Vicki Levidiotis

New Approaches to the Treatment of Dense Deposit Disease

The development of clinical treatment protocols usually relies on evidence-based guidelines that focus on randomized, controlled trials. For rare renal diseases, such stringent requirements can represent a significant challenge. Dense deposit disease (DDD; also known as membranoproliferative glomerulonephritis type II) is a prototypical rare disease. It affects only two to three people per million and leads to renal failure within 10 yr in 50% of affected children. On the basis of pathophysiology, this article presents a diagnostic and treatment algorithm for patients with DDD. Diagnostic tests should assess the alternative pathway of complement for abnormalities. Treatment options include aggressive BP control and reduction of proteinuria, and on the basis of pathophysiology, animal data, and human studies, plasma infusion or exchange, rituximab, sulodexide, and eculizumab are additional options. Criteria for treatment success should be prevention of progression as determined by maintenance or improvement in renal function. A secondary criterion should be normalization of activity levels of the alternative complement pathway as measured by C3/C3d ratios and C3NeF levels. Outcomes should be reported to a central repository that is now accessible to all clinicians. As the understanding of DDD increases, novel therapies should be integrated into existing protocols for DDD and evaluated using an open-label Bayesian study design.

Heparanase Is Involved in the Pathogenesis of Proteinuria as a Result of Glomerulonephritis

The beta-D-endoglycosidase heparanase has been proposed to be important in the pathogenesis of proteinuria by selectively degrading the negatively charged side chains of heparan sulfate proteoglycans within the glomerular basement membrane. A loss of negatively charged heparan sulfate proteoglycans may result in alteration of the permselective properties of the glomerular basement membrane, loss of glomerular epithelial and endothelial cell anchor points, and liberation of growth factors. In this study, therefore, the role of heparanase in passive Heymann nephritis (PHN) was examined. Normal glomeruli showed low-level heparanase expression as determined by immunohistochemistry and Western blot analysis. Days 5, 14, and 28 of PHN were associated with an increase in endothelial and glomerular epithelial cell heparanase. Reverse transcription-PCR confirmed a significant increase in mRNA at day 21 of disease (P < 0.0004). Furthermore, urinary and glomerular heparanase activities were significantly increased at days 5 and 21 of disease, respectively. Western blot analysis of isolated glomeruli separated into membrane- and cytosol-enriched protein fractions showed that the active 58-kD heparanase species was increased but restricted to the cytosol of diseased glomeruli at day 21. The inactive 65-kD precursor, however, was found in membrane and cytosol-diseased fractions, suggesting cell membrane processing. Complement depletion prevented glomerular heparanase expression; in addition, administration of a polyclonal anti-heparanase antibody significantly reduced urinary protein excretion at day 5 of disease to 62 +/- 11 mg/d compared with 203 +/- 43 and 159 +/- 18 mg/d in the normal rabbit serum- and normal saline-treated experimental groups, respectively (P < 0.002). Proteinuria was reduced in the absence of any altered glomerular C5b-9 activity, sheep IgG deposition, or rat anti-sheep antibody titers. These data suggest that heparanase contributes to the pathogenesis of proteinuria in PHN.

Pregnancy and Maternal Outcomes Among Kidney Transplant Recipients

Fertility rates, pregnancy, and maternal outcomes are not well described among women with a functioning kidney transplant. Using data from the Australian and New Zealand Dialysis and Transplant Registry, we analyzed 40 yr of pregnancy-related outcomes for transplant recipients. This analysis included 444 live births reported from 577 pregnancies; the absolute but not relative fertility rate fell during these four decades. Of pregnancies achieved, 97% were beyond the first year after transplantation. The mean age at the time of pregnancy was 29 +/- 5 yr. Compared with previous decades, the mean age during the last decade increased significantly to 32 yr (P < 0.001). The proportion of live births doubled during the last decade, whereas surgical terminations declined (P < 0.001). The fertility rate (or live-birth rate) for this cohort of women was 0.19 (95% confidence interval 0.17 to 0.21) relative to the Australian background population. We also matched 120 parous with 120 nulliparous women by year of transplantation, duration of transplant, age at transplantation +/-5 yr, and predelivery creatinine for parous women or serum creatinine for nulliparous women; a first live birth was not associated with a poorer 20-yr graft or patient survival. Maternal complications included preeclampsia in 27% and gestational diabetes in 1%. Taken together, these data confirm that a live birth in women with a functioning graft does not have an adverse impact on graft and patient survival.

