Systemic gene therapy with thymosin ß4 alleviates glomerular injury in mice.

Plasma ultrafiltration in the kidney occurs across glomerular capillaries, which are surrounded by epithelial cells called podocytes. Podocytes have a unique shape maintained by a complex cytoskeleton, which becomes disrupted in glomerular disease resulting in defective filtration and albuminuria. Lack of endogenous thymosin ß4 (TB4), an actin sequestering peptide, exacerbates glomerular injury and disrupts the organisation of the podocyte actin cytoskeleton, however, the potential of exogenous TB4 therapy to improve podocyte injury is unknown. Here, we have used Adriamycin (ADR), a toxin which injures podocytes and damages the glomerular filtration barrier leading to albuminuria in mice. Through interrogating single-cell RNA-sequencing data of isolated glomeruli we demonstrate that ADR injury results in reduced levels of podocyte TB4. Administration of an adeno-associated viral vector encoding TB4 increased the circulating level of TB4 and prevented ADR-induced podocyte loss and albuminuria. ADR injury was associated with disorganisation of the podocyte actin cytoskeleton in vitro, which was ameliorated by treatment with exogenous TB4. Collectively, we propose that systemic gene therapy with TB4 prevents podocyte injury and maintains glomerular filtration via protection of the podocyte cytoskeleton thus presenting a novel treatment strategy for glomerular disease.

The Biological Significance and Implications of Planar Cell Polarity for Nephrology.

The orientation of cells in two-dimensional and three-dimensional space underpins how the kidney develops and responds to disease. The process by which cells orientate themselves within the plane of a tissue is termed planar cell polarity. In this Review, we discuss how planar cell polarity and the proteins that underpin it govern kidney organogenesis and pathology. The importance of planar cell polarity and its constituent proteins in multiple facets of kidney development is emphasised, including ureteric bud branching, tubular morphogenesis and nephron maturation. An overview is given of the relevance of planar cell polarity and its proteins for inherited human renal diseases, including congenital malformations with unknown aetiology and polycystic kidney disease. Finally, recent work is described outlining the influence of planar cell polarity proteins on glomerular diseases and highlight how this fundamental pathway could yield a new treatment paradigm for nephrology.

Vangl2, a planar cell polarity molecule, is implicated in irreversible and reversible kidney glomerular injury.

Planar cell polarity (PCP) pathways control the orientation and alignment of epithelial cells within tissues. Van Gogh-like 2 (Vangl2) is a key PCP protein that is required for the normal differentiation of kidney glomeruli and tubules. Vangl2 has also been implicated in modifying the course of acquired glomerular disease, and here, we further explored how Vangl2 impacts on glomerular pathobiology in this context. Targeted genetic deletion of Vangl2 in mouse glomerular epithelial podocytes enhanced the severity of not only irreversible accelerated nephrotoxic nephritis but also lipopolysaccharide-induced reversible glomerular damage. In each proteinuric model, genetic deletion of Vangl2 in podocytes was associated with an increased ratio of active-MMP9 to inactive MMP9, an enzyme involved in tissue remodelling. In addition, by interrogating microarray data from two cohorts of renal patients, we report increased VANGL2 transcript levels in the glomeruli of individuals with focal segmental glomerulosclerosis, suggesting that the molecule may also be involved in certain human glomerular diseases. These observations support the conclusion that Vangl2 modulates glomerular injury, at least in part by acting as a brake on MMP9, a potentially harmful endogenous enzyme. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Gastric adenocarcinoma revealed by atypical pulmonary lymphangitic carcinomatosis.

Pulmonary lymphangitic carcinomatosis (PLC) is a well-known form of tumour metastasis to the pulmonary lymphatic system or to the adjacent interstitial tissue resulting in thickening of the bronchovascular bundle and septa. Another type of tumour metastasis to the lung involves the pulmonary vascular system and is known as pulmonary tumour thrombotic microangiopathy (PTTM). In this article, we will describe the unusual case of a young Chinese woman with gastric adenocarcinoma revealed by atypical radiographic lesions consistent with both PLC and PTTM. We will discuss the existing evidence and hypotheses about the pathophysiology of both conditions.

Heterozygous Vangl2Looptail mice reveal novel roles for the planar cell polarity pathway in adult lung homeostasis and repair.

