Angiotensin converting enzyme inhibition blocks interstitial hyaluronan dissipation in the neonatal rat kidney via hyaluronan synthase 2 and hyaluronidase 1.

A functional renin-angiotensin system (RAS) is required for normal kidney development. Neonatal inhibition of the RAS in rats results in long-term pathological renal phenotype and causes hyaluronan (HA), which is involved in morphogenesis and inflammation, to accumulate. To elucidate the mechanisms, intrarenal HA content was followed during neonatal completion of nephrogenesis with or without angiotensin converting enzyme inhibition (ACEI) together with mRNA expression of hyaluronan synthases (HAS), hyaluronidases (Hyal), urinary hyaluronidase activity and cortical lymphatic vessels, which facilitate the drainage of HA from the tissue. In 6-8days old control rats cortical HA content was high and reduced by 93% on days 10-21, reaching adult low levels. Medullary HA content was high on days 6-8 and then reduced by 85% to 12-fold above cortical levels at day 21. In neonatally ACEI-treated rats the reduction in HA was abolished. Temporal expression of HAS2 corresponded with the reduction in HA content in the normal kidney. In ACEI-treated animals cortical HAS2 remained twice the expression of controls. Medullary Hyal1 increased in controls but decreased in ACEI-treated animals. Urine hyaluronidase activity decreased with time in control animals while in ACEI-treated animals it was initially 50% lower and did not change over time. Cells expressing the lymphatic endothelial mucoprotein podoplanin in ACEI-treated animals were increased 18-fold compared to controls suggesting compensation. In conclusion, the high renal HA content is rapidly reduced due to reduced HAS2 and increased Hyal1 mRNA expressions. Normal angiotensin II function is crucial for inducing these changes. Due to the extreme water-attracting and pro-inflammatory properties of HA, accumulation in the neonatally ACEI-treated kidneys may partly explain the pathological renal phenotype of the adult kidney, which include reduced urinary concentration ability and tubulointerstitial inflammation.

Renal hyaluronan content during experimental uncontrolled diabetes in rats.

With diabetes mellitus, the ability of the kidneys to maintain fluid balance is affected. Hyperglycaemia increases production of hyaluronan in cultured kidney cells implying that diabetes promotes induction of hyaluronan in the kidney. The aim of the present study was to determine if the interstitial matrix component hyaluronan is differently distributed within the kidney in diabetic rats compared to non-diabetic rats. Furthermore, to test if diabetic rats are able to respond with diuresis upon a hypotonic fluid load. The normal heterogeneous intrarenal distribution of hyaluronan was confirmed in non-diabetic control rats, with 60-fold more in the papilla than in the cortex. In diabetic animals, the cortical hyaluronan was unaffected but the papillary hyaluronan content was 3-fold higher than in non-diabetic rats. This increase correlated with a more than three-fold induction of the papillary hyaluronan-synthase 2 mRNA expression. In non-diabetic animals, 2 h water loading increased papillary hyaluronan (+93%) and diuresis (17-fold). In diabetic animals, baseline diuresis was 8-fold higher than in non-diabetic animals, which correlated with hyperglycaemia, glucosuria and proteinuria. Water loading in diabetic animals did not further increase papillary hyaluronan or diuresis: the urine flow rate decreased. To conclude, papillary hyaluronan is elevated in diabetic rats, which coincides with induction of hyaluronan-synthase 2 mRNA, hyperglycaemia, glucosuria, proteinuria and overt diuresis. The inability to respond to a water load with further diuresis may be related to the already elevated papillary hyaluronan and the inability to change hyaluronan during water loading.

Hormonal regulation of renomedullary hyaluronan.

AIM: Hyaluronan (HA) is involved in renomedullary water handling through its water-binding capacity. This study addressed the effect of hormones involved in regulating fluid-electrolyte homeostasis on renomedullary HA content in vivo and in vitro. METHODS: The kidneys from rats treated with L-NAME, indomethacin, vasopressin (AVP) or methylprednisolone (MP) during euvolaemia or water loading were analysed for HA by RIA, ELISA and histochemical staining. HA was measured in renomedullary interstitial cells treated with AVP, angiotensin II (Ang II) or a combination of AVP and Ang II. RESULTS: Baseline renal cortical and medullary HA content was unaffected by 2 h of intravenous treatment with L-NAME (NOS inhibitor) or indomethacin (cyclo-oxygenase inhibitor), whereas AVP reduced medullary HA by 33%. During 2 h of acute water loading, diuresis was accompanied by an increase in renomedullary HA (+45%), but cortical HA was unaffected. In both L-NAME- and indomethacin-treated animals, the water loading-induced increase in renomedullary HA was absent, indicating involvement of NO and prostaglandins. After 7 days of MP treatment, medullary HA was reduced by 40%, but the water loading-induced elevation in HA remained. In cultured renomedullary interstitial cells, AVP reduced the HA content in the supernatant by 63%, and simultaneous treatment with Ang II reduced the HA content even further (95%). CONCLUSION: AVP reduces HA content, and NO and prostaglandins are needed for the increase in HA during water loading.

Antibody responses to xenogenic antigens--a study in the mouse-to-rat system.

Antibodies play a crucial role in the rejection of an organ that has been transplanted between different animal species, i.e. xenotransplantation. In previous work, we have induced a state of humoral tolerance where mouse-to-rat heart grafts continued to beat under ciclosporine A monotherapy. Initially, a combined treatment with ciclosporine A and 15-deoxyspergualin was given. This state of tolerance could not be reproduced when the vascularised heart graft was replaced with a free tissue graft or xenogeneic blood transfusions. To gain further insight into the humoral response against mouse antigens, we studied the antibody production in naive rats and rats challenged with heart transplants, heart cells, mononuclear cells (MNC) and erythrocytes from mice. Rats not challenged with any mouse cells or organs had a moderate amount of antibodies targeted against mouse MNC as well as rosette-forming cells in the spleen targeted against mouse erythrocytes. A challenge with either mouse MNC or erythrocytes lead to immunisation with antibodies of both IgM and IgG subtype directed against both MNC and erythrocytes. Antibody titres against mouse erythrocytes in animals challenged with MNC were not detectable until day 7, whereas antibody titres against mouse MNC in animals challenged with erythrocytes were detected on day 1. Immunisation with mouse erythrocytes raised the titre of rosette-forming cells in the spleen compared with naive rats (P < 0.05). Our data indicate that different xenogeneic antigens in the mouse-to-rat system are shared between heart cells, MNC and erythrocytes; however, the immunisation patterns differ regarding the time when antibodies are first detected.

Monotherapy with the vitamin D analogue MC1288 does not result in prolonged kidney allograft survival in rhesus monkeys.

The active form of vitamin D3, 1,25(OH)2D3, has pronounced immunoregulatory properties and is a potential treatment of immune-based disorders. However, the central role of this hormone in calcium and bone metabolism complicates its long-term use as an immunomodulator. Some newly developed vitamin D3-derived analogues, such as MC1288, have an improved immunoregulatory potential and prolong allograft survival in rodent models. Such compounds might be a valuable component of immunosuppressive treatment regimen in transplantation and autoimmunity. The rhesus monkey provides a useful model for the preclinical validation of new therapeutic strategies for transplantation. The present study shows that MC1288 inhibits both proliferation and interferon-gamma production by rhesus peripheral blood mononuclear cells in a mixed lymphocyte reaction. We have tested the maximum tolerated dose of MC1288 in a rhesus monkey model of kidney transplantation. The observed effects on serum calcium and parathyroid hormone confirm the in vivo activity of MC1288. However, as a monotherapy, MC1288 did not cause prolongation of the kidney allograft survival in rhesus monkeys.

Macrophage uptake of ultra-small iron oxide particles for magnetic resonance imaging in experimental acute cardiac transplant rejection.

PURPOSE: To discriminate between acutely rejecting and non-rejecting transplanted hearts using a blood pool contrast agent and T2* magnetic resonance imaging (MRI) in a clinical 1.5T scanner. MATERIAL AND METHODS: Allogeneic and syngeneic heterotopic heart transplantations were performed in rats. One allogeneic and one syngeneic group each received either the ultra-small iron oxide particle (USPIO), at two different doses, or no contrast agent at all. MRI was performed on postoperative day 6. Immediately after the MR scanning, contrast agent was injected and a further MRI was done 24 h later. Change in T2* was calculated. RESULTS: No significant difference in change in T2* could be seen between rejecting and non-rejecting grafts in either of the doses, or in the control groups. There was a difference between the allogeneic group that received the higher contrast agent dose and the allogeneic group that did not receive any contrast agent at all. CONCLUSION: In our rat model, measurements of T2* after myocardial macrophage uptake of AMI-227 in a clinical 1.5T scanner were not useful for the diagnosis of acute rejection.

Effects of commonly used immunosuppressants on graft-derived fibroblasts.

In acute rejection of transplanted organs intragraft fibroblasts increase their production of hyaluronan. Hyaluronan has strong water binding capacity and an increased tissue content of hyaluronan thus contributes to the development of interstitial oedema. The present study examined the effects of commonly used immunosuppressants (prednisolone, cyclosporin, tacrolimus, mycophenolic acid and sirolimus) on fibroblast proliferation, hyaluronan production and cell surface receptor expression. Fibroblasts isolated from rejecting tissue and from normal, non-transplanted tissue were studied in parallel. All substances investigated, except tacrolimus, were found to affect fibroblasts in one way or another. The most striking effect was the almost total inhibition of fibroblast proliferation in the presence of mycophenolic acid. Cyclosporin reduced the proliferation by about 50% and prednisolone had an inhibiting effect on hyaluronan production (50% reduction). These effects were observed on fibroblasts isolated from rat cardiac allografts undergoing rejection as well as on fibroblasts obtained from normal heart tissue. In contrast, sirolimus was found to stimulate the proliferation of fibroblasts from rejecting tissue (100% increase), but not that of normal fibroblasts. The majority of the fibroblasts expressed the hyaluronan receptor CD44, with a more intense expression in cultures of fibroblasts derived at rejection. None of the immunosuppressants affected the staining pattern (number of positive cells or intensity). The inhibitory effects of prednisolone, cyclosporin and mycophenolic acid on fibroblasts may contribute to the overall beneficial effects of these drugs when used for prevention or treatment of rejection.

Pretransplant xenogeneic blood transfusions reduce the humoral response in a mouse-to-rat heart transplantation model.

A mouse heart transplanted to a rat is rejected promptly 3 days after transplantation, independent of whether cyclosporin A (CyA) is used as an immunosuppressant or not. Adding a short course of deoxyspergualin (DSG) initially, in addition to continuous CyA treatment, results in long-term graft survival and permits retransplantation during CyA monotherapy. In this paper, we have explored the possibility of substituting the initial heart transplant with blood transfusions. Lymphocyte-enriched blood transfusions combined with CyA and an initial course of DSG proved to lower or eliminate the haemagglutinating antibody titre normally seen in acute vascular xenorejection. The therapy, however, did not prolong the mean survival of the cardiac xenograft, but the same treatment protocol could result in either hyperacute rejection or prolonged survival of up to 11 days. In conclusion, this and earlier studies propose that a humoral unresponsiveness can be induced if the recipient vascular circulation is exposed to a xenoantigen in a mouse-to-rat combination.

Synthesis and biological evaluation of technetium(III) mixed-ligand complexes with high affinity for the cerebral 5-HT(1A) receptor and the alpha1-adrenergic receptor.

Tc(III) and Re(III) complexes [M(NS(3))(CNR)] (M = Re, 99mTc, NS(3) = 2,2',2"-nitrilotris(ethanethiol), CNR = functionalized isocyanide bearing a derivative of WAY 100635) have been synthesized and characterized. Re was used as Tc surrogate for chemical characterization and in vitro receptor-binding studies. For two representatives subnanomolar affinities for the 5-HT(1A) as well as for the alpha1-adrenergic receptor were reached. Biodistribution studies in rats of the 99mTc complexes showed brain uptakes between 0.3 and 0.5% ID/organ (5 min p.i.). In vitro autoradiography of one 99mTc representative in sections of post mortem human brain indicate its accumulation in 5-HT(1A) receptor-rich brain regions. However, addition of the specific 5-HT(1A) receptor agonist 8-OH-DPAT as well as the alpha1-adrenoceptor antagonist prazosin could not substantially block this tracer accumulation. A preliminary SPET study in a monkey showed negligible brain uptake.

Renal cortical accumulation of hyaluronan in adult rats exposed neonatally to angiotensin-converting enzyme inhibition.

Neonatal inhibition of the renin-angiotensin system [angiotensin-converting enzyme (ACE) inhibition] in the rat results in long-term abnormal renal morphology and function, including interstitial inflammation and fibrosis. Hyaluronan (hyaluronic acid, HA) has pathological implications in inflammatory diseases and renal ischaemia-reperfusion injury. The present study aimed at determining if renal cortical HA in the adult rat is correlated to the abnormal morphology and function in rats treated neonatally with the ACE inhibitor enalapril. In adult control rats (23 weeks old), the cortical HA content was very low [about 5 microg g(-1) dry weight (d.w.)] and about 1% of the papillary HA content. In rats treated neonatally with enalapril (days 3-13), the cortical HA level was 15 times that in control rats already at 21 days after birth, and it persisted at this level during adulthood (at 23 weeks). At 13 weeks the enalapril-treated animals showed markedly reduced ability (-53%) to concentrate urine during 24-h thirst provocation. At 21 days as well as at 23 weeks the enalapril-treated kidneys displayed morphological changes, such as papillary atrophy, dilation of the tubules and cellular infiltration of the cortical tissue. Histochemical staining confirmed the HA quantification assay and revealed a patchy staining for HA located in the same regions as the infiltrating cells. In conclusion, neonatal treatment with the ACE inhibitor enalapril results in renal morphological and functional abnormalities during adulthood. Cortical HA levels are already seriously elevated at day 21 and coexist with infiltrating cells. Besides the known effects of angiotensin II in development, the accumulation of HA in these kidneys may be involved in the genesis of at least the cortical abnormalities in enalapril-treated animals because of the proinflammatory effects and water-binding properties of HA.

Bacterial translocation from defunctionalized rat small bowel.

Bacterial translocation from the intestine may cause severe infectious complications in a number of clinical situations, including the short bowel syndrome and after small bowel transplantation. The aim of the present study was to develop a simplified model for the study of bacterial translocation from a defunctionalized intestine. An ileal segment from untreated or cyclosporine-treated rats was exteriorized as a Thiry-Vella loop. After 1, 3. or 7 days, bacterial translocation and distribution of immunocompetent cells were assessed. The data obtained were compared with data from animals subjected to intestinal transplantation. Translocation to the mesenteric lymph nodes was detected in 60% of the Thiry-Vella loop animals on day 1. in 100% on day 3, and in 83% on day 7: concomitantly, the number of macrophages and T-cells in the mesenteric lymph nodes increased from day I until day 7. The degree of bacterial translocation on days 3 and 7 in animals with a Thiry-Vella loop was comparable with that observed 7 days after intestinal transplantation. Furthermore, treatment with cyclosporine A enhanced the number of translocating bacteria. In the model presented here bacterial translocation occurs from the small bowel to the mesenteric lymph nodes. The model offers possibilities to study the mechanisms and immunological phenomena associated with microbial translocation.

Influence of laser irradiation on endogenous hyaluronan in rabbit iris and aqueous humor.

PURPOSE: To monitor changes of endogenous hyaluronan in the iris tissue and aqueous humor after an isolated trauma to the iris by argon laser irradiation of the anterior surface of the iris. METHODS: Iris and aqueous hyaluronan concentrations in rabbit were measured with a radiometric assay at different time points after laser irradiation. RESULTS: Total hyaluronan content in iris tissue increased 3-fold to a peak concentration of 71-72 microg/g at 1 and 2 days after laser treatment. Aqueous hyaluronan increased to a maximum of about 1.6 microg/ml at 2 h and 12 h after laser irradiation of the iris. CONCLUSIONS: The iris tissue responds with increased hyluronan synthesis to an isolated iris argon laser irradiation and it seems to be the most important source of aqueous hyaluronan.

Ventricular proliferation zones in the brain of an adult teleost fish and their relation to neuromeres and migration (secondary matrix) zones.

