Identification of novel pathogenic variants of CUBN in patients with isolated proteinuria.

BACKGROUND: Although proteinuria is long recognized as an independent risk factor for progressive chronic kidney diseases, not all forms of proteinuria are detrimental to kidney function, one of which is isolated proteinuria caused by cubilin (CUBN)-specific mutations. CUBN encodes an endocytic receptor, initially found to be responsible for the Imerslund-Gräsbeck syndrome (IGS; OMIM #261100) characterized by a combined phenotype of megaloblastic anemia and proteinuria. METHODS: After analyzing their clinical and pathological characterizations, next-generation sequencing for renal disease genes or whole-exome sequencing (WES) was performed on four patients with non-progressive isolated proteinuria. CUBN biallelic pathogenic variants were identified and further analyzed by cDNA-PCR sequencing, immunohistochemistry, minigene assay, and multiple in silico prediction tools, including 3D protein modeling. RESULTS: Here, we present four patients with isolated proteinuria caused by CUBN C-terminal biallelic pathogenic variants, all of which showed no typical IGS symptoms, such as anemia and vitamin B12 deficiency. Their urine protein levels fluctuated between +~++ and estimated glomerular filtration rate (eGFR) were normal or slightly higher. Mild mesangial hypercellularity was found in three children's renal biopsies. A homozygous splice-site variant of CUBN (c.6821+3 (IVS44) A>G) was proven to result in the exon 44 skipping and premature translation termination by cDNA sequencing and immunohistochemistry. Compound heterozygous mutations were identified among the other three children, including another novel splice-site variant (c.10764+1 (IVS66) G>A) causing the retention of first 4 nucleotides in intron 66 by minigene assay, two unreported missense mutations (c.4907G>A (p.R1636Q); c. 9095 A>G (p.Y3032C)), and two reported missense mutations in China (c.8938G>A (p.D2980N); c. 9287T>C (p.L3096P)), locating behind the vitamin B12-binding domain, affecting CUB11, CUB16, CUB22, CUB23, and CUB27 domains, respectively. CONCLUSION: These results demonstrate that above CUBN mutations may cause non-progressive and isolated proteinuria, expanding the variant spectrum of CUBN and benefiting our understanding of proteinuria and renal function.

Kidney tubular injury induced by valproic acid: systematic literature review.

BACKGROUND: Valproic acid is prescribed for epilepsy and as prophylaxis for bipolar disorder and migraine headaches. It has also been implicated as a cause of a kidney tubular injury. METHODS: We undertook a review of the literature to characterize the biochemical and histopathological features of the overt kidney tubular injury and to evaluate the possible existence of a pauci-symptomatic injury. The pre-registered review (CRD42022360357) was performed following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) methodology. Searches were conducted in Excerpta Medica, the National Library of Medicine, and Web of Science. The gray literature was also considered. RESULTS: For the final analysis, we retained 36 articles: 28 case reports documented 48 individuals with epilepsy on valproic acid for 7 months or more and presenting with features consistent with an overt kidney tubular injury. The following disturbances were noted: hypophosphatemia (N = 46), normoglycemic glycosuria (N = 46), total proteinuria (N = 45), metabolic acidosis (N = 36), hypouricemia (N = 27), tubular proteinuria (N = 27), hypokalemia (N = 23), and hypocalcemia (N = 8). A biopsy, obtained in six cases, disclosed altered proximal tubular cells with giant and dysmorphic mitochondria. Eight case series addressed the existence of a pauci- or even asymptomatic kidney injury. In the reported 285 subjects on valproic acid for 7 months or more, an isolated tubular proteinuria, mostly N-acetyl-ß-glucosaminidase, was often noted. CONCLUSIONS: Valproic acid may induce an overt kidney tubular injury, which is associated with a proximal tubular mitochondrial toxicity. Treatment for 7 months or more is often associated with a pauci- or oligosymptomatic kidney tubular injury. A higher resolution version of the Graphical abstract is available as Supplementary information.

Population-based identification and temporal trend of children with primary nephrotic syndrome: The Kaiser Permanente nephrotic syndrome study.

