Interplay between gut microbiota, bone health and vascular calcification in chronic kidney disease.

Deregulations in gut microbiota may play a role in vascular and bone disease in chronic kidney disease (CKD). As glomerular filtration rate declines, the colon becomes more important as a site of excretion of urea and uric acid, and an increased bacterial proteolytic fermentation alters the gut microbial balance. A diet with limited amounts of fibre, as well as certain medications (eg phosphate binders, iron supplementation, antibiotics) further contribute to changes in gut microbiota composition among CKD patients. At the same time, both vascular calcification and bone disease are common in patients with advanced kidney disease. This narrative review describes emerging evidence on gut dysbiosis, vascular calcification, bone demineralization and their interrelationship termed the 'gut-bone-vascular axis' in progressive CKD. The role of diet, gut microbial metabolites (ie indoxyl sulphate, p-cresyl sulphate, trimethylamine N-oxide (TMAO) and short-chain fatty acids (SCFA)), vitamin K deficiency, inflammatory cytokines and their impact on both bone health and vascular calcification are discussed. This framework may open up novel preventive and therapeutic approaches targeting the microbiome in an attempt to improve cardiovascular and bone health in CKD.

Exhaled Hydrogen as a Marker of Intestinal Fermentation Is Associated with Diarrhea in Kidney Transplant Recipients.

BACKGROUND: Diarrhea is common among kidney transplant recipients (KTR). Exhaled hydrogen (H2) is a surrogate marker of small bowel dysbiosis, which may drive diarrhea. We studied the relationship between exhaled H2 and diarrhea in KTR, and explored potential clinical and dietary determinants. METHODS: Clinical, laboratory, and dietary data were analyzed from 424 KTR participating in the TransplantLines Biobank and Cohort Study (NCT03272841). Fasting exhaled H2 concentration was measured using a model DP Quintron Gas Chromatograph. Diarrhea was defined as fast transit time (types 6 and 7 according to the Bristol Stool Form Scale, BSFS) of 3 or more episodes per day. We studied the association between exhaled H2 and diarrhea with multivariable logistic regression analysis, and explored potential determinants using linear regression. RESULTS: KTR (55.4 ± 13.2 years, 60.8% male, mean eGFR 49.8 ± 19.1 mL/min/1.73 m2) had a median exhaled H2 of 11 (5.0-25.0) ppm. Signs of small intestinal bacterial overgrowth (exhaled H2 ≥ 20 ppm) were present in 31.6% of the KTR, and 33.0% had diarrhea. Exhaled H2 was associated with an increased risk of diarrhea (odds ratio 1.51, 95% confidence interval 1.07-2.14 per log2 ppm, p = 0.02). Polysaccharide intake was independently associated with higher H2 (std. ß 0.24, p = 0.01), and a trend for an association with proton-pump inhibitor use was observed (std. ß 0.16 p = 0.05). CONCLUSION: Higher exhaled H2 is associated with an increased risk of diarrhea in KTR. Our findings set the stage for further studies investigating the relationship between dietary factors, small bowel dysbiosis, and diarrhea after kidney transplantation.

Ppia is the most stable housekeeping gene for qRT-PCR normalization in kidneys of three Pkd1-deficient mouse models.

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is the most common inherited renal disorder, characterized by renal cyst development leading to end-stage renal disease. Although the appropriate choice of suitable reference is critical for quantitative RNA analysis, no comparison of frequently used "housekeeping" genes is available. Here, we determined the validity of 7 candidate housekeeping genes (Actb, Actg1, B2m, Gapdh, Hprt, Pgam1 and Ppia) in kidney tissues from mouse models orthologous to ADPKD, including a cystic mice (CY) 10-12 weeks old (Pkd1flox/flox:Nestincre/Pkd1flox/-:Nestincre, n = 10) and non-cystic (NC) controls (Pkd1flox/flox/Pkd1flox/-, n = 10), Pkd1-haploinsufficient (HT) mice (Pkd1+/-, n = 6) and wild-type (WT) controls (Pkd1+/+, n = 6) and a severely cystic (SC) mice 15 days old (Pkd1V/V, n = 7) and their controls (CO, n = 5). Gene expression data were analyzed using six distinct statistical softwares. The estimation of the ideal number of genes suggested the use of Ppia alone as sufficient, although not ideal, to analyze groups altogether. Actb, Hprt and Ppia expression profiles were correlated in all samples. Ppia was identified as the most stable housekeeping gene, while Gapdh was the least stable for all kidney samples. Stat3 expression level was consistent with upregulation in SC compared to CO when normalized by Ppia expression. In conclusion, present findings identified Ppia as the best housekeeping gene for CY + NC and SC + CO groups, while Hprt was the best for the HT + WT group.

Urine and stone analysis for the investigation of the renal stone former: a consensus conference.

The Consensus Group deliberated on a number of questions concerning urine and stone analysis over a period of months, and then met to develop consensus. The Group concluded that analyses of urine and stones should be routine in the diagnosis and treatment of urinary stone diseases. At present, the 24-h urine is the most useful type of urine collection, and accepted methods for analysis are described. Patient education is also important for obtaining a proper urine sample. Graphical methods for reporting urine analysis results can be helpful both for the physician and for educating the patient as to proper dietary changes that could be beneficial. Proper analysis of stones is also essential for diagnosis and management of patients. The Consensus Group also agreed that research has shown that evaluation of urinary crystals could be very valuable, but the Group also recognizes that existing methods for assessment of crystalluria do not allow this to be part of stone treatment in many places.

Phyllanthus niruri as a promising alternative treatment for nephrolithiasis.

In spite of considerable efforts to identify effective treatments for urolithiasis, this is a goal yet to be achieved. This review summarizes experimental and clinical data evaluating the effect of the plant Phyllanthus niruri, a plant with worldwide distribution, as a potential agent to prevent and/or to treat urolithiasis The review is based on data from the literature and on the results obtained by our group from either in vivo/in vitro experiments or clinical studies. Phyllanthus niruri has been shown to interfere with many stages of stone formation, reducing crystals aggregation, modifying their structure and composition as well as altering the interaction of the crystals with tubular cells leading to reduced subsequent endocytosis. The clinical beneficial effects of Phyllanthus niruri may be related to ureteral relaxation, helping to eliminate calculi or to clear fragments following lithotripsy, or also to a putative reduction of the excretion of urinary crystallization promoters such as calcium. No adverse renal, cardiovascular, neurological or toxic effects have been detected in either of these studies. Altogether, these studies suggest a preventive effect of Phyllanthus niruri in stone formation or elimination, but still longer-term randomized clinical trials are necessary to confirm its therapeutic properties.

Dietary Recommendations for Bariatric Patients to Prevent Kidney Stone Formation.

Bariatric surgery (BS) is one of the most common and efficient surgical procedures for sustained weight loss but is associated with long-term complications such as nutritional deficiencies, biliary lithiasis, disturbances in bone and mineral metabolism and an increased risk of nephrolithiasis, attributed to urinary metabolic changes resultant from low urinary volume, hypocitraturia and hyperoxaluria. The underlying mechanisms responsible for hyperoxaluria, the most common among all metabolic disturbances, may comprise increased intestinal oxalate absorption consequent to decreased calcium intake or increased dietary oxalate, changes in the gut microbiota, fat malabsorption and altered intestinal oxalate transport. In the current review, the authors present a mechanistic overview of changes found after BS and propose dietary recommendations to prevent the risk of urinary stone formation, focusing on the role of dietary oxalate, calcium, citrate, potassium, protein, fat, sodium, probiotics, vitamins D, C, B6 and the consumption of fluids.

Searching for mutations in the HNF1B gene in a Brazilian cohort with renal cysts and hyperglycemia.

OBJECTIVE: To verify the presence of variants in HNF1B in a sample of the Brazilian population selected according to the presence of renal cysts associated with hyperglycemia. SUBJECTS AND METHODS: We evaluated 28 unrelated patients with clinical suspicion of HNF1B mutation because of the concomitant presence of diabetes mellitus (DM) or prediabetes and renal cysts. Genotyping was accomplished using Sanger sequencing or multiplex ligation-dependent probe amplification (MLPA). In positive cases, available relatives were recruited. RESULTS: We found two patients with HNF1B mutations. The first presented the variant p.Pro328Leufs*48(c.983delC) and had DM, renal cysts, and hypomagnesemia. The second presented a heterozygous whole gene deletion in HNF1B, DM, renal cysts, body and tail pancreatic agenesis, and hypomagnesemia; this alteration was also found in his two siblings and his father. CONCLUSION: The recruitment of suspected cases of HNF1B gene mutations in Brazilians due to hyperglycemia and renal cysts presents two positive cases. Our cases contribute to the annotation of clinical and biochemical phenotypes of this rare form of maturity-onset diabetes of the young (MODY).

