Salivary iodide status as a measure of whole body iodine homoeostasis?

Iodine is a trace element required to produce the thyroid hormones, which are critical for development, growth and metabolism. To ensure appropriate population iodine nutrition, convenient and accurate methods of monitoring are necessary. Current methods for determining iodine status either involve a significant participant burden or are subject to considerable intra-individual variation. The continuous secretion of iodide in saliva potentially permits its use as a convenient, non-invasive assessment of status in populations. To assess its likely effectiveness, we reviewed studies analysing the association between salivary iodide concentration (SIC) and dietary iodine intake, urinary iodide concentration (UIC) and/or 24-h urinary iodide excretion (UIE). Eight studies conducted in different countries met the inclusion criteria, including data for 921 subjects: 702 healthy participants and 219 with health conditions. SIC correlated positively with UIC and/or UIE in four studies, with the strength of relationship ranging from r = 0·19 to r = 0·90 depending on sampling protocol, age, and if salivary values were corrected for protein concentration. Additionally, SIC positively correlated with dietary intake, being strongest when saliva was collected after dinner. SIC varied with external factors, including thyroid function, use of some medications, smoking and overall health status. Evidence provided here supports the use of SIC as a viable, low-burden method for determining iodine status in populations. However, small sample sizes and high variability indicates the need for more extensive analyses across age groups, ethnicities, disease states and dietary groups to clarify the relative accuracy and reliability in each case and standardise procedure.

Systematic review and meta-analysis of iodine nutrition in modern vegan and vegetarian diets.

Vegan and vegetarian diets are widely supported and adopted, but individuals following such diets remain at greater risk of iodine deficiency. This systematic review and meta-analysis was conducted to assess the iodine intake and status in adults following a vegan or vegetarian diet in the modern day. A systematic review and quality assessment were conducted from October 2020 to December 2022 according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) and Meta-analysis of Observational Studies in Epidemiology (MOOSE) guidance. Studies were identified in Ovid MEDLINE, Web of Science, PubMed, and Scopus. Eleven articles were eligible for review containing 4421 adults (aged ≥ 18 years). Vegan groups had the lowest median urinary iodine concentration (mUIC) (12·2/l). None of the dietary groups had mUIC within the optimal range for iodine status (100-200 µg/l) (WHO). Vegan diets had the poorest iodine intake (17·3 µg/d) and were strongly associated with lower iodine intake (P = < 0·001) compared with omnivorous diets. Lower intake in vegan diets was influenced by sex (P = 0·007), the presence of voluntary or absence of Universal Salt Iodisation (USI) programmes (P = 0·01 & P = < 0·001), and living in a country with adequate iodine nutrition (P = < 0·001). Vegetarians and particularly vegans living in countries with no current USI programme continue to have increased risk of low iodine status, iodine deficiency and inadequate iodine intake. Further research into the usefulness of mandatory fortification of vegan appropriate foods is required.

Remote effects of acute kidney injury in a porcine model.

Acute kidney injury (AKI) is a common and serious condition with no specific treatment. An episode of AKI may affect organs distant from the kidney, further increasing the morbidity associated with AKI. The mechanism of organ cross talk after AKI is unclear. The renal and immune systems of pigs and humans are alike. Using a preclinical animal (porcine) model, we tested the hypothesis that early effects of AKI on distant organs is by immune cell infiltration, leading to inflammatory cytokine production, extravasation, and edema. In 29 pigs exposed to either sham surgery or renal ischemia-reperfusion (control, n = 12; AKI, n = 17), we assessed remote organ (liver, lung, brain) effects in the short (from 2- to 48-h reperfusion) and longer term (5 wk later) using immunofluorescence (for leukocyte infiltration, apoptosis), a cytokine array, tissue elemental analysis (e.g., electrolytes), blood hematology and chemistry (e.g., liver enzymes), and PCR (for inflammatory markers). AKI elicited significant, short-term (∼24 h) increments in enzymes indicative of acute liver damage (e.g. , AST: ALT ratio; P = 0.02) and influenced tissue biochemistry in some remote organs (e.g., lung tissue [Ca(2+)] increased; P = 0.04). These effects largely resolved after 48 h, and no further histopathology, edema, apoptosis, or immune cell infiltration was noted in the liver, lung, or hippocampus in the short and longer term. AKI has subtle biochemical effects on remote organs in the short term, including a transient increment in markers of acute liver damage. These effects resolved by 48 h, and no further remote organ histopathology, apoptosis, edema, or immune cell infiltration was noted.

Remote conditioning or erythropoietin before surgery primes kidneys to clear ischemia-reperfusion-damaged cells: a renoprotective mechanism?

Acute kidney injury is common, serious with no specific treatment. Ischemia-reperfusion is a common cause of acute kidney injury (AKI). Clinical trials suggest that preoperative erythropoietin (EPO) or remote ischemic preconditioning may have a renoprotective effect. Using a porcine model of warm ischemia-reperfusion-induced AKI (40-min bilateral cross-clamping of renal arteries, 48-h reperfusion), we examined the renoprotective efficacy of EPO (1,000 iu/kg iv.) or remote ischemic preconditioning (3 cycles, 5-min inflation/deflation to 200 mmHg of a hindlimb sphygmomanometer cuff). Ischemia-reperfusion induced significant kidney injury at 24 and 48 h (χ(2), 1 degree of freedom, >10 for 6/7 histopathological features). At 2 h, a panel of biomarkers including plasma creatinine, neutrophil gelatinase-associated lipocalin, and IL-1ß, and urinary albumin:creatinine could be used to predict histopathological injury. Ischemia-reperfusion increased cell proliferation and apoptosis in the renal cortex but, for pretreated groups, the apoptotic cells were predominantly intratubular rather than interstitial. At 48-h reperfusion, plasma IL-1ß and the number of subcapsular cells in G2-M arrest were reduced after preoperative EPO, but not after remote ischemic preconditioning. These data suggest an intrarenal mechanism acting within cortical cells that may underpin a renoprotective function for preoperative EPO and, to a limited extent, remote ischemic preconditioning. Despite equivocal longer-term outcomes in clinical studies investigating EPO as a renoprotective agent in AKI, optimal clinical dosing and administration have not been established. Our data suggest further clinical studies on the potential renoprotective effect of EPO and remote ischemic preconditioning are justified.

Protein restriction in the pregnant mouse modifies fetal growth and pulmonary development: role of fetal exposure to {beta}-hydroxybutyrate.

Maternal undernutrition during sensitive periods of pregnancy results in offspring predisposed towards the development of a number of diseases of adulthood, including hypertension and diabetes. In order to determine the nature of any gross alterations in fetal growth during early organogenesis, