Impact of exercise training associated with enalapril treatment on blood pressure variability and renal dysfunctions in an experimental model of arterial hypertension and postmenopause.

OBJECTIVE: In this study, we aimed to investigate the effects of the concurrent exercise training (CET) associated with the enalapril maleate on blood pressure variability (BPV) and renal profile in an experimental model of arterial hypertension (AH) and postmenopause. METHODS: Female ovariectomized spontaneously hypertensive rats (SHR) were distributed into 4 groups (n = 8/group): sedentary (SO), sedentary + enalapril (SOE), trained (TO) and trained + enalapril (TOE). Both enalapril (3mg/kg) and CET (3 days/week) were conducted during 8 weeks. Blood pressure (BP) was directly recorded for BPV analyses. Renal function, morphology, inflammation and oxidative stress were assessed. RESULTS: The SOE, TO e TOE groups presented decreased systolic BP compared with SO. Both trained groups (TO and TOE) presented lower BPV and increased baroreflex sensitivity (TO: 0.76 ± 0.20 and TOE: 1.02 ± 0.40 vs. SO: 0.40 ± 0.07 ms/mmHg) compared with SO group, with additional improvements in TOE group. Creatinine and IL-6 levels were reduced in SOE, TO and TOE compared with SO group, while IL-10 was increased only in TOE group (vs. SO). Enalapril combined with CET promote reduction in lipoperoxidation (TOE: 1.37 ± 0.26 vs. SO: 2.08 ± 0.48 and SOE: 1.84 ± 0.35 µmol/mg protein) and hydrogen peroxide (TOE: 1.89 ± 0.40 vs. SO: 3.70 ± 0.19 and SOE: 2.73 ± 0.70 µM), as well as increase in catalase activity (vs. sedentary groups). The tubulointerstitial injury was lower in interventions groups (SOE, TO and TOE vs. SO), with potentialized benefits in the trained groups. CONCLUSIONS: Enalapril combined with CET attenuated BPV and baroreflex dysfunctions, probably impacting on end-organ damage, as demonstrated by attenuation in the AH-induced renal inflammations, oxidative stress and morphofunctional impairments in postmenopausal rats.

Sex modifies the renal consequences of high fructose consumption introduced after weaning.

After lactation, many children consume fructose-rich processed foods. However, overconsumption of these foods can predispose individuals to non-communicable chronic diseases, which can have different repercussions depending on the sex. Thus, we evaluated the effects of fructose overload introduced after weaning on the renal function of young rats of both sexes. Methods: After weaning, male and female offspring of Wistar rats were assigned to drink water (the male/water and female/water groups) or 20% D-fructose solution (male/fructose and female/fructose groups). Food and water or fructose solution was offered ad libitum. Rats were evaluated at 4 months. Parameters analyzed: blood pressure, body weight, triglyceride levels, glomerular filtration rate, sodium, potassium, calcium, and magnesium excretion, macrophage infiltration, and eNOS and 8OHdG expression in renal tissue. CEUA-UNIFESP: 2757270117. Results: Fructose intake affected the blood pressure, body weight, and plasma triglyceride in all rats. Glomerular filtration rate was significantly reduced in males that received fructose when compared to that of the control group. Sodium and potassium excretion decreased in all fructose-treated rats; however, the excreted load of these ions was significantly higher in females than in males. In the female control group, calcium excretion was higher than that of the male control group. Fructose overload increased magnesium excretion in females, and also increased macrophage infiltration and reduced eNOS expression in both males and females. Conclusion: Fructose overload introduced after weaning caused metabolic and renal changes in rats. Renal function was more affected in males; however, several significant alterations were also observed in the female-fructose group.

Disfunção autonômica cardiovascular e dano renal: papel da inflamação e do estresse oxidativo / Cardiovascular autonomic dysfunction e renal damage: role of inflammation and oxidative stress

Evidências demonstram que não só a elevação da pressão arterial, mas também o aumento da variabilidade da pressão arterial (VPA) pode contribuir para a piora no dano renal e redução da sobrevida em portadores doença renal crônica. Este artigo tem como objetivo revisar o impacto da disfunção autonômica cardiovascular (hiperatividade simpática, prejuízo nasensibilidade dos barorreceptores e/ou aumento da VPA) e sua associação com inflamação e estresse oxidativo no desenvolvimento e progressão do dano renal (AU).

Evidence shows that not only the increase in blood pressure, but also the increase in blood pressure variability (BPV) can contribute to worsening kidney damage and reduced survival in patients with chronic kidney disease. This article aims to review the impact of cardiovascular autonomic dysfunction (sympathetic hyperactivity, impaired baroreflex sensitivity and/or increased BPV) and its association with inflammation and oxidative stress on the development and progression of renal damage (AU).

Effects of maternal fructose intake on the offspring's kidneys.

Fructose overload is associated with cardiovascular and metabolic disorders. During pregnancy, these alterations may affect the maternal environment and predispose offspring to diseases. AIMS: To evaluate the renal morphology and function of offspring of dams that received fructose overload during pregnancy and lactation. METHODS: Female Wistar rats were divided into the control (C) and fructose (F) groups. C received food and water ad libitum, and F received food and d-fructose solution (20%) ad libitum. The d-fructose offer started 1 week before mating and continued during pregnancy and lactation. The progeny were designated as control (C) or fructose (F); after weaning, half of the F received water to drink (FW), and half received d-fructose (FF). Blood pressure (BP) and renal function were evaluated. The expression of sodium transporters (NHE3-exchanger, NKCC2 and NCC-cotransporters, and ENaC channels) and markers of renal dysfunction, including ED1 (macrophage), eNOS, 8OHdG (oxidative stress), renin, and ACE 1 and 2, were evaluated. CEUA-UNIFESP: 2757270117. The FF group presented with reduced glomerular filtration rate and urinary osmolarity, increased BP, proteinuria, glomerular hypertrophy, macrophage infiltration, and increased expression of transporters (NHE3, NCC, and ENaC), 8OHdG, renin, and ACE1. The FW group did not show increased BP and renal functional alterations; however, it presented glomerular hypertrophy, macrophage infiltration, and increased expression of the transporters (NHE3, NKCC2, NCC, and ENaC), renin, and ACE1. These data suggest that fructose overload during fetal development alters renal development, resulting in the increased expression of renin, ACE1, and sodium transporters, thus predisposing to hypertension and renal dysfunction.

Cross-sex testosterone therapy modifies the renal morphology and function in female rats and might underlie increased systolic pressure.

Testosterone esters are hormones commonly used for affirming gender identity in transmen. The present study evaluates the effect of testosterone on renal morphology and function in an animal model submitted to cross-sex hormone therapy used for transmen. Two-month-old Wistar rats were divided into three groups: male control (MC), female control (FC), and female on testosterone therapy (FTT). The FTT group received testosterone cypionate (3.0 mg/kg, i.m.), and the MC and MF groups received vehicle oil every 10 days for 4 months. Renal function and indirect systolic blood pressure (SBP) measurements were evaluated at 6 months of age. Plasma and urine concentrations of urea, creatinine, sodium, potassium, osmolality, and glomerular filtration rate (GFR) were measured. The kidneys were weighed, paraffin-embedded, and histological sections were prepared to evaluate the glomerular area. We verified that the FTT group, in comparison to FC, had increased kidney weight [MC, 3.2 ± 0.05; FC, 1.8 ± 0.04; FTT, 2.2 ± 0.06; g], decreased urine osmolarity [MC, 486.9 ± 18.3; FC, 1012.0 ± 5.4; FTT, 768.2 ± 40.3 mOsm/L/g kw], reduced GFR [MC, 0.77 ± 0.04; FC, 0.78 ± 0.02; FTT, 0.67 ± 0.03; mL/min/g kw], larger glomerular area [MC, 9334 ± 120.8; FC, 7884 ± 112.8; FTT, 9078 ± 133.4 µm2 ], and higher SBP [MC, 126 ± 3.4; FC, 119 ± 1.0; FTT, 131 ± 1.4; mmHg]. Sodium excretion was higher in FC and FTT in comparison to MC [MC, 0.34 ± 0.05; FC, 0.56 ± 0.06; FTT, 0.54 ± 0.04; mEq/24 h/g kw]. Cross-sex hormone therapy with testosterone in female rats induces renal morphofunctional changes and may underlie increased systolic pressure, suggesting an adaptation similar to what is observed in transmen on long-term testosterone therapy.

Programmed Adult Kidney Disease: Importance of Fetal Environment.