Kidneys and women's health: key challenges and considerations.

The theme of World Kidney Day 2018 is 'kidneys and women's health: include, value, empower'. To mark this event, Nature Reviews Nephrology asked four leading researchers to discuss key considerations related to women's kidney health, including specific risk factors, as well as the main challenges and barriers to care for women with kidney disease and how these might be overcome. They also discuss policies and systems that could be implemented to improve the kidney health of women and their offspring and the areas of research that are needed to improve the outcomes of kidney disease in women.

Defining metabolic activity of nephrolithiasis - Appropriate evaluation and follow-up of stone formers.

Considering the variation in metabolic evaluation and medical management of kidney stone disease, this consensus review was created to discuss the metabolic activity of nephrolithiasis, define the difference between single and recurrent stone formers, and develop a schema for metabolic and radiologic follow-up. A systematic review of the literature was performed to identify studies of metabolic evaluation and follow-up of patients with nephrolithiasis. Both single and recurrent stone formers share many similarities in metabolic profiles. The study group determined that based on an assessment of risk for stone recurrence and metabolic activity, single and recurrent stone formers should be evaluated comprehensively, including two 24 h urine studies on a random diet. Targeted medication and dietary recommendations are effective for many patients in reducing the risk of stone recurrence. Follow-up of those with stone disease should be obtained depending on the level of metabolic activity of the patient, the risk of chronic kidney disease and the risk of osteoporosis/osteopenia. A standard scheme includes a baseline metabolic profile, a repeat study 3-6 months after initiation of treatment, and then yearly when stable, with abdominal imaging obtained every 1-2 years.

Fish Oil Supplementation Reduces Inflammation but Does Not Restore Renal Function and Klotho Expression in an Adenine-Induced CKD Model.

BACKGROUND: Chronic kidney disease and inflammation promote loss of Klotho expression. Given the well-established anti-inflammatory effects of omega-3 fatty acids, we aimed to investigate the effect of fish oil supplementation in a model of CKD. METHODS: Male C57BL/6 mice received supplementation with an adenine-enriched diet (AD, n = 5) or standard diet (CTL, n = 5) for 10 days. Two other experimental groups were kept under the adenine diet for 10 days. Following adenine withdrawal on the 11th day, the animals returned to a standard diet supplemented with fish oil (Post AD-Fish oil, n = 9) or not (Post AD-CTL, n = 9) for an additional period of 7 days. RESULTS: Adenine mice exhibited significantly higher mean serum urea, creatinine, and renal expression of the pro-inflammatory markers Interleukin-6 (IL-6), C-X-C motif chemokine 10 (CXCL10), and Interleukin-1ß (IL-1ß), in addition to prominent renal fibrosis and reduced renal Klotho gene expression compared to the control. Post AD-Fish oil animals demonstrated a significant reduction of IL-6, C-X-C motif chemokine 9 (CXCL9), and IL-1ß compared to Post AD-CTL animals. However, serum creatinine, renal fibrosis, and Klotho were not significantly different in the fish oil-treated group. Furthermore, renal histomorphological changes such as tubular dilatation and interstitial infiltration persisted despite treatment. CONCLUSIONS: Fish oil supplementation reduced renal pro-inflammatory markers but was not able to restore renal function nor Klotho expression in an adenine-induced CKD model.

Hyperoxaluria in a Model of Mini-Gastric Bypass Surgery in Rats.