Zones containing actively dividing cells (proliferation zones: PZs), in the brain of adult three-spined sticklebacks, were identified by autoradiographic detection of (3)H-thymidine and immunocytochemical detection of the thymidine analogue 5'-bromodeoxyuridine (BrdU), singly or in combination, and by immunocytochemical detection of proliferating cell nuclear antigen (PCNA) by monoclonal antibodies. The PZs are associated with boundaries between adult brain regions, as well as with defined morphofunctional subdivisions. PZs are located at the border between the telencephalon and diencephalon, and at the border between the mesencephalon and the rhombencephalon. In the midbrain, the PZ follows the dorsomedial, caudal, and ventrolateral aspects of each tectal hemisphere, extending over the caudal aspect of the torus semicircularis to the nucleus lateralis valvulae. In the hindbrain, the major PZ apparently represents the persisting embryonic secondary matrix layer of the developing cerebellum. In the forebrain, the PZs are associated with the ventricular zones of the olfactory bulbs and ventral telencephalic area ("subpallium"), dorsal telencephalic area ("pallium"), preoptic region, ventral thalamus, dorsal thalamus, epithalamus, pretectum, posterior tuberculum, and the hypothalamus. The diencephalic PZs are parcellated according to a neuromeric organisation (a synencephalic, a posterior, and an anterior parencephalic neuromere: p1, p2, and p3). The PZs of the secondary prosencephalon (telencephalon and hypothalamus) thus would belong to neuromeres p4-6, but do not form an immediately recognised serial pattern. The prosencephalic PZs correlate well with parts of embryonic migration areas as defined by Bergquist and Källén ([1954] J. Comp. Neurol. 100:627-659), morphogenetic fields from which postmitotic neurones migrate to their final destination.

Renomedullary interstitial cells in culture; the osmolality and oxygen tension influence the extracellular amounts of hyaluronan and cellular expression of CD44.

Our previous studies have suggested a role for renomedullary interstitial cells (RMICs) and renal medullary hyaluronan (HA) in water homeostasis. In the present study, cultured rat RMICs were used to examine the relationship of osmolality and oxygen tension on the extracellular amount of HA in the culture and to the cellular immunoreactivity to CD44, a HA binding protein. Under isotonic (330 mOsm(.)kg(-1) H(2)O), normoxic (20% O(2)) conditions, supernatant from sub-confluent RMICs contained 120+/-37 pg 10(4) cells(-1) 24 h(-1) of HA. Under hyperosmotic conditions (630 mOsm kg(-1) H(2)O), HA in the supernatant was decreased by 42% and under hypoosmotic conditions (230 mOsm kg(-1) H(2)O) it was doubled. Under hypoxic, iso-osmolar conditions (5% and 1% O(2), 330 mOsm kg(-1) H(2)O) this HA content was decreased by 56 and 48%, respectively, compared with normoxic, iso-osmolal conditions. Expression of CD44 on sub-confluent cells increased with increasing osmolality, as shown by immunostaining and flow cytometric analysis. The increases in CD44 from 330 to 630, 930 and 1230 mOsm kg(-1) H(2)O amounted to 5, 142 and 212%, respectively. Low oxygen tension (5% O(2)) decreased the intensity of CD44 immunofluorescence by 31%. Cell viability was similar at all conditions studied. In summary, these data indicate that cultured RMICs produce HA and are immunoreactive to CD44. In the supernatant of RMICs, the HA content decreases under hyperosmotic, hypoxic conditions. Conversely, CD44 immunoreactivity increases under hyperosmotic conditions. These results may explain our previous in vivo findings of a decreased renal papillary HA content during anti-diuresis and an increased content during water diuresis. The results support the concept that RMICs play an important role in renal water handling.

Characterization of Forssman and other antigen/antibody systems in vascularized mouse heart to rat xenotransplantation.