The Barker hypothesis strongly supported the influence of fetal environment on the development of chronic diseases in later life. Multiple experimental and human studies have identified that the deleterious effect of fetal programming commonly leads to alterations in renal development. The interplay between environmental insults and fetal genome can induce epigenetic changes and lead to alterations in the expression of renal phenotype. In this review, we have explored the renal development and its functions, while focusing on the epigenetic findings and functional aspects of the renin-angiotensin system and its components.

Effects of sleep restriction during pregnancy on lipids and glucose homeostasis of female offspring submitted to ovariectomy.

Sleep shortening during pregnancy may alter the mother's environment, affecting the offspring. Thus, the present study evaluated the metabolic profile of female offspring from sleep-restricted rats during the last week of pregnancy. Pregnant Wistar rats were distributed into two groups: control (C) and sleep restriction (SR). The SR was performed 20 h/day, from 14th to 20th day of pregnancy. At 2 months, half of the offspring were subjected to ovariectomy (OVX); the others, to sham surgery. Studied groups were Csham, Covx, SRsham and SRovx. Cholesterol (HDL, LDL and C-total), triglycerides (TG) and glucose and insulin tolerance tests (GTT-ITT) were evaluated at 8 months. RSsham presented higher values of TG, while SRovx presented higher TG, LDL and C-total. Basal glucose concentration was increased in SRsham and SRovx. These data suggest that SR during pregnancy may be a risk factor for the development of diseases in adult female offspring.

Effect of maternal diabetes on female offspring / Efeito do diabetes materno na prole feminina

Objective To evaluate the effect of maternal diabetes on the blood pressure and kidney function of female offspring, as well as if such changes exacerbate during pregnancy. Methods Diabetes mellitus was induced in female rats with the administration of streptozotocin in a single dose, one week before mating. During pregnancy, blood pressure was measured through plethysmography. On the 20th day of pregnancy, the animals were placed for 24 hours in metabolic cages to obtain urine samples. After the animals were removed from the cages, blood samples were withdrawn. One month after pregnancy, new blood and urine sample were collected. Kidney function was evaluated through proteinuria, plasma urea, plasma creatinine, creatinine excretion rate, urinary flow, and creatinine clearance. Results The female offspring from diabetic mothers showed an increase in blood pressure, and a decrease in glomerular filtration rate in relation to the control group. Conclusion Hyperglycemia during pregnancy was capable of causing an increase in blood pressure and kidney dysfunction in the female offspring. .

Objetivo Avaliar o efeito do diabetes materno sobre a pressão arterial e a função renal da prole feminina, bem como verificar se as alterações observadas se exacerbam durante a prenhez. Métodos O diabetes mellitus foi induzido em ratas com a administração de estreptozocina em dose única, uma semana antes do cruzamento. Durante a prenhez, foram feitas medidas da pressão arterial por pletismografia. No 20o dia da prenhez, os animais foram colocados durante 24 horas em gaiolas metabólicas para obtenção de amostras de urina. Após a retirada dos animais das gaiolas, foram obtidas amostras de sangue. Um mês após a prenhez, foram obtidas novas amostras de sangue e urina para as determinações. A função renal foi avaliada por meio de proteinúria, ureia plasmática, creatinina plasmática, carga excretada de creatinina, fluxo urinário e clearance de creatinina. Resultados As fêmeas da prole de mães diabéticas apresentaram elevação da pressão arterial e redução do ritmo de filtração glomerular em relação ao grupo controle. Conclusão A hiperglicemia durante a gestação foi capaz de causar elevação da pressão arterial e disfunção renal na prole de sexo feminino. .

Effect of maternal diabetes on female offspring.

OBJECTIVE: To evaluate the effect of maternal diabetes on the blood pressure and kidney function of female offspring, as well as if such changes exacerbate during pregnancy. METHODS: Diabetes mellitus was induced in female rats with the administration of streptozotocin in a single dose, one week before mating. During pregnancy, blood pressure was measured through plethysmography. On the 20th day of pregnancy, the animals were placed for 24 hours in metabolic cages to obtain urine samples. After the animals were removed from the cages, blood samples were withdrawn. One month after pregnancy, new blood and urine sample were collected. Kidney function was evaluated through proteinuria, plasma urea, plasma creatinine, creatinine excretion rate, urinary flow, and creatinine clearance. RESULTS: The female offspring from diabetic mothers showed an increase in blood pressure, and a decrease in glomerular filtration rate in relation to the control group. CONCLUSION: Hyperglycemia during pregnancy was capable of causing an increase in blood pressure and kidney dysfunction in the female offspring.