INTRODUCTION: Limited population-based data exist about children with primary nephrotic syndrome (NS). METHODS: We identified a cohort of children with primary NS receiving care in Kaiser Permanente Northern California, an integrated healthcare delivery system caring for >750,000 children. We identified all children <18 years between 1996 and 2012 who had nephrotic range proteinuria (urine ACR>3500 mg/g, urine PCR>3.5 mg/mg, 24-hour urine protein>3500 mg or urine dipstick>300 mg/dL) in laboratory databases or a diagnosis of NS in electronic health records. Nephrologists reviewed health records for clinical presentation and laboratory and biopsy results to confirm primary NS. RESULTS: Among 365 cases of confirmed NS, 179 had confirmed primary NS attributed to presumed minimal change disease (MCD) (72%), focal segmental glomerulosclerosis (FSGS) (23%) or membranous nephropathy (MN) (5%). The overall incidence of primary NS was 1.47 (95% Confidence Interval:1.27-1.70) per 100,000 person-years. Biopsy data were available in 40% of cases. Median age for patients with primary NS was 6.9 (interquartile range:3.7 to 12.9) years, 43% were female and 26% were white, 13% black, 17% Asian/Pacific Islander, and 32% Hispanic. CONCLUSION: This population-based identification of children with primary NS leveraging electronic health records can provide a unique approach and platform for describing the natural history of NS and identifying determinants of outcomes in children with primary NS.

Roles of glomerular endothelial hyaluronan in the development of proteinuria.

Vascular endothelial cells are covered with glycocalyx comprising heparan sulfate, hyaluronan, chondroitin sulfate, and associated proteins. Glomerular endothelial glycocalyx is involved in protecting against induction of proteinuria and structural damage, but the specific components in glycocalyx that represent therapeutic targets remain unclear. Anti-vascular endothelial growth factor (VEGF) therapy is associated with an increased risk of glomerular endothelial injury. This study investigated whether hyaluronan could provide a therapeutic target to protect against proteinuria. We conducted ex vivo and in vivo experiments to explore the effects of degrading glomerular hyaluronan by administering hyaluronidase and of supplementation with hyaluronan. We investigated hyaluronan expression using biotin-labeled hyaluronan-binding protein (HABP) in human kidney specimens or serum hyaluronan in endothelial injuries under inhibition of VEGF signaling. We directly demonstrated hyaluronan in glomerular endothelial layers using HABP staining. Ex vivo and in vivo experiments showed the development of proteinuria after digestion of hyaluronan in glomerular capillaries. Supplementation with hyaluronan after hyaluronidase treatment suppressed proteinuria. Mice in the in vivo study developed albuminuria after intraperitoneal injection of hyaluronidase with decreased glomerular hyaluronan and increased serum hyaluronan. In human kidneys with endothelial cell dysfunction and proteinuria due to inhibition of VEGF, glomerular expression of hyaluronan was reduced even in normal-appearing glomeruli. Serum hyaluronan levels were elevated in patients with pre-eclampsia with VEGF signaling inhibition. Our data suggest that hyaluronan itself plays crucial roles in preventing proteinuria and preserving the integrity of endothelial cells. Hyaluronan could provide a therapeutic target for preventing glomerular endothelial glycocalyx damage, including VEGF signaling inhibition.

Preclinical Testing of Viral Therapeutic Efficacy in Pristane-Induced Lupus Nephritis and Diffuse Alveolar Hemorrhage Mouse Models.

Systemic lupus erythematosus (SLE) is a multifactorial and heterogeneous autoimmune disease involving multiple organ systems and tissues. Lupus nephritis occurs in approximately 60% of patients with SLE and is the leading cause of morbidity. Diffuse alveolar hemorrhage (DAH) is a rare but very serious complication of SLE with a greater than 50% associated mortality. The etiology of SLE is unclear but has proposed genetic, hormonal, and environmental aspects. Pristane is a saturated terpenoid alkane and has become the most popular laboratory model for inducing lupus in mice. The pristane model of SLE has the capacity to reproduce many components of the human presentation of the disease. Previous studies have demonstrated that virus-derived immune-modulating proteins have the potential to control inflammatory and autoimmune disorders. Serp-1, a 55 kDa secreted and highly glycosylated immune modulator derived from myxoma virus (MYXV), has potent immunomodulatory activity in models of vasculitis, viral sepsis, collagen-induced arthritis, and transplant rejection. This chapter describes the mouse preclinical pristane lupus model as a method to examine virus-derived protein efficacy for treating autoimmune diseases and specifically lupus nephritis and DAH.