BACKGROUND: Bariatric surgery is associated with hyperoxaluria hence predisposing to nephrolithiasis. The present study aimed to investigate the underlying mechanisms contributing to increased urinary oxalate in a mini-gastric bypass (MGB) surgery model in rats under different dietary conditions. The expression of intestinal oxalate transporters was also evaluated. METHODS: Male rats underwent MGB (n = 21) or Sham procedure (n = 21) and after recovery were fed a standard or high-fat diet with or without oxalate for 8 weeks. Stool and urine were collected before surgery (baseline) and at the end of protocol (final), when intestinal fragments were harvested for expression of Slc26a3 and Slc26a6 oxalate transporters. RESULTS: MGB groups fed with fat, irrespective of oxalate supplementation, presented steatorrhea. In MGB animals fed with fat and oxalate (Fat + Ox), final values of urinary oxalate and calcium oxalate supersaturation risk were markedly and significantly increased versus baseline or Sham animals under the same diet, as well as MGB groups under other diets. Slc26a3 was decreased in biliopancreatic limbs of MGB rats, probably reflecting a physiological adaptation to the restriction of food passage. Slc26a6 was not altered in any harvested intestinal fragment. CONCLUSIONS: A high-fat and oxalate diet induced hyperoxaluria and elevation in calcium oxalate supersaturation risk in a MGB rat model. The presence of fat malabsorption and increased dietary oxalate absorption, but not modifications of Slc26a3 and Slc26a6 oxalate transporters, accounted for these findings, suggesting that bariatric patients may benefit from a low-fat and low-oxalate diet.

Effects of creatine supplementation on body composition and renal function in rats.

BACKGROUND: The aim of the present study was to evaluate the long-term effects of oral creatine supplementation on renal function and body composition (fat and lean mass) in an experimental model. METHODS: Male Wistar rats were supplemented with creatine (2 g.kg(-1) of food) for 10 wk in combination with treadmill exercise, 12 m.min(-1), 1 h.d(-1) (CREAT + EX, N = 12) or not (CREAT, N = 10), and compared with exercised animals without creatine supplementation (EX, N = 7) and CONTROL animals, N = 7. Body composition and bone mineral density (BMD) were determined by dual x-ray absorptiometry and glomerular filtration rate (GFR) and renal plasma flow (RPF) were measured by inulin and paraaminohippurate clearance, respectively. RESULTS: At the end of the study (post), CREAT+EX presented higher lean mass and lower fat mass than CREAT, EX or CONTROL (349.7 +/- 19.7 vs 313.3 +/- 20.3, 311.9 +/- 30.8, 312.4 +/- 21.0 g and 5.7 +/- 2.3 vs 10.0 +/- 3.3, 9.8 +/- 1.5, 10.0 +/- 3.5%, P < 0.05, respectively). Post lean/fat mass ratio was higher than baseline only in CREAT + EX (18.9 +/- 7.2 vs 8.6 +/- 1.8, P < 0.05). Post BMD was significantly higher than baseline in all groups. GFR and RPF were lower in CREAT versus CONTROL (0.5 +/- 0.1 vs 1.0 +/- 0.1 and 1.5 +/- 0.2 vs 2.4 +/- 0.5 mL.min(-1), P < 0.05, respectively). CONCLUSION: Creatine supplement in combination with exercise increased the proportion of lean mass more than EX or CREAT alone. The use of creatine alone induced an important and significant reduction of both RPF and GFR.

Stop dietary calcium restriction in kidney stone-forming patients.

Epidemiologic studies have demonstrated an increased incidence of kidney stones in individuals with low calcium intake. More recently, a 5-year clinical study found the recurrence of kidney stones was higher in stone-formers on a low dietary calcium treatment. Considering these important new findings, and the known adverse effect on bone mass, dietary calcium restriction should be avoided in kidney stone-forming individuals.

Searching for mutations in the HNF1B gene in a Brazilian cohort with renal cysts and hyperglycemia

ABSTRACT Objective To verify the presence of variants in HNF1B in a sample of the Brazilian population selected according to the presence of renal cysts associated with hyperglycemia. Subjects and methods We evaluated 28 unrelated patients with clinical suspicion of HNF1B mutation because of the concomitant presence of diabetes mellitus (DM) or prediabetes and renal cysts. Genotyping was accomplished using Sanger sequencing or multiplex ligation-dependent probe amplification (MLPA). In positive cases, available relatives were recruited. Results We found two patients with HNF1B mutations. The first presented the variant p.Pro328Leufs*48(c.983delC) and had DM, renal cysts, and hypomagnesemia. The second presented a heterozygous whole gene deletion in HNF1B, DM, renal cysts, body and tail pancreatic agenesis, and hypomagnesemia; this alteration was also found in his two siblings and his father. Conclusion The recruitment of suspected cases of HNF1B gene mutations in Brazilians due to hyperglycemia and renal cysts presents two positive cases. Our cases contribute to the annotation of clinical and biochemical phenotypes of this rare form of maturity-onset diabetes of the young (MODY).