Lung diseases impose a huge economic and health burden worldwide. A key aspect of several adult lung diseases, such as idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD), including emphysema, is aberrant tissue repair, which leads to an accumulation of damage and impaired respiratory function. Currently, there are few effective treatments available for these diseases and their incidence is rising. The planar cell polarity (PCP) pathway is critical for the embryonic development of many organs, including kidney and lung. We have previously shown that perturbation of the PCP pathway impairs tissue morphogenesis, which disrupts the number and shape of epithelial tubes formed within these organs during embryogenesis. However, very little is known about the role of the PCP pathway beyond birth, partly because of the perinatal lethality of many PCP mouse mutant lines. Here, we investigate heterozygous Looptail (Lp) mice, in which a single copy of the core PCP gene, Vangl2, is disrupted. We show that these mice are viable but display severe airspace enlargement and impaired adult lung function. Underlying these defects, we find that Vangl2Lp/+ lungs exhibit altered distribution of actin microfilaments and abnormal regulation of the actin-modifying protein cofilin. In addition, we show that Vangl2Lp/+ lungs exhibit many of the hallmarks of tissue damage, including an altered macrophage population, abnormal elastin deposition and elevated levels of the elastin-modifying enzyme, Mmp12, all of which are observed in emphysema. In vitro, disruption of VANGL2 impairs directed cell migration and reduces the rate of repair following scratch wounding of human alveolar epithelial cells. Moreover, using population data from a birth cohort of young adults, all aged 31, we found evidence of an interactive effect between VANGL2 and smoking on lung function. Finally, we show that PCP genes VANGL2 and SCRIB are significantly downregulated in lung tissue from patients with emphysema. Our data reveal an important novel role for the PCP pathway in adult lung homeostasis and repair and shed new light on the genetic factors which may modify destructive lung diseases such as emphysema.

Planar cell polarity genes Celsr1 and Vangl2 are necessary for kidney growth, differentiation, and rostrocaudal patterning.

The mammalian kidney contains nephrons comprising glomeruli and tubules joined to ureteric bud-derived collecting ducts. It has a characteristic bean-like shape, with near-complete rostrocaudal symmetry around the hilum. Here we show that Celsr1, a planar cell polarity (PCP) gene implicated in neural tube morphogenesis, is required for ureteric tree growth in early development and later in gestation prevents tubule overgrowth. We also found an interaction between Celsr1 and Vangl2 (another PCP gene) in ureteric tree growth, most marked in the caudal compartment of the kidneys from compound heterozygous mutant mice with a stunted rump. Furthermore, these genes together are required for the maturation of glomeruli. Interestingly, we demonstrated patients with CELSR1 mutations and spina bifida can have significant renal malformations. Thus, PCP genes are important in mammalian kidney development and have an unexpected role in rostrocaudal patterning during organogenesis.

Effective treatment with rituximab for the maintenance of remission in frequently relapsing minimal change disease.

AIM: Treatment of frequently relapsing or steroid-dependent minimal change disease (MCD) in children and adults remains challenging. Glucocorticoids and/or other immunosuppressive agents are the mainstay of treatment, but patients often experience toxicity from prolonged exposure and may either become treatment dependent and/or resistant. Increasing evidence suggests that rituximab (RTX) can be a useful alternative to standard immunosuppression and allow withdrawal of maintenance immunosuppressants; however, data on optimal treatment regimens, long-term efficacy and safety are still limited. METHODS: We undertook a prospective study of RTX to allow immunosuppression minimization in 15 young adults with frequently relapsing or steroid-dependent, biopsy-proven MCD. All patients were in remission at the start of treatment and on a calcineurin inhibitor. Two doses of RTX (1 gr) were given 6 months apart. A subset of patients also received an additional dose 12 months later, in order to examine the benefit of re-treatment. Biochemical and clinical parameters were monitored over an extended follow-up period of up to 43 months. RESULTS: Median steroid-free survival after RTX was 25 months (range 4-34). Mean relapse frequency decreased from 2.60 ± 0.28 to 0.4 ± 0.19 (P < 0.001) after RTX. Seven relapses occurred, five of which (71%) when CD19 counts were greater than 100 µ. Immunoglobulin levels remained unchanged, and no major side effects were observed throughout the follow-up period. CONCLUSIONS: Rituximab therapy is effective at maintaining prolonged steroid-free remission and reducing relapse frequency in this group of patients. Our study lends further support for the role of RTX in the treatment of patients with frequently relapsing or steroid-dependent MCD.