The Therapeutic Effect of Active Vitamin D Supplementation in Preventing the Progression of Diabetic Nephropathy in a Diabetic Mouse Model.

BACKGROUND: Diabetic nephropathy (DN) is one of the most common microvascular complications of diabetes and is the leading cause of end-stage renal disease (ESRD) and replacement therapy worldwide. Vitamin D levels in DN patients are very low due to the decrease in the synthesis and activity of 1-α hydroxylase in the proximal tubule cells and decrease in the vitamin D receptor abundance. To date, few studies have shown the antioxidant effects of 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] on hyperglycemia-induced renal injury. The selective activator of the vitamin D receptor, paricalcitol, reduces proteinuria and slows the progression of kidney injury. The precise mechanism through which vitamin D affects diabetic status and provides kidney protection remains to be determined. METHODS: Diabetes mellitus (DM) was induced in 94 8-week-old DBA/2J mice by intraperitoneal injection of streptozotocin (STZ). DM mice were randomly divided into receiving vehicle or treatment with paricalcitol, the active vitamin D analog, 1 week after DM induction or paricalcitol treatment 3 weeks after DM induction. An additional control group of healthy wild-type mice was not treated. Urine albumin, blood urea nitrogen, and creatinine levels were measured before and at the end of the paricalcitol treatment. Periodic acid-Schiff, immunohistochemistry staining, and western blot of the renal tissues of vitamin D receptor, villin, nephrin, and podocin expressions, were analyzed. RESULTS: Paricalcitol treatment restored villin, nephrin, and podocin protein levels that were downregulated upon DM induction, and reduced fibronectin protein level. Vitamin D receptor activation by paricalcitol may reduce proteinuria of DN in mice and alleviate high-glucose-induced injury of kidney podocytes by regulating the key molecules such nephrin-podocin. CONCLUSIONS: Paricalcitol treatment was associated with improved structural changes in type 1 diabetic mice including upregulation of vitamin D receptor expression, and decreased fibrosis markers such as fibronectin. These effects may contribute to the consistent benefit of vitamin D analog to slow the deterioration in glomerular function and reduce the risk of ESRD in patients with type 1 and 2 diabetes mellitus. Our results suggest that additional use of paricalcitol may be beneficial in treating patients with diabetes under standard therapeutic strategies.

Effects of BSF on Podocyte Apoptosis via Regulating the ROS-Mediated PI3K/AKT Pathway in DN.

Diabetic nephropathy (DN) is the leading cause of end-stage renal disease (ESRD). The ROS-mediated PI3K/AKT pathway plays a key role in podocyte apoptosis and DN progression. Our previous study demonstrated that Baoshenfang (BSF) can decrease proteinuria and attenuate podocyte injury. However, the effects of BSF on podocyte apoptosis induced by the ROS-mediated PI3K/AKT pathway remain unclear. Herein, in vivo and in vitro studies have been performed. In our in vivo study, BSF significantly decreased 24-h urinary protein, serum creatinine, and blood urea nitrogen levels in DN mice. Meanwhile, BSF significantly inhibited oxidative stress and podocyte apoptosis in our in vivo and in vitro studies. Moreover, BSF significantly decreased the inhibition of the PI3K/AKT pathway induced by HG in DN. More importantly, the effects of BSF on podocyte apoptosis were reversed by PI3K siRNA transfection. In conclusion, BSF can decrease proteinuria and podocyte apoptosis in DN, in part through regulating the ROS-mediated PI3K/AKT pathway.

Effect of shenyan xiaobai granule on nephrin and podocin of adriamycin-induced renal injury: A randomised controlled trial.

ETHNOPHARMACOLOGICAL RELEVANCE: ShenYanXiaoBai granules is a traditional Chinese herbal medicine, It is used widely for the treatment of proteinuria caused by various kidney diseases. AIM OF THE STUDY: This study investigated the mechanism of Shenyan Xiaobai Granule in the treatment of nephritis proteinuria. MATERIALS AND METHODS: 100 male wistar rats were divided into a blank group (n = 20) and a nephropathy group (n = 80) using random number table after 1 week adaptive feeded. Rats were injected with adriamycin (6.5 mg/kg) via the tail vein to induce nephropathy except for blank group. Every rat's urine protein was checked with urine protein dipstick test after three days that showed all rats in nephropathy group were successful modelled. Nephropathy group was divided into model group, benazepril group, ShenYanXiaoBai low dose group, ShenYanXiaoBai high dose group equally. Blank and model group were given distilled water 2 ml as control, then benazepril group received benazepril 0.90 mg/kg, ShenYanXiaoBai low dose group received ShenYanXiaoBai granules 1.80 g/kg as high dose group was given 3.60 g/kg, gavage for 6 days a week last for seven weeks. Urinary albumin/urinary creatinine were measured in seventh day every week. Three rats were randomly selected from each group to be executed in 3th and 5th weekend to detect the mRNA and protein expression level in kidney. The rest rats were as well. CONCLUSIONS: The therapeutic effect of ShenYanXiaoBai high dose group was better than the two other treated groups from the 5th week to the 7th week, the comparison had a significant difference. The therapeutic effect of benazepril group was better than the ShenYanXiaoBai low dose group in the 7 weeks and the comparison had a significant difference.

Inhibition of the ERK1/2-mTORC1 axis ameliorates proteinuria and the fibrogenic action of transforming growth factor-ß in Adriamycin-induced glomerulosclerosis.

Transforming growth factor-ß (TGF-ß) plays crucial roles in the development of focal segmental glomerulosclerosis, but key molecular pathways remain unknown. Here, we identified the regulation of mammalian target of rapamycin complex1 (mTORC1) by TGF-ß via ERK1/2 in the Adriamycin-induced murine model of focal segmental glomerulosclerosis. Adriamycin administration elicited early activation of TGF-ß-ERK1/2-mTORC1 in podocytes, which persisted at later stages of albuminuria and glomerulosclerosis. Phosphorylation of the TGF-ß receptor-I (TGF-ßRI), Smad3, ERK1/2 and ribosomal protein S6 were evident in the glomeruli of adriamycin-treated mice. Targeting TGFß-RI and mTORC1 with pharmacological inhibitors suppressed TGF-ß signaling in glomeruli and significantly reduced albuminuria, glomerulosclerosis, protein levels of collagen 4α3, plasminogen activator inhibitor-1, and vimentin and restored mRNA levels of podocyte markers. Low dose US Food and Drug Administration (FDA)-approved MEK/ERK inhibitor trametinib/GSK1120212 blunted TGF-ß1-induced mTORC1 activation in podocytes, ameliorated up-regulation of TGF-ß, plasminogen activator inhibitor-1, monocyte chemoattractant protein-1, fibronectin and α-smooth muscle actin and prevented albuminuria and glomerulosclerosis with improved serum albumin. In cultured podocytes, this pathway was found to be associated with translation of fibrogenic collagen 4α3 and plasminogen activator inhibitor-1, without influencing their transcription. Notably, rapamycin suppressed upstream p-TGF-ßRI, p-Smad3 and p-ERK1/2, and trametinib down-regulated upstream p-Smad3 in ex vivo and in vivo studies, indicating that harmful paracrine signaling among glomerular cells amplified the TGF-ß-ERK1/2-mTORC1 axis by forming a positive feedback loop. Thus, an accentuated TGF-ß-ERK1/2-mTORC1 pathway is suggested as a central upstream mediator to develop proteinuria and glomerulosclerosis. Hence, preventing activation of this vicious loop by trametinib may offer a new therapeutic strategy for glomerular disease treatment.