Myeloid-derived suppressor cells and associated events in urethane-induced lung cancer.

OBJECTIVES: Myeloid-derived suppressor cells contribute to the immunosuppressive microenvironment during tumor development and limit the efficacy of cancer immunotherapy. Identifying myeloid-derived suppressor cells and associated factors is the first step in creating strategies to reverse the suppressive effects of these cells on the immune system. METHODS: To induce lung cancer, we administered 2 doses of urethane to BALB/c mice and observed these animals for 120 days. After this period, we evaluated the percentage of myeloid-derived suppressor cells in the blood, lung and bone marrow. The expression of alpha-smooth muscle actin, transforming growth factor-ß, Toll-like receptor 2, Toll-like receptor 4, and interleukin-6 was also determined in the lung tissue. RESULTS: Myeloid-derived suppressor cells were increased in all evaluated tissues after lung cancer development in association with increased Toll-like receptor 4 expression and decreased interleukin-6 expression in the lung. We observed alpha-smooth muscle actin and transforming growth factor-ß expression in lung nodules. CONCLUSIONS: We believe that the early diagnosis of cancer through determining the blood levels of myeloid-derived suppressor cells followed by the depletion of these cells should be further investigated as a possible approach for cancer treatment.

Myeloid-derived suppressor cells and associated events in urethane-induced lung cancer

OBJECTIVES: Myeloid-derived suppressor cells contribute to the immunosuppressive microenvironment during tumor development and limit the efficacy of cancer immunotherapy. Identifying myeloid-derived suppressor cells and associated factors is the first step in creating strategies to reverse the suppressive effects of these cells on the immune system. METHODS: To induce lung cancer, we administered 2 doses of urethane to BALB/c mice and observed these animals for 120 days. After this period, we evaluated the percentage of myeloid-derived suppressor cells in the blood, lung and bone marrow. The expression of alpha-smooth muscle actin, transforming growth factor-β, Toll-like receptor 2, Toll-like receptor 4, and interleukin-6 was also determined in the lung tissue. RESULTS: Myeloid-derived suppressor cells were increased in all evaluated tissues after lung cancer development in association with increased Toll-like receptor 4 expression and decreased interleukin-6 expression in the lung. We observed alpha-smooth muscle actin and transforming growth factor-β expression in lung nodules. CONCLUSIONS: We believe that the early diagnosis of cancer through determining the blood levels of myeloid-derived suppressor cells followed by the depletion of these cells should be further investigated as a possible approach for cancer treatment. .

Increased renal sympathetic nerve activity leads to hypertension and renal dysfunction in offspring from diabetic mothers.

The exposure of the fetus to a hyperglycemic environment promotes the development of hypertension and renal dysfunction in the offspring at adult age. We evaluated the role of renal nerves in the hypertension and renal changes seen in offspring of diabetic rats. Diabetes was induced in female Wistar rats (streptozotocin, 60 mg/kg ip) before mating. Male offspring from control and diabetic dams were studied at an age of 3 mo. Systolic blood pressure measured by tail cuff was increased in offspring of diabetic dams (146 ± 1.6 mmHg, n = 19, compared with 117 ± 1.4 mmHg, n = 18, in controls). Renal function, baseline renal sympathetic nerve activity (rSNA), and arterial baroreceptor control of rSNA were analyzed in anesthetized animals. Glomerular filtration rate, fractional sodium excretion, and urine flow were significantly reduced in offspring of diabetic dams. Two weeks after renal denervation, blood pressure and renal function in offspring from diabetic dams were similar to control, suggesting that renal nerves contribute to sodium retention in offspring from diabetic dams. Moreover, basal rSNA was increased in offspring from diabetic dams, and baroreceptor control of rSNA was impaired, with blunted responses to infusion of nitroprusside and phenylephrine. Thus, data from this study indicate that in offspring from diabetic mothers, renal nerves have a clear role in the etiology of hypertension; however, other factors may also contribute to this condition.