Regulation of the renal-specific Na+–K+–2Cl− co-transporter NKCC2 by AMP-activated protein kinase (AMPK)

The renal-specific NKCC2 (Na+-K+-2Cl- co-transporter 2) is regulated by changes in phosphorylation state, however, the phosphorylation sites and kinases responsible have not been fully elucidated. In the present study, we demonstrate that the metabolic sensing kinase AMPK (AMP-activated protein kinase) phosphorylates NKCC2 on Ser126 in vitro. Co-precipitation experiments indicated that there is a physical association between AMPK and the N-terminal cytoplasmic domain of NKCC2. Activation of AMPK in the MMDD1 (mouse macula densa-derived 1) cell line resulted in an increase in Ser126 phosphorylation in situ, suggesting that AMPK may phosphorylate NKCC2 in vivo. The functional significance of Ser126 phosphorylation was examined by mutating the serine residue to an alanine residue resulting in a marked reduction in co-transporter activity when exogenously expressed in Xenopus laevis oocytes under isotonic conditions. Under hypertonic conditions no significant change of activity was observed. Therefore the present study identifies a novel phosphorylation site that maintains NKCC2-mediated transport under isotonic or basal conditions. Moreover, the metabolic-sensing kinase, AMPK, is able to phosphorylate this site, potentially linking the cellular energy state with changes in co-transporter activity.

Acute kidney injury in the rat causes cardiac remodelling and increases angiotensin-converting enzyme 2 expression

Patients with kidney failure are at high risk of a cardiac death and frequently develop left ventricular hypertrophy (LVH). The mechanisms involved in the cardiac structural changes that occur in kidney failure are yet to be fully delineated. Angiotensin‐converting enzyme (ACE) 2 is a newly described enzyme that is expressed in the heart and plays an important role in cardiac function. This study assessed whether ACE2 plays a role in the cardiac remodelling that occurs in experimental acute kidney injury (AKI). Sprague–Dawley rats had sham (control) or subtotal nephrectomy surgery (STNx). Control rats received vehicle (n= 10), and STNx rats received the ACE inhibitor (ACEi) ramipril, 1 mg kg−1 day−1 (n= 15) or vehicle (n= 13) orally for 10 days after surgery. Rats with AKI had polyuria (P < 0.001), proteinuria (P < 0.001) and hypertension (P < 0.001). Cardiac structural changes were present and characterized by LVH (P < 0.001), fibrosis (P < 0.001) and increased cardiac brain natriuretic peptide (BNP) mRNA (P < 0.01). These changes occurred in association with a significant increase in cardiac ACE2 gene expression (P < 0.01) and ACE2 activity (P < 0.05). Ramipril decreased blood pressure (P < 0.001), LVH (P < 0.001), fibrosis (P < 0.01) and BNP mRNA (P < 0.01). These changes occurred in association with inhibition of cardiac ACE (P < 0.05) and a reduction in cardiac ACE2 activity (P < 0.01). These data suggest that AKI, even at 10 days, promotes cardiac injury that is characterized by hypertrophy, fibrosis and increased cardiac ACE2. Angiotensin‐converting enzyme 2, by promoting the production of the antifibrotic peptide angiotensin(1–7), may have a cardioprotective role in AKI, particularly since amelioration of adverse cardiac effects with ACE inhibition was associated with normalization of cardiac ACE2 activity.

A synthetic heparanase inhibitor reduces proteinuria in passive Heymann nephritis

The beta-D-endoglycosidase heparanase has been proposed to be important in the pathogenesis of proteinuria by acting to selectively degrade the negatively charged side chains of heparan sulfate proteoglycans (HSPG) within the glomerular basement membrane (GBM). A loss of the negatively charged HSPG may result in alteration of the permselective properties of the GBM, loss of glomerular epithelial and endothelial cell anchor points, and liberation of growth factors. This study examined the effect of PI-88, a sulfated oligosaccharide heparanase inhibitor, on renal function, glomerular ultrastructure, and proteinuria. Continuous PI-88 infusion at 25 mg/kg per d did not adversely affect animal behavior, growth, or GFR. Cortical tubular vacuolation, however, was observed by light microscopy, and GBM thickness was significantly reduced in these animals (P < 0.0002). Tissue distribution studies using [(35)S]-labeled PI-88 revealed high levels of radioactivity in the kidney after a single subcutaneous injection of 25 mg/kg, suggesting protracted accumulation; moreover, active PI-88 was detected in urine. In passive Heymann nephritis, PI-88 delivered as a continuous infusion at 25 mg/kg per d significantly reduced autologous-phase proteinuria, at day 14 (P < 0.009), in the absence of altered sheep antibody deposition, C5b-9 deposition, and circulating rat anti-sheep antibody titers. Glomerular vascular endothelial growth factor and fibroblast growth factor expression was unaffected by PI-88 administration. However, PI-88 administration significantly prevented glomerular HSPG loss as demonstrated by quantitative immunofluorescence studies (P < 0.0001) in the absence of altered agrin distribution. These data therefore confirm the importance of heparanase in the development of proteinuria.