Rhodojaponin II attenuates kidney injury by regulating TGF-ß1/Smad pathway in mice with adriamycin nephropathy.

ETHNOPHARMACOLOGICAL RELEVANCE: Rhododendron molle G. Don (Ericaceae) (RM) is a natural medicinal plant. Its root extracts have been applied in clinic and proved to be effective in chronic glomerulonephritis and rheumatoid arthritis in China. Surprising, little is understood about the key compound of RM and the exact mechanisms underlying its treatment on kidney diseases. In this study, we will explore whether rhodojaponin II (R-II), as the important compound of RM, also exerts the major effect. MATERIALS AND METHODS: Mouse model of focal segmental glomerulosclerosis was induced by single dose of adriamycin injection. Induced adriamycin nephropathy (ADRN) mice were treated individually with RM root extract (5 mg/kg, n = 5), RM root extract (60 mg/kg, n = 5), R-II (0.04 mg/kg, n = 6) or captopril (30 mg/kg, n = 5) for five weeks. Podocyte marker (nephrin and podocin) expressions were examined by immunohistochemical staining and Western Blot analysis. Fibronectin level was evaluated by immunohistochemical staining and Western Blot analysis. Interstitial infiltrated inflammatory cells (CD4+ T cells, CD8+ T cells, and CD68+ macrophages) were examined with immunohistochemical staining. The expressions of NF-ĸB p-p65 and TGF-ß1/Smad pathway associated key proteins, such as TGF-ß1, Smad3, phosphorylated-Smad3 (p-Smad3), and Smad7, were analyzed respectively by Western Blot analysis. RESULTS: RM root extract (5 mg/kg) and its important compound R-II (0.04 mg/kg) significantly ameliorated proteinuria, podocyte injury, and glomerulosclerosis, meanwhile, they hampered interstitial fibrosis in mice with ADRN. R-II significantly reduced NF-ĸB p65 phosphorylation, interstitial infiltrated CD4+ T cells, CD8+ T cells, and CD68+ macrophages, at the same time, down-regulated TGF-ß1 and p-Smad3 protein expressions in mice with ADRN. CONCLUSION: RM root extract, R-II, could effectively ameliorate proteinuria and kidney injury in ADRN, related to its anti-inflammatory effects, as well as suppression of TGF-ß1/Smad signaling pathway.

Therapeutic and antiproteinuric effects of salvianolic acid A in combined with low-dose prednisone in minimal change disease rats: Involvement of PPARγ/Angptl4 and Nrf2/HO-1 pathways.

Danshen (Salvia miltiorrhiza) and prednisone are extensively applied in the treatment of kidney disease. Salvianolic acid A (SAA), the major biologically active component of Danshen, which has various biological effects. Our previous findings have demonstrated the renoprotective effect of SAA in various kidney disease rodent models. Here, we explore the therapeutic potential and possible mechanisms of SAA in combination with low-dose prednisone in adriamycin (ADR)-induced minimal change disease (MCD) rat model and mouse podocyte injury cell model. SAA was injected via tail vein at 10 mg/kg/day and prednisone at 5 mg/kg/day via gavage. Each drug was administered daily alone or in combination for 3 weeks. Combination therapy showed significant therapeutic efficacy as manifested by relieved urinary proteins, improved blood biochemical indicators including serum total protein, albumin, triglyceride, cholesterol, the indices of renal function i.e. blood urea nitrogen and serum creatinine levels, and ameliorated pathological lesions. Particularly, co-administration showed a significant anti-proteinuria effect in MCD rats. Further studies suggested that co-administration effectively ameliorated the podocyte injury as indicated by the reduction of podocyte foot processes fusion, up-regulation of synaptopodin and down-regulation of desmin. These beneficial effects are accompanied by activation of the Nrf2/HO-1 and PPARγ/Angptl4 pathways in vivo, and the effect of SAA on PPARγ/Angptl4 is also demonstrated in vitro. These findings suggested that SAA exerted podocyte-protection against MCD injury through PPARγ/Angptl4 and Nrf2/HO-1 pathways, and combined with low-dose prednisone possessed a significant anti-proteinuria and therapeutic effects in MCD rats.