S100A8/A9 (calprotectin) is critical for development of glomerulonephritis and promotes inflammatory leukocyte-renal cell interactions.

Glomerulonephritis is a common cause of end-stage renal disease. Infiltrating leukocytes interacting with renal cells play a critical role during the initiation and progression of glomerulonephritis, but the exact mechanisms are not clearly defined. By using the murine model of nephrotoxic nephritis, we investigated the role of S100A8/A9 [myeloid-related protein (MRP) 8/14, calprotectin] in promoting glomerulonephritis. In nephrotoxic nephritis, wild-type (WT) mice with glomerulonephritis have elevated serum levels of S100A8/A9, whereas mice deficient in MRP14 (S100a9(-/-)), and hence S100A8/A9, are significantly protected from disease. By using bone marrow transplants, we showed that MRP14 deficiency is required in both the hemopoietic and intrinsic cells for the protective effect. In vitro, both the WT bone marrow-derived macrophages and renal mesangial cells stimulated with S100A8/A9 secrete IL-6, CXCL1, and tumor necrosis factor α; however, Mrp14(-/-) cells exhibit significantly blunted proinflammatory responses. The interaction of WT bone marrow-derived macrophages with renal microvascular endothelial cells results in increased levels of monocyte chemotactic protein 1, IL-8, and IL-6 cytokines, which is attenuated in Mrp14(-/-) bone marrow-derived macrophages. Data shows that S100A8/A9 plays a critical role during glomerulonephritis, exerting and amplifying autocrine and paracrine proinflammatory effects on bone marrow-derived macrophages, renal endothelial cells, and mesangial cells. Therefore, complete S100A8/A9 blockade may be a new therapeutic target in glomerulonephritis.

Atmin mediates kidney morphogenesis by modulating Wnt signaling.

The DNA damage protein and transcription factor Atmin (Asciz) is required for both lung tubulogenesis and ciliogenesis. Like the lungs, kidneys contain a tubular network that is critical for their function and in addition, renal ciliary dysfunction has been implicated in the pathogenesis of cystic kidney disease. Using the Atmin mouse mutant Gasping6 (Gpg6), we investigated kidney development and found it severely disrupted with reduced branching morphogenesis, resulting in fewer epithelial structures being formed. Unexpectedly, transcriptional levels of key cilia associated genes were not altered in Atmin(Gpg6/Gpg6) kidneys. Instead, Gpg6 homozygous kidneys exhibited altered cytoskeletal organization and modulation of Wnt signaling pathway molecules, including ß-catenin and non-canonical Wnt/planar cell polarity (PCP) pathway factors, such as Daam2 and Vangl2. Wnt signaling is important for kidney development and perturbation of Wnt signaling pathways can result in cystic, and other, renal abnormalities. In common with other PCP pathway mutants, Atmin(Gpg6/Gpg6) mice displayed a shortened rostral-caudal axis and mis-oriented cell division. Moreover, intercrosses between Atmin(Gpg6/+) and Vangl2(Lp/+) mice revealed a genetic interaction between Atmin and Vangl2. Thus we show for the first time that Atmin is critical for normal kidney development and we present evidence that mechanistically, Atmin modifies Wnt signaling pathways, specifically placing it as a novel effector molecule in the non-canonical Wnt/PCP pathway. The identification of a novel modulator of Wnt signaling has important implications for understanding the pathobiology of renal disease.

Planar cell polarity and the kidney.

Planar cell polarity (PCP) is the uniform orientation and alignment of a group of cells orthogonal to the apical-basal axis within a tissue. Originally described in insects, it is now known that PCP is required for many processes in vertebrates, including directional cell movement, polarized cell division, ciliary orientation, neural tube closure, heart development and lung branching. In this review, we outline the evidence implicating PCP in kidney development and disease focusing initially on the function of PCP in ureteric bud branching and elongation. We then describe how defects in PCP may lead to polycystic kidney disease and discuss a newly identified role for PCP in the kidney filtration barrier.

Albuminuria is associated with too few glomeruli and too much testosterone.