Heparanase inhibition reduces proteinuria in a model of accelerated anti-glomerular basement membrane antibody disease

Background:  The β‐ d‐endoglycosidase, heparanase, is emerging as an important contributor to the pathogenesis of proteinuria. The purpose of the present study therefore was to examine the role of heparanase in a model of accelerated anti‐glomerular basement disease (anti‐GBM).

New insights into the molecular biology of the glomerular filtration barrier and associated disease

SUMMARY:  The glomerular filtration barrier of the kidney can no longer be considered as an inert and adynamic structure, viewed by electron microscopy. Molecular biology, medical genetics and protein chemistry have enabled us to further understand the complex structure and function of this highly specialized barrier of the kidney. Minor aberrations of physiology can lead to fatal disease. Recent advances in the understanding of the physiology of endothelial cells, glomerular epithelial cells and the glomerular basement membrane and its components, and how these relate to disease, will be considered systematically.

Reduction in renal ACE2 expression in subtotal nephrectomy in rats is ameliorated with ACE inhibition.

Alterations within the RAS (renin–angiotensin system) are pivotal for the development of renal disease. ACE2 (angiotensin-converting enzyme 2) is expressed in the kidney and converts the vasoconstrictor AngII (angiotensin II) into Ang-(1–7), a peptide with vasodilatory and anti-fibrotic actions. Although the expression of ACE2 in the diabetic kidney has been well studied, little is known about its expression in non-diabetic renal disease. In the present study, we assessed ACE2 in rats with acute kidney injury induced by STNx (subtotal nephrectomy). STNx and Control rats received vehicle or ramipril (1 mg·kg−1 of body weight·day−1), and renal ACE, ACE2 and mas receptor gene and protein expression were measured 10 days later. STNx rats were characterized by polyuria, proteinuria, hypertension and elevated plasma ACE2 activity (all P<0.01) and plasma Ang-(1–7) (P<0.05) compared with Control rats. There was increased cortical ACE binding and medullary mas receptor expression (P<0.05), but reduced cortical and medullary ACE2 activity in the remnant kidney (P<0.05 and P<0.001 respectively) compared with Control rats. In STNx rats, ramipril reduced blood pressure (P<0.01), polyuria (P<0.05) and plasma ACE2 (P<0.01), increased plasma Ang-(1–7) (P<0.001), and inhibited renal ACE (P<0.001). Ramipril increased both cortical and medullary ACE2 activity (P<0.01), but reduced medullary mas receptor expression (P<0.05). In conclusion, our results show that ACE2 activity is reduced in kidney injury and that ACE inhibition produced beneficial effects in association with increased renal ACE2 activity. As ACE2 both degrades AngII and generates the vasodilator Ang-(1–7), a decrease in renal ACE2 activity, as observed in the present study, has the potential to contribute to the progression of kidney disease.

Activators of the energy sensing kinase AMPK inhibit random cell movement and chemotaxis in U937 cells

AMP‐activated protein kinase (AMPK) is a key energy sensor, known to regulate energy metabolism in diverse cell types. Hypoxia is encountered frequently in the microenvironments of inflammatory lesions and is a critical regulator of function in inflammatory cells. Energy deficiency is one of the consequences of hypoxia, but its potential role in modulating leucocyte function has received little attention. Using micropore chemotaxis assays to assess migratory responses of the monocyte‐like cell line U937, it was found that the AMPK activators AICAR and phenformin rapidly reduced random migration (chemokinesis) as well as chemotaxis due to stromal cell‐derived factor (SDF)1α. There was an approximate 50% reduction in both chemokinesis and chemotaxis following 30 min preincubation with both AICAR and phenformin (P < 0.01), and this continued with up to 24 h preincubation. The binding of SDF1α to its receptor CXCR4 was unaltered, suggesting AMPK was acting on downstream intracellular signalling pathways important in cell migration. As AMPK and statins are known to inhibit HMG CoA reductase, and both reduce cell migration, the effect of mevastatin on U937 cells was compared with AMPK activators. Mevastatin inhibited cell migration but required 24 h preincubation. As expected, the inhibitory effect of mevastatin was associated with altered subcellular localization of the Rho GTPases, RhoA and cdc42, indicating decreased prenylation of these molecules. Although the effect of AMPK activation was partially reversed by mevalonate, this was not associated with altered subcellular localization of Rho GTPases. The data suggest that activation of AMPK has a general effect on cell movement in U937 cells, and this is not due to inhibition of HMG CoA reductase. These are the first data to show an effect of AMPK on cell movement, and suggest a fundamental role for energy deficiency in regulating cellular behaviour.