D-α -tocopherol reduces renal damage in hypertensive rats

This study investigated the beneficial effects of D-α-tocopherol supplementation in protecting against the renal morphological and functional changes caused by hypertension. Spontaneously hypertensive (SHR) and normotensive control (WKY) rats received D-α-tocopherol (80 mg/kg by gavage) or vehicle (mineral oil) every other day for 60 days, from the age of 2 months. After this treatment period, all animals were assessed for renal morphological and functional parameters. The glomerular hypertrophy, increased interlobular wall thickness and enlarged renal vascular resistance found in SHR were reduced by D-α-tocopherol treatment. Sodium and volume retention observed in SHR were also decreased by D-α-tocopherol treatment. Moreover, D-α-tocopherol supplementation significantly reduced arterial pressure in SHR but not in WKY. D-α-tocopherol also reduced the excretion of thiobarbituric acid-reactive substances (TBARS), a marker of oxidative stress, in SHR. These results suggest that D-α-tocopherol supplementation can reduce kidney damage induced by hypertension.

O presente estudo avaliou os efeitos benéficos da suplementação com D-α-tocopherol sobre as alterações morfológicas e funcionais renais causadas pela hipertensão arterial. A partir de dois meses de idade, ratos espontaneamente hipertensos (SHR) e controles normotensos (WKY) receberam D-α-tocoferol (80 mg/kg por gavagem) ou veículo (óleo mineral) em dias alternados, por 60 dias. Após este período foram avaliados os parâmetros morfofuncionais dos rins. Os animais SHR apresentaram: hipertrofia glomerular, aumento da espessura da parede das arterias interlobulares e elevação da resistência vascular renal. Estas alterações foram menores nos animais SHR suplementados com D-α-tocoferol. A retenção de sódio e de volume encontrada nos SHR também foi reduzida com o tratamento com D-α-tocoferol. Além disso, a suplementação com D-α-tocoferol reduziu significativamente a pressão arterial em SHR, mas não em WKY. Houve, também, redução da excreção de ácido tiobarbitúrico (TBARS), um marcador de estresse oxidativo, nos animais SHR tratados com D-α-tocoferol. Os resultados sugerem que suplementação com D-α-tocoferol é capaz de reduzir importantes alterações renais causadas pela hipertensão arterial.

Sleep restriction during pregnancy: hypertension and renal abnormalities in young offspring rats.

STUDY OBJECTIVES: Because the maternal environment can affect several physiological functions of the newborn, the aim of the present study was to examine the impact of sleep restriction during pregnancy on renal morphology and function in young offspring. DESIGN: Female 3-month-old Wistar rats were divided in 2 experimental groups: C (control) and SR (sleep restriction between the 14th and 20th day of pregnancy). Pregnancy was confirmed by vaginal smear. SR females were subjected to sleep restriction by the multiple platform technique for 20 h daily. After birth, only male litters (6 for each mother) were selected and designated OC (offspring from C) and OSR (offspring from SR). At 2 months of age, blood pressure (BP) was measured by tail plethysmography; at 3 months the renal plasma flow (RPF), glomerular filtration rate (GFR), glomerular area, and number of glomeruli per mm3 were evaluated. MEASUREMENTS AND RESULTS: Offspring from SR had higher systolic blood pressure than OC. In this group (OSR), we also observed significant increase in RPF and GFR, enlarged glomeruli diameter, and reduced number of glomeruli per mm3 of renal tissue. CONCLUSIONS: Our data suggest that sleep restriction during pregnancy is able to modify renal development, resulting in morphologic and functional alterations in young offspring.

Micronutrient prenatal supplementation prevents the development of hypertension and vascular endothelial damage induced by intrauterine malnutrition.