In the present study, the nature of hyperacute xenograft rejection was closely studied in a vascularized mouse-to-rat transplantation model. Antibodies against mouse heart, erythrocytes and lymphocytes and against the Forssman antigen were raised in the rat. Upon heterotopic heart transplantation the respective antisera were intravenously (i.v.) injected. Passive transfer of antiheart, antierythrocyte or antilymphocyte serum resulted in hyperacute rejection of the transplanted mouse heart. Subfractionation of the antiheart serum showed that the capacity to induce hyperacute rejection was carried by the immunoglobulin (Ig)G fraction. When antierythrocyte serum adsorbed with mouse erythrocytes was administered the cardiac grafts remained beating. To the contrary, antilymphocyte serum adsorbed with erythrocytes still had the capacity to induce hyperacute rejection. None of the rats that had previously been challenged with the Forssman antigen rejected their grafts hyperacutely. Subsequent investigations by electron microscopy revealed that the Forssman antigen is expressed on dendritic cells (DC) adjacent to the vessels, but not on the vascular endothelium, thus explaining the inability of the anti-Forssman serum to induce hyperacute rejection. Taken together, we have demonstrated the existence of several xenoantigens that can be targets for antibody-mediated rejection, suggesting that more than one relevant xenoantigen exists also in more distantly related combinations, such as the pig-to-human combination.

Concordant xenotransplantation--non-vascularized pancreatic islets are more difficult to regraft than the vascularized heart.

We have previously demonstrated that it is possible to perform retransplantation of a xenogeneic heart (mouse-to-rat) using cyclosporine A as monotherapy, provided that the first heart is transplanted under a short course of deoxyspergualin (DSG). If DSG is omitted, the first heart is rejected within four days and the second heart succumbs to hyperacute rejection within minutes. A mouse heart as first graft does not protect a consecutive pancreatic islet graft, although the heart continues to function after rejection of the cellular graft. One explanation for this discrepancy may be the fact that cellular grafts, as pancreatic islets, lack an endothelial lining. We have, therefore, further investigated possible differences between vascularized and non-vascularized xenografts regarding their capacity to induce unresponsiveness. The use of pancreatic islets as primary graft neither accelerated nor decelerated the speed of rejection of the vascularized heart used as secondary graft. Furthermore, hemagglutinating and cytotoxic antibody titres responded in the same manner as in naive rats transplanted with a mouse heart. Retransplantation with pancreatic islets also resulted in complete rejection of both the primary and secondary grafts. Thus, the lack of unresponsiveness cannot simply be explained by differences, between the pancreatic and cardiac tissues, in antigen expression. In addition, intraperitoneal transplantation of mouse heart cells as primary graft resulted in rejection of a secondary cardiac graft after three days. However, it cannot be totally excluded that the time of antigen exposure had an impact on these results. In conclusion, our previous and present studies suggest that the presence of an intact vascular bed, both in the first and second graft, is necessary to create a state of unresponsiveness. Because the pancreatic islets lack an endothelial lining, they do not benefit from an unresponsiveness of the immune system. Neither are they able to induce such an unresponsiveness.

Role of hyaluronan in acute pancreatitis.

BACKGROUND: The connective tissue component hyaluronan is accumulated locally in the damaged tissue during various inflammatory conditions. Owing to the strong water-binding capacity of this glycosaminoglycan, increased tissue content of hyaluronan is paralleled by the development of interstitial edema. The aim with the current experiment was to investigate whether hyaluronan is accumulated in acute pancreatitis and if increased levels of hyaluronan can be correlated to the inflammation of the pancreatic tissue. METHODS: Acute pancreatitis was induced in Sprague-Dawley rats by the administration of supramaximal doses of the cholecystokinin analogue caerulein. The animals were followed for 5 hours (n = 4), 24 hours (n = 6), or 48 hours (n = 5), and the pancreata were then investigated for hyaluronan and water content, hyaluronan distribution, general morphology and the presence of CD44-positive cells, macrophages, and T lymphocytes. RESULTS: Hyaluronan accumulated in the edematous interstitium during acute pancreatitis. Twenty-four hours after the induction of pancreatitis, the hyaluronan content of the pancreata had increased by more than 100%. Simultaneously, CD44-positive cells infiltrated the tissue. However, no correlation between hyaluronan and water was seen at any time point. CONCLUSIONS: This study shows that acute pancreatitis is associated with a strong but transient increase in interstitial hyaluronan and an infiltration of CD44-positive cells located mainly in the same region as the accumulated hyaluronan.