Normally, the glomerular filtration barrier almost completely excludes circulating albumin from entering the urine. Genetic variation and both pre- and postnatal environmental factors may affect albuminuria in humans. Here we determine whether glomerular gene expression in mouse strains with naturally occurring variations in albuminuria would allow identification of proteins deregulated in relatively 'leaky' glomeruli. Albuminuria increased in female B6 to male B6 to female FVB/N to male FVB/N mice, whereas the number of glomeruli/kidney was the exact opposite. Testosterone administration led to increased albuminuria in female B6 but not female FVB/N mice. A common set of 39 genes, many expressed in podocytes, were significantly differentially expressed in each of the four comparisons: male versus female B6 mice, male versus female FVB/N mice, male FVB/N versus male B6 mice, and female FVB/N versus female B6 mice. The transcripts encoded proteins involved in oxidation/reduction reactions, ion transport, and enzymes involved in detoxification. These proteins may represent novel biomarkers and even therapeutic targets for early kidney and cardiovascular disease.

Comparison of volume status in asymptomatic haemodialysis and peritoneal dialysis outpatients.

BACKGROUND: The majority of haemodialysis (HD) patients gain weight between dialysis sessions and thereby become volume overloaded, whereas peritoneal dialysis (PD) is a more continuous technique. Cardiovascular mortality and hypertension is increased with both treatment modalities. We therefore wished to compare volume status in PD and HD to determine whether PD patients are chronically volume overloaded, as a risk factor for cardiovascular mortality. Study Design, Setting and Participants:We retrospectively audited 72 healthy HD patients and 115 healthy PD patients attending a university hospital dialysis centre for routine outpatient treatment, who had multi-frequency bioimpedance measurements of extracellular water to total body water (ECW/TBW). RESULTS: The groups were well matched for age, sex, weight and ethnicity, PD patients had greater urine output [1,075 (485-1,613) vs. 42.5 (0-1,020) ml/day, p < 0.001], but there was no difference in antihypertensive prescription (63.5 vs. 76.4%), mean arterial blood pressure (post-dialysis 101.6 ± 1.5 mm Hg vs. pre-dialysis 102 ± 2.4 mm Hg), although post-dialysis arterial blood pressure was lower than in PD patients (96.4 ± 3.1 mm Hg, p < 0.05). The ratio of ECW/TBW fell after HD (pre-dialysis 0.394 ± 0.001 vs. post-dialysis 0.389 ± 0.004, p < 0.001) and was similar in the PD group to the group before HD (0.393 ± 0.001), and greater than that in the group after HD (p < 0.001). ECW/TBW was greater than the normal reference range in 30% PD patients, 28% patients before HD and 20% patients after HD. CONCLUSIONS: Overhydration is common in healthy stable PD outpatients, and ECW volumes in PD patients are not dissimilar to those of pre-dialysis HD patients. The role of chronic volume overload as a risk factor for cardiovascular disease needs further investigation.

Is N-terminal probrain-type natriuretic peptide a clinically useful biomarker of volume overload in peritoneal dialysis patients?

BACKGROUND: Brain natriuretic peptide (BNP) has been reported to be a powerful predictor of peritoneal dialysis patient survival. However, it is unclear as to whether this is related to cardiac dysfunction or chronic volume overload. METHODS: To investigate the relationship between BNP, cardiac function and fluid volume overload, we reviewed multifrequency bioimpedance, transthoracic echocardiography and serum N-terminal probrain-type natriuretic peptide (NTproBNP) in 115 stable peritoneal dialysis outpatients attending for assessment of peritoneal dialysis and transport status. RESULTS: In this cross-sectional study, the median NTproBNP was 251 (118-605) pmol/L. On simple univariate analysis, NTproBNP was associated with markers of residual renal function, volume overload, hypertension and hypertensive cardiac disease and inflammation [reduced serum albumin and raised C-reactive protein]. However, on multivariate logistical regression analysis, the strongest association for log NTproBNP was with the estimated right ventricular end-systolic pressure (ß = 0.02, F = 11.5, P = 0.001), followed by log 24-h urine volume (ß = -0.19, F = 10.7, P = 0.002), extracellular/total body water ratio (ß = 13.5, F = 6.1, P = 0.017) and the number of different antihypertensive medications prescribed (ß = 0.15, F = 8.7, P = 0.005). CONCLUSION: In this cross-sectional study, although NTproBNP was associated with residual renal function, cardiac hypertrophy, volume overload and inflammation on simple univariate analysis, on further examination NTproBNP was predominantly affected by factors associated with volume overload, and these results require confirmation in a prospective study.