Salidroside ameliorates Adriamycin nephropathy in mice by inhibiting ß-catenin activity.

Salidroside is a major phenylethanoid glycoside in Rhodiola rosea L., a traditional Chinese medicine, with multiple biological activities. It has been shown that salidroside possesses protective effects for alleviating diabetic renal dysfunction, contrast-induced-nephropathy and other kidney diseases. However, the involved molecular mechanism was still not understood well. Herein, we examined the protective effects of salidroside in mice with Adriamycin (ADR)-induced nephropathy and the underlying molecular mechanism. The results showed that salidroside treatment ameliorates proteinuria; improves expressions of nephrin and podocin; and reduces kidney fibrosis and glomerulosclerosis induced by ADR. Mechanistically, ADR induces a robust accumulation of ß-catenin in the nucleus and stimulates its downstream target gene expression. The application of salidroside largely abolishes the nuclear translocation of ß-catenin and thus inhibits its activity. Furthermore, the activation of ß-catenin almost completely counteracts the protective roles of salidroside in ADR-injured podocytes. Taken together, our data indicate that salidroside ameliorates proteinuria, renal fibrosis and podocyte injury in ADR nephropathy, which may rely on inhibition of ß-catenin signalling pathway.

Effects of Tripterygium wilfordii Induction Therapy to IgA Nephropathy Patients with Heavy Proteinuria.

Although some new drugs have been developed, Tripterygium wilfordii HOOK F. (TWHF) has the merits of relatively lower price and fewer side effects. Unfortunately, the efficacy and safety of the TWHF (especially dosage 120 mg/d) in the immunoglobulin A (IgA) nephropathy (IgAN) are still lacking. A cohort study including 49 IgAN patients with heavy proteinuria who received induction therapy was undertaken. Patients were divided into three groups: Prednisone (PRE), conventional-dose TWHF (CTW) and double-dose TWHF (DTW). The clinical features, laboratory data, histological manifestations and outcomes of the groups were compared. We found that urinary protein excretion and rates of elevated n-acetyl-ß-D-glucosaminidase (NAG) and retinol binding protein (RBP) were prominent in all groups. Neither histopathological changes nor the rates of renal insufficiency were significantly different among groups. Patients in the PRE (69.2%) and DTW groups (87.5%) achieved complete remission; none of the CTW group did. Furthermore, the total remission rate of the DTW group was substantially higher than that of the CTW group. The degree of hypoproteinemia, improved considerably in the PRE and DTW groups. Treatment was well tolerated in all patients, and no serious adverse events were observed. Our findings suggested that induction therapy with double dose TWHF significantly improved response rates in IgAN patients with heavy proteinuria, and did not considerably increase side effects.

Aconitine: A potential novel treatment for systemic lupus erythematosus.

BACKGROUND: Aconitum plants have been widely used in China for thousands of years. Recent evidences indicate that aconitine, the main active ingredient of Aconitum, has immunomodulatory properties that might be useful for treating autoimmune diseases, such as rheumatoid arthritis. In this study, we conducted a pilot study to explore the effect and mechanisms of aconitine on the treatment of systemic lupus erythematosus. METHODS: A pristane-induced murine model was used. The pristane-induced mice were treated with aconitine (25, 75 µg kg-1 d-1, po) for 9 weeks. Every three weeks, proteinuria was detected to monitor the kidney damage and blood was collected to measure serum levels of autoantibodies, besides the kidney pathological examination. The major B cell activating factor and major pro-inflammatory mediators, PGE2, IL-17a and IL-6, were also detected. RESULTS: We found that aconitine significantly improved the mouse health, decreased the elevated blood leukocyte counts, reduced the serum level of anti-double-stranded DNA (anti-dsDNA) antibody, greatly ameliorated renal histopathologic damage and reduced IgG deposit in glomerular. Furtherly, the levels of PGE2, IL-17a and IL-6, were found to have decreased in aconitine treated mice. CONCLUSION: We have demonstrated that aconitine can inhibit the progression of disease and ameliorate the pathologic lesion of systemic lupus erythematosus.