Optimum nutrition for kidney stone disease.

We summarize the data regarding the associations of individual dietary components with kidney stones and the effects on 24-hour urinary profiles. The therapeutic recommendations for stone prevention that result from these studies are applied where possible to stones of specific composition. Idiopathic calcium oxalate stone-formers are advised to reduce ingestion of animal protein, oxalate, and sodium while maintaining intake of 800 to 1200 mg of calcium and increasing consumption of citrate and potassium. There are few data regarding dietary therapy of calcium phosphate stones. Whether the inhibitory effect of citrate sufficiently counteracts increasing urine pH to justify more intake of potassium and citrate is not clear. Reduction of sodium intake to decrease urinary calcium excretion would also be expected to decrease calcium phosphate stone recurrence. Conversely, the most important urine variable in the causation of uric acid stones is low urine pH, linked to insulin resistance as a component of obesity and the metabolic syndrome. The mainstay of therapy is weight loss and urinary alkalinization provided by a more vegetarian diet. Reduction in animal protein intake will reduce purine ingestion and uric acid excretion. For cystine stones, restriction of animal protein is associated with reduction in intake of the cystine precursor methionine as well as cystine. Reduction of urine sodium results in less urine cystine. Ingestion of vegetables high in organic anion content, such as citrate and malate, should be associated with higher urine pH and fewer stones because the amino acid cystine is soluble in more alkaline urine. Because of their infectious origin, diet has no definitive role for struvite stones except for avoiding urinary alkalinization, which may worsen their development.

Evaluation of nephrolithiasis in autosomal dominant polycystic kidney disease patients.

BACKGROUND AND OBJECTIVES: Nephrolithiasis (LIT) is more prevalent in patients with autosomal dominant polycystic kidney disease (ADPKD) than in the general population. Renal ultrasonography may underdetect renal stones because of difficulties imposed by parenchymal and/or cyst wall calcifications. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: A total of 125 patients with ADPKD underwent ultrasonography and unenhanced computed tomography (CT) scan, routine blood chemistry, and spot and 24-h urine collections. RESULTS: CT scan detected calculi in 32 patients, including 20 whose previous ultrasonography revealed no calculi. The percentage of hypocitraturia was high but not statistically different between patients with ADPKD+LIT or ADPKD. Hyperuricosuria and distal renal tubular acidosis were less prevalent but also did not differ between groups, whereas hyperoxaluria was significantly higher in the former. Hypercalciuria was not detected. Renal volume was significantly higher in patients with ADPKD+LIT versus ADPKD, and a stepwise multivariate logistic regression analysis showed that a renal volume >or=500 ml was a significant predictor of LIT in patients with ADPKD and normal renal function, after adjustments for age and hypertension. CONCLUSIONS: CT scan was better than ultrasonography to detect LIT in patients with ADPKD. Larger kidneys from patients with ADPKD were more prone to develop stones, irrespective of the presence of metabolic disturbances.

Bone disease in idiopathic hypercalciuria.

PURPOSE OF REVIEW: Decreased bone mineral density and increased prevalence of bone fractures have been found in patients with idiopathic hypercalciuria. The purpose of this review is to summarize the recent published evidence that supports a potential role of the bone, and its link to the kidney and intestine, in the pathogenesis of idiopathic hypercalciuria. The effects of hypercalciuria on bone and the implications for treatment are also reviewed. RECENT FINDINGS: Evidence suggests that the incidence of a first fracture in kidney stone patients is fourfold higher than the control population. Support for the role of bone in the pathophysiology of hypercalciuria has been corroborated. New studies have detailed the effects of several cytokines - increased number and sensitivity of vitamin D receptors, and increased acid production - upon the bone acting cells. Similarly, recent clinical and experimental studies have suggested that genetic factors confer a predisposition to the formation of renal calcium stones and bone demineralization. SUMMARY: Whether hypercalciuria is the result of a primary bone disorder, a consequence of a persisting negative calcium balance or a combination of both still remains to be determined. Nevertheless, bone status must be evaluated and followed up in patients with idiopathic hypercalciuria.

