Nathalie Caron

PKD1 Haploinsufficiency Causes a Syndrome of Inappropriate Antidiuresis in Mice

Mutations in PKD1 are associated with autosomal dominant polycystic kidney disease. Studies in mouse models suggest that the vasopressin (AVP) V2 receptor (V2R) pathway is involved in renal cyst progression, but potential changes before cystogenesis are unknown. This study used a noncystic mouse model to investigate the effect of Pkd1 haploinsufficiency on water handling and AVP signaling in the collecting duct (CD). In comparison with wild-type littermates, Pkd1(+/-) mice showed inappropriate antidiuresis with higher urine osmolality and lower plasma osmolality at baseline, despite similar renal function and water intake. The Pkd1(+/-) mice had a decreased aquaretic response to both a water load and a selective V2R antagonist, despite similar V2R distribution and affinity. They showed an inappropriate expression of AVP in brain, irrespective of the hypo-osmolality. The cAMP levels in kidney and urine were unchanged, as were the mRNA levels of aquaporin-2 (AQP2), V2R, and cAMP-dependent mediators in kidney. However, the (Ser256) phosphorylated AQP2 was upregulated in Pkd1(+/-) kidneys, with AQP2 recruitment to the apical plasma membrane of CD principal cells. The basal intracellular Ca(2+) concentration was significantly lower in isolated Pkd1(+/-) CD, with downregulated phosphorylated extracellular signal-regulated kinase 1/2 and decreased RhoA activity. Thus, in absence of cystic changes, reduced Pkd1 gene dosage is associated with a syndrome of inappropriate antidiuresis (positive water balance) reflecting decreased intracellular Ca(2+) concentration, decreased activity of RhoA, recruitment of AQP2 in the CD, and inappropriate expression of AVP in the brain. These data give new insights in the potential roles of polycystin-1 in the AVP and Ca(2+) signaling and the trafficking of AQP2 in the CD.

Synthesis and fragmentation of hyaluronan in renal ischaemia

BACKGROUND The turnover of hyaluronan (HA), especially the production of low-molecular-weight fragments of HA, was examined in a model of unilateral renal ischaemia-reperfusion (IR) in rats. METHODS HA was extracted from the outer and inner stripe of the outer medulla (OSOM and ISOM) at different times following IR. Its fragmentation was measured using membrane filtration and size-exclusion chromatography. Quantitative reverse transcription-polymerase chain reaction, zymography and immunohistochemistry were used to assess the expression and localization of various forms of HA synthase (HAS) and hyaluronidase (HYAL). Macrophage infiltration was evaluated using immunohistochemistry. RESULTS HA accumulated at Day 1 mostly as high-molecular-weight (HMW) species with an elution profile similar to a reference 2500 kDa HA and at Day 14 mostly as medium- to low-size fragments. Within 1 day, HAS1 messenger RNA was up-regulated > 50- and 35-fold in OSOM and ISOM, respectively. Thereafter, HAS1 tended to normalize, while HAS2 increased steadily. Both synthetic enzymes were localized around tubules and in the interstitium. Conversely, HYAL1, HYAL2 and global hyaluronidase activity were repressed during the first 24 h. The patterns were identical in the OSOM and ISOM despite markedly different amounts of HA at baseline. There was no obvious correlation between HA deposits and macrophage infiltration. CONCLUSIONS In the post-ischaemic kidney, HA starts to accumulate at Day 1 mostly as HMW species. Later on, a large proportion becomes degraded into smaller fragments. This pattern is explained by coordinated changes in the expression of HA synthases and hyaluronidases, especially an early induction of HAS1. The current data open the door to timed pharmacological interventions blocking the production of HA fragments.

Lack of hyaluronidases exacerbates renal post-ischemic injury, inflammation, and fibrosis.

Renal ischemia-reperfusion injury (IRI) is a pathological process that may lead to acute renal failure and chronic dysfunction in renal allografts. During IRI, hyaluronan (HA) accumulates in the kidney, but suppression of HA accumulation during IRI protects the kidney from ischemic insults. Here we tested whether Hyal1-/- and Hyal2-/- mice display exacerbated renal damage following unilateral IRI due to a higher HA accumulation in the post-ischemic kidney compared with that in the kidney of wild-type mice. Two days after IRI in male mice there was accumulation of HA and CD44 in the kidney, marked tubular damage, infiltration, and increase creatininemia in wild-type mice. Knockout mice exhibited higher amounts of HA and higher creatininemia. Seven days after injury, wild-type mice had a significant decrease in renal damage, but knockout mice still displayed exacerbated inflammation. HA and CD44 together with α-smooth muscle actin and collagen types I and III expression were increased in knockout compared with wild-type mice 30 days after IRI. Thus, both HA-degrading enzymes seem to be protective against IRI most likely by reducing HA accumulation in the post-ischemic kidney and decreasing the inflammatory processes. Deficiency in either HYAL1 or HYAL2 leads to enhanced HA accumulation in the post-ischemic kidney and consequently worsened inflammatory response, increased tubular damage, and fibrosis.