Btk inhibition treats TLR7/IFN driven murine lupus.

Bruton's tyrosine kinase (Btk) is expressed in a variety of immune cells and previous work has demonstrated that blocking Btk is a promising strategy for treating autoimmune diseases. Herein, we utilized a tool Btk inhibitor, M7583, to determine the therapeutic efficacy of Btk inhibition in two mouse lupus models driven by TLR7 activation and type I interferon. In BXSB-Yaa lupus mice, Btk inhibition reduced autoantibodies, nephritis, and mortality. In the pristane-induced DBA/1 lupus model, Btk inhibition suppressed arthritis, but autoantibodies and the IFN gene signature were not significantly affected; suggesting efficacy was mediated through inhibition of Fc receptors. In vitro studies using primary human macrophages revealed that Btk inhibition can block activation by immune complexes and TLR7 which contributes to tissue damage in SLE. Overall, our results provide translational insight into how Btk inhibition may provide benefit to a variety of SLE patients by affecting both BCR and FcR signaling.

Protective Effect of Salvia Przewalskii Extract on Puromycin-Induced Podocyte Injury.

BACKGROUND: To determine the effect of Salvia przewalskii extract (SPE) from total phenolic acids on puromycin aminonucleoside (PAN)-induced rat podocyte injury. METHODS: The rats were divided into groups that were treated with either PAN only or PAN followed by tacrolimus or SPE. We evaluated the effects of SPE on podocyte injury 5, 10, 15 and 21 days following treatment. RESULTS: (1) Proteinuria was observed starting on day 5 in all groups. The peak levels of proteinuria differed among the groups with tacrolimus and high-dose SPE, which significantly decreased proteinuria relative to the PAN and low- and medium-dose SPE groups. The proteinuria in each group decreased by day 15 and returned to a normal level by day 21. (2) H&E and PAS staining revealed no abnormality in glomerular morphology. With electron microscopy, we observed foot process effacement in the rats of all groups starting on day 5, but rats in the tacrolimus and high-dose SPE groups exhibited a lower degree. (3) IHC staining of nephrin and podocin revealed unaffected expression and better linear distributions in the high-dose SPE and tacrolimus groups. Western blot analysis confirmed that SPE could improve the expression of proteins. (4) The mRNA levels of nephrin and podocin in the tacrolimus and high-dose SPE groups were significantly higher than that in the others. CONCLUSION: In our study, we first demonstrated the ability of SPE to reduce proteinuria, preserve the morphology and structure of podocytes and retain the levels of slit diaphragm proteins on PAN-induced rat podocytes injury.

Sialic acid supplementation ameliorates puromycin aminonucleoside nephrosis in rats.

Defects in sialylation are known to have serious consequences on podocyte function leading to collapse of the glomerular filtration barrier and the development of proteinuria. However, the cellular processes underlying aberrant sialylation in renal disease are inadequately defined. We have shown in cultured human podocytes that puromycin aminonucleoside (PAN) downregulates enzymes involved in sialic acid metabolism and redox homeostasis and these can be rescued by co-treatment with free sialic acid. The aim of the current study was to ascertain whether sialic acid supplementation could improve renal function and attenuate desialylation in an in vivo model of proteinuria (PAN nephrosis) and to delineate the possible mechanisms involved. PAN nephrotic rats were supplemented with free sialic acid, its precursor N-acetyl mannosamine or the NADPH oxidase inhibitor apocynin. Glomeruli, urine, and sera were examined for evidence of kidney injury and therapeutic efficacy. Of the three treatment regimens, sialic acid had the broadest efficacy in attenuating PAN-induced injury. Proteinuria and urinary nephrin loss were reduced. Transmission electron microscopy revealed that podocyte ultrastructure, exhibited less severe foot process effacement. PAN-induced oxidative stress was ameliorated as evidenced by a reduction in glomerular NOX4 expression and a downregulation of urine xanthine oxidase levels. Sialylation dysfunction was improved as indicated by reduced urinary concentrations of free sialic acid, restored electrophoretic mobility of podocalyxin, and improved expression of a sialyltransferase. These data indicate that PAN induces alterations in the expression of enzymes involved in redox control and sialoglycoprotein metabolism, which can be ameliorated by sialic acid supplementation possibly via its properties as both an antioxidant and a substrate for sialylation.