Immobilization and hypercalciuria in children.

Intermediate-term immobilization may lead to an increase in serum and urinary calcium. In order to test this hypothesis, we evaluated 46 children, 21 with Legg-Calvé-Perthes disease (LCP; 7.2+/-1.8 years old) and 25 with developmental dysplasia of the hip joint (DDH; 10+/-5 months of age), submitted to immobilization for up to 16 weeks. These two conditions require intermediate-term immobilization as treatment modality, and no studies evaluating calcium metabolism in these groups of patients have been conducted. In LCP patients, blood and 24-h urine samples were obtained before the beginning of treatment and after 1, 6, 8, 14 and 16 weeks of immobilization, while in DDH patients, blood and spot urine samples were collected before treatment and after 6 and 14 weeks of treatment. Urinary calcium, creatinine, potassium and sodium as well as serum calcium, phosphorus, parathyroid hormone, creatinine and alkaline phosphatase were determined in those samples. Renal ultrasound was performed before and after treatment. A mean increase of 2.3 times baseline values of urinary calcium was observed in 40% of previously normocalciuric LCP patients after only 1 week of immobilization. Among the DDH children, who had never previously ambulated, there was no significant variation in the urinary calcium excretion. None of the serum parameters changed in either group throughout the study. Urinary stones were not evidenced by renal ultrasound. Therefore, the present data suggested that intermediate-term immobilization led to a transient increase in urinary calcium in 40% of LCP patients. Complications such as urinary stones were not observed. In conclusion, this modality of treatment does not impose an increased risk of urinary stone formation in LCP and DDH patients.

Phyllanthus niruri as a promising alternative treatment for nephrolithiasis

In spite of considerable efforts to identify effective treatments for urolithiasis, this is a goal yet to be achieved. This review summarizes experimental and clinical data evaluating the effect of the plant Phyllanthus niruri, a plant with worldwide distribution, as a potential agent to prevent and/or to treat urolithiasis The review is based on data from the literature and on the results obtained by our group from either in vivo/in vitro experiments or clinical studies. Phyllanthus niruri has been shown to interfere with many stages of stone formation, reducing crystals aggregation, modifying their structure and composition as well as altering the interaction of the crystals with tubular cells leading to reduced subsequent endocytosis. The clinical beneficial effects of Phyllanthus niruri may be related to ureteral relaxation, helping to eliminate calculi or to clear fragments following lithotripsy, or also to a putative reduction of the excretion of urinary crystallization promoters such as calcium. No adverse renal, cardiovascular, neurological or toxic effects have been detected in either of these studies. Altogether, these studies suggest a preventive effect of Phyllanthus niruri in stone formation or elimination, but still longer-term randomized clinical trials are necessary to confirm its therapeutic properties.

Infecçäo do trato urinário no pós-transplante renal em crianças / Urinary tract infecction after renal transplant in children

O presente estudo teve como objetivo avaliar os aspectos clínicos-laboratoriais e os fatores de risco encolvidos na UTI em crianças transplantadas renais. Fatores de risco relacionados à infecçäodo trato urinário(ITU) no período de pós-transplante renal, tais como:sexo, idade, tipo de doador, doença de base, tempo detratamento dialítico prévio ao transplante e nível deimunossupressäo foram avaliados em 62 crianças. Foramtambém analisados perda do enxerto e funçäo renal após ITU. A ITU foi identificada em 20/62 (32 porcento) dos pacientes, observando-se recorrência em 45 porcento dos casos, 25 porcento dos quais reinfecçöes. Em 84 porcento dos casos, as ITU foram assintomáticas. A bactéria mais frequêntemente isolada foi Escherichia coli, tanto nos 63 porcentodos episódios de ITU domiciliar, quanto nos 37 porcento de ITU hospitalar. As 20 crianças apresentaram 38 episódios de ITU, sendo 60 porcento no período precose (até três meses após-transplante) e 40 porcento no período tardio. Em resumo, os autores abservaram maior frequência de ITU precose no pós-tansplante näo associada a idade, sexo, tipo de doador, tempo prévio em diáis, doença de base e perda da funçä renal. Crianças com ITU näo evoluíram com maior taxa d perda do enxerto em relaçäo ao grupo controle durante o período avaliado (AU>