AIMS: The premise that intrauterine malnutrition plays an important role in the development of cardiovascular and renal diseases implies that these disorders can be programmed during fetal life. Here, we analyzed the hypothesis that supplementation with mixed antioxidant vitamins and essential mineral in early life could prevent later elevation of blood pressure and vascular and renal dysfunction associated with intrauterine malnutrition. MAIN METHODS: For this, female Wistar rats were randomly divided into three groups on day 1 of pregnancy: control fed standard chow ad libitum; restricted group fed 50% of the ad libitum intake and a restricted plus micronutrient cocktail group treated daily with a combination of micronutrient (selenium, folate, vitamin C and vitamin E) by oral gavage. KEY FINDINGS: In adult offspring, renal function and glomerular number were impaired by intrauterine malnutrition, and the prenatal micronutrient treatment did not prevent it. However, increased blood pressure and reduced endothelium-dependent vasodilation were prevented by the micronutrient prenatal treatment. Intrauterine malnutrition also led to reduced NO production associated with increased superoxide generation, and these parameters were fully normalized by this prenatal treatment. SIGNIFICANCE: Our current findings indicate that programming alterations during fetal life can be prevented by interventions during the prenatal period, and that disturbance in availability of both antioxidant vitamins and mineral may play a crucial role in determining the occurrence of long-term cardiovascular injury.

Effect of sodium overload on renal function of offspring from diabetic mothers.

The aim if this study was to evaluate the effect of sodium overload on blood pressure and renal function in the offspring of diabetic rat mothers. Diabetes was induced with a single dose of streptozotocin before mating. Experimental groups were control (C), offspring from diabetic mother (D), control with sodium chloride (NaCl) overload (CS), and offspring from diabetic mother submitted to NaCl overload (DS). After weaning, all groups received food ad libitum; groups C and D had water ad libitum, and CS and DS received NaCl 0.15 M as drinking water. Renal morphology and function were evaluated in 3-month-old rats. Glomerular area, macrophage infiltration, interlobular artery wall thickness, and renal vascular resistance were significantly increased in CS, D, and DS compared with C. Renal plasma flow (RPF) and glomerular filtration rate (GFR) were decreased in CS and D compared with C. In DS, GFR and fractional filtration were increased, suggesting a state of hyperfiltration. Hypertension was observed in groups D, CS, and DS from 2 months on and was more severe in DS. Our data suggest that diabetes during intrauterine development and salt overload beginning at an early age can cause hypertension and renal injury. When these conditions were associated, morphological and functional changes were much more intense, suggesting acceleration in the process of kidney injury.

The influence of L-arginine on blood pressure, vascular nitric oxide and renal morphometry in the offspring from diabetic mothers.

The present study was designed to evaluate the effects of L-arginine (L-arg) supplementation on blood pressure, vascular nitric oxide content, and renal morphometry in the adult offspring from diabetic mothers. Diabetes mellitus was induced in female rats with a single dose of streptozotocin (50 mg/kg), before mating. The offspring was divided into four groups: group C (controls); group DO (diabetic offspring); group CA (controls receiving 2% L-arg solution dissolved in 2% sucrose in the drinking water) and group DA (DO receiving the L-arg solution). Oral supplementation began after weaning and continued until the end of the experiments. In DO, hypertension was observed, from 3 mo on. In DA, pressure levels were not different from C and CA. In 6-mo-old animals, basal NO production (assessed by DAF-2) was significantly depressed in DO in comparison to controls. The NO production was significantly increased after stimulation with Ach or BK in all groups, the increase being greater in control than in DO rats. L-arg was able to improve the NO production and to prevent the glomerular hypertrophy in the DO. Our data suggest that the bioavailability of NO is reduced in the DO, because L-arg corrected both the hypertension and glomerular hypertrophy.

Maternal diabetes mellitus--early consequences for the offspring.

Although several studies have focused on the effects of nutritional status during intrauterine development, few have addressed the impact of maternal diabetes mellitus on renal function and morphology in the young offspring. In the present study, renal morpho-functional aspects were studied in the offspring of diabetic rats. Diabetes was induced in female rats with a single dose of streptozotocyn (STZ), 10 days before mating. After weaning, the offspring (DO) had free access to food and water. Arterial blood pressure was measured, by tail plethysmography, from 2 months on. Renal function was evaluated in 2- and 3-month-old rats in the DO group and in controls (C). Analysis of renal morphology was carried out in newborn and in 1-, 2- and 3-month-old rats in both groups. Although the nephron number was not changed in the DO group, glomerular hypertrophy was observed from 2 months on. At the same age, the glomerular filtration rate was significantly reduced in DO, and blood pressure was significantly increased, when compared to C. Glucose tolerance test (GTT) from DO showed a different profile when compared to C. The number of PCNA positive cells in renal tissue was similar in both groups. Our data suggests that exposure to intrauterine diabetes may be an important cause of both impaired renal function and hypertension in offspring, without changes in the nephron number.