A1M Ameliorates Preeclampsia-Like Symptoms in Placenta and Kidney Induced by Cell-Free Fetal Hemoglobin in Rabbit.

Preeclampsia is one of the most serious pregnancy-related diseases and clinically manifests as hypertension and proteinuria after 20 gestational weeks. The worldwide prevalence is 3-8% of pregnancies, making it the most common cause of maternal and fetal morbidity and mortality. Preeclampsia lacks an effective therapy, and the only "cure" is delivery. We have previously shown that increased synthesis and accumulation of cell-free fetal hemoglobin (HbF) in the placenta is important in the pathophysiology of preeclampsia. Extracellular hemoglobin (Hb) and its metabolites induce oxidative stress, which may lead to acute renal failure and vascular dysfunction seen in preeclampsia. The human endogenous protein, α1-microglobulin (A1M), removes cell-free heme-groups and induces natural tissue repair mechanisms. Exogenously administered A1M has been shown to alleviate the effects of Hb-induced oxidative stress in rat kidneys. Here we attempted to establish an animal model mimicking the human symptoms at stage two of preeclampsia by administering species-specific cell-free HbF starting mid-gestation until term, and evaluated the therapeutic effect of A1M on the induced symptoms. Female pregnant rabbits received HbF infusions i.v. with or without A1M every second day from gestational day 20. The HbF-infused animals developed proteinuria and a significantly increased glomerular sieving coefficient in kidney that was ameliorated by co-administration of A1M. Transmission electron microscopy analysis of kidney and placenta showed both intracellular and extracellular tissue damages after HbF-treatment, while A1M co-administration resulted in a significant reduction of the structural and cellular changes. Neither of the HbF-treated animals displayed any changes in blood pressure during pregnancy. In conclusion, infusion of cell-free HbF in the pregnant rabbits induced tissue damage and organ failure similar to those seen in preeclampsia, and was restored by co-administration of A1M. This study provides preclinical evidence supporting further examination of A1M as a potential new therapy for preeclampsia.

Primary coenzyme Q10 deficiency presenting as fatal neonatal multiorgan failure.

Coenzyme Q10 deficiency is a clinically and genetically heterogeneous disorder, with manifestations that may range from fatal neonatal multisystem failure, to adult-onset encephalopathy. We report a patient who presented at birth with severe lactic acidosis, proteinuria, dicarboxylic aciduria, and hepatic insufficiency. She also had dilation of left ventricle on echocardiography. Her neurological condition rapidly worsened and despite aggressive care she died at 23 h of life. Muscle histology displayed lipid accumulation. Electron microscopy showed markedly swollen mitochondria with fragmented cristae. Respiratory-chain enzymatic assays showed a reduction of combined activities of complex I+III and II+III with normal activities of isolated complexes. The defect was confirmed in fibroblasts, where it could be rescued by supplementing the culture medium with 10 µM coenzyme Q10. Coenzyme Q10 levels were reduced (28% of controls) in these cells. We performed exome sequencing and focused the analysis on genes involved in coenzyme Q10 biosynthesis. The patient harbored a homozygous c.545T>G, p.(Met182Arg) alteration in COQ2, which was validated by functional complementation in yeast. In this case the biochemical and morphological features were essential to direct the genetic diagnosis. The parents had another pregnancy after the biochemical diagnosis was established, but before the identification of the genetic defect. Because of the potentially high recurrence risk, and given the importance of early CoQ10 supplementation, we decided to treat with CoQ10 the newborn child pending the results of the biochemical assays. Clinicians should consider a similar management in siblings of patients with CoQ10 deficiency without a genetic diagnosis.