Expression of Nestin, Vimentin, and NCAM by Renal Interstitial Cells after Ischemic Tubular Injury

This work explores the distribution of various markers expressed by interstitial cells in rat kidneys after ischemic injury (35 minutes) during regeneration of S3 tubules of outer stripe of outer medulla (OSOM). Groups of experimental animals (n = 4) were sacrificed every two hours during the first 24 hours post-ischemia as well as 2, 3, 7, 14 days post-ischemia. The occurrence of lineage markers was analyzed on kidney sections by immunohistochemistry and morphometry during the process of tubular regeneration. In postischemic kidneys, interstitial cell proliferation, assessed by 5-bromo-2′-deoxyuridine (BrdU) and Proliferating Cell Nuclear Antigen (PCNA) labeling, was prominent in outer medulla and reach a maximum between 24 and 72 hours after reperfusion. This population was characterized by the coexpression of vimentin and nestin. The density of -Neural Cell Adhesion Molecule (NCAM) positive interstitial cells increased transiently (18–72 hours) in the vicinity of altered tubules. We have also localized a small population of α-Smooth Muscle Actin (SMA)-positive cells confined to chronically altered areas and characterized by a small proliferative index. In conclusion, we observed in the postischemic kidney a marked proliferation of interstitial cells that underwent transient phenotypical modifications. These interstitial cells could be implicated in processes leading to renal fibrosis.

Inhibition of hyaluronan is protective against renal ischaemia–reperfusion injury

BACKGROUND Ischaemia-reperfusion injury (IRI) to the kidney is a complex pathophysiological process that leads to acute renal failure and chronic dysfunction in renal allografts. It was previously demonstrated that during IRI, hyaluronan (HA) accumulates in the cortical and external medullary interstitium along with an increased expression of its main receptor, CD44, on inflammatory and tubular cells. The HA-CD44 pair may be involved in persistent post-ischaemic inflammation. Thus, we sought to determine the role of HA in the pathophysiology of ischaemia-reperfusion (IR) by preventing its accumulation in post-ischaemic kidney. METHODS C57BL/6 mice received a diet containing 4-methylumbelliferone (4-MU), a potent HA synthesis inhibitor. At the end of the treatment, unilateral renal IR was induced and mice were euthanized 48 h or 30 days post-IR. RESULTS 4-MU treatment for 14 weeks reduced the plasma HA level and intra-renal HA content at 48 h post-IR, as well as CD44 expression, creatininemia and histopathological lesions. Moreover, inflammation was significantly attenuated and proliferation was reduced in animals treated with 4-MU. In addition, 4-MU-treated mice had a significantly reduced expression of α-SMA and collagen types I and III, i.e. less renal fibrosis, 30 days after IR compared with untreated mice. CONCLUSION Our results demonstrate that HA plays a significant role in the pathogenesis of IRI, perhaps in part through reduced expression of CD44. The suppression of HA accumulation during IR may protect renal function against ischaemic insults.

An Integrated View of Aristolochic Acid Nephropathy: Update of the Literature

The term “aristolochic acid nephropathy” (AAN) is used to include any form of toxic interstitial nephropathy that is caused either by ingestion of plants containing aristolochic acids (AA) as part of traditional phytotherapies (formerly known as “Chinese herbs nephropathy”), or by the environmental contaminants in food (Balkan endemic nephropathy). It is frequently associated with urothelial malignancies. Although products containing AA have been banned in most of countries, AAN cases remain regularly reported all over the world. Moreover, AAN incidence is probably highly underestimated given the presence of AA in traditional herbal remedies worldwide and the weak awareness of the disease. During these two past decades, animal models for AAN have been developed to investigate underlying molecular and cellular mechanisms involved in AAN pathogenesis. Indeed, a more-in-depth understanding of these processes is essential to develop therapeutic strategies aimed to reduce the global and underestimated burden of this disease. In this regard, our purpose was to build a broad overview of what is currently known about AAN. To achieve this goal, we aimed to summarize the latest data available about underlying pathophysiological mechanisms leading to AAN development with a particular emphasis on the imbalance between vasoactive factors as well as a focus on the vascular events often not considered in AAN.

DIFFERENTIAL REGULATION OF ANGIOTENSIN II RECEPTORS DURING RENAL INJURY AND COMPENSATORY HYPERTROPHY IN THE RAT

1. The renin‐angiotensin system may be involved in the compensatory adaptations occurring after the reduction of renal mass and during the consecutive changes leading to chronic renal failure. We therefore investigated the regulation of angiotensin II receptors in two models of renal hypertrophy in the rat: hypertrophy following uninephrectomy (UNx) or subtotal nephrectomy (STNx). The level of angiotensin type 1 (AT1A‐R and AT1B‐R) and type 2 (AT2‐R) receptor mRNA was quantified by competitive reverse transcription‐polymerase chain reaction (RT‐PCR) in specific renal zones and the intrarenal distribution of angiotensin II receptors was analysed by immunohistochemistry.

Protective effect of nitric oxide in aristolochic acid‐induced toxic acute kidney injury: an old friend with new assets

What is the central question of this study? Despite the fact that the pathogenesis of aristolochic acid (AA) nephropathy is still unclear, we sought to determine whether nitric oxide is involved in the underlying mechanism of AA‐induced acute kidney injury (AKI). What is the main finding and its importance? Using a model of progressive tubulointerstitial nephritis, in which AA nephropathy exhibits two interconnected phases, an acute phase and a chronic phase of injury, we demonstrated that maintenance of nitric oxide bioavailability is essential to improve the outcome of AA‐induced AKI.

Measurement of Changes in Glomerular Filtration Rate Induced by Atrial Natriuretic Peptide in the Rat Kidney

This study was undertaken to improve the measurement of glomerular filtration rate (GFR) during the acute diuretic phase induced by atrial natriuretic peptide (ANP), which may indeed alter the renal clearance of inulin (GFRCL) due to dead space error. A technique to measure GFR without urine collections was therefore developed in anaesthetized rats prepared as for micropuncture. To do so, arterial blood was periodically collected and renal venous blood was withdrawn simultaneously from a catheter inserted into the left suprarenal vein to determine the renal extraction coefficient of inulin (CEIN). In addition, renal blood flow (RBF) was continuously measured with an electromagnetic flow transducer fitted around the left renal artery to estimate renal plasma flow (RPF). GFR (GFRCE) was then calculated as the product of RPF and CEIN. To study the effects of ANP on GFR, rats were injected i.v. with 10 μl of saline without (n= 6; vehicle) or with 1 μg ANP (n= 6; ANP) and GFRCE and GFRCL were compared before and after each treatment. They did not differ significantly during baseline measurements in each experimental group and were not modified after vehicle. Similarly, RBF remained constant. In contrast, RBF and GFRCE increased rapidly and simultaneously 90 s after ANP, from 9·07 ± 0·25 to 10·07 ± 0·35 (12%) and from 1·209 ± 0·188 to 1·715 ± 0·190 ml min−1 (42%), respectively (P < 0·05). GFRCL increased to an even greater extent (88%). Moreover, the peak enhancement of GFRCL was delayed and occurred 180 s after ANP. The renal clearance of inulin was thus unduly elevated due to sudden changes in the dead space induced by the diuretic effect of ANP. In conclusion, determination of glomerular filtration rate by the method of renal extraction of inulin provided more reliable results than those achieved using the classical method of renal clearance of inulin. Moreover, it was sufficiently sensitive to detect small and transient changes in GFR induced by the injection of 1 μg ANP.

A hybrid assembly by encapsulation of human cells within mineralised beads for cell therapy

Background The design of new technologies for treatment of human disorders such as protein deficiencies is a complex and difficult task. Particularly, the construction of artificial organs, based on the immunoisolation of protein-secreting cells, requires the use of suitable materials which have to be biocompatible with the immunoisolated cells and avoid any inappropriate host response. Methodology/Principal Findings This work investigates the in vivo behavior of mechanically resistant hybrid beads which can be considered as a model for artificial organ for cell therapy. This hybrid system was designed and fabricated via the encapsulation of living cells (HepG2) within alginate-silica composites. Two types of beads (alginate-silica hybrid (AS) or alginate/silica hybrid subsequently covered by an external layer of pure alginate (ASA)), with or without HepG2 cells, were implanted into several female Wistar rats. After four weeks, the potential inflammatory local response that might be due to the presence of materials was studied by histochemistry. The results showed that the performance of ASA beads was quite promising compared to AS beads, where less abnormal rat behaviour and less inflammatory cells in histological sections were observed in the case of ASA beads. Conclusions/Significance The current study highlights that alginate-silica composite materials coated with an extra-alginate shell offer much promise in the development of robust implantation devices and artificial organs.