeled strongly for VPF mRNA and protein. CONCLUSIONS The strong expression of VPF in prostate and seminal vesicle and the high concentration of VPF in semen suggest an important role for this cytokine in male fertility.

Acceptance of allogeneic fibroblasts in skin equivalent transplants

Living skin equivalents (SE) were prepared by combining cultured fibroblasts with a collagen matrix and overlaying this lattice with keratinocytes. SEs prepared using allogeneic female rat fibroblasts or xenogeneic rabbit or human fibroblasts and keratinocytes isogeneic to the graft recipient were transplanted to recipient male rats. Biopsies of some of these SE grafts were examined histologically at intervals ranging from 5 days to 2 months. Biopsies of other grafts were done, and fibroblasts grown from them were karyotyped to determine the percentage of donor fibroblasts remaining in the graft. SEs containing xenogeneic fibroblasts were rejected. Allografted fibroblasts in SEs were accepted by recipient rats after a transient mononuclear cell response. A second SE allograft from the same donor strain did not provoke rejection either in the original allograft or in the challenge allograft. A secondary graft of allogeneic skin did not provoke rejection in the original SE graft, although the skin graft was rejected. Grafting the recipient first with allogeneic skin and then with the SE allograft led to rejection of the skin but not of the SE graft, ruling out the possibility that suppressor T cells were responsible for SE allograft acceptance. Allografted fibroblasts in SEs do not provoke a rejection response, even in presensitized animals, do not render the recipient tolerant to allogeneic skin, and do not act as targets when active rejection is taking place. We propose that cells bearing class I antigens may be acceptable graft constitutents if incorporated in a tissue equivalent excluding cells with class II antigens.

Localization of carbonic anhydrase activity in the vertebrate retina

Abstract Carbonic anhydrase activity was localized histochemically in the retinas of five species: turtle ( Pseudemys scripta ), toad ( Bufo marinus ), New Zealand white rabbit, Sprague-Dawley albino rat, and human. By light microscopy, activity was present in Muller cells of all species and in pigment epithelium of the turtle, rat, and rabbit. In the human, the only mammal studied with numerous cones, the cones, but not the rods, displayed carbonic anhydrase activity. By electron microscopy, the reaction product was a finely granular precipitate, localized to the Muller cell cytoplasm and the cytoplasm associated with the pigment epithelial basilar infoldings and apical villous projections. Depending on the species studied, the histochemical reaction was completely inhibited by acetazolamide at concentrations of 0·5−5 × 10 −6 m in the incubation medium. The mean carbonic anhydrase activities of the rabbit and turtle retinas were biochemically quantitated to be 991 units and 504 units/g dry weight, respectively, and 8·9−10·3% of the original activity was retained after fixing for 2 hr in 3%, 0·17 m cacodylate-buffered glutaraldehyde.

Localization of carbonic anhydrase activity in the vertebrate nephron.

SynopsisThe localization of carbonic anhydrase activity in the vertebrate nephron has been examined with particular reference to the proximal tubule and collecting duct. In all species studied, activity was present in the proximal tubular epithelium. In the pigeon and turtle, distinctive and similar patterns of staining were observed in the glomerulus and first portion of the proximal tubule. In the rat and rhesus monkey, the entire proximal tubule exhibited activity; in these species it has been shown previously with micropuncture techniques that there is a high absorptive capacity of this nephron segment for bicarbonate. In contrast, large portions of the dog proximal tubule were inactive; similar studies in this animal have shown tubular concentrations of bicarbonate only slightly lower than plasma levels. In the rat and dog, the entire length of the collecting duct was diffusely and intensely active; in contrast, pigeon collecting duct showed no activity. An alternating pattern of inactive and intensely active cells was observed in the collecting ducts of the toad, turtle, rabbit and monkey. A similar pattern has been described in the turtle and toad bladder, tissues utilized forin vitro studies of ion transport and H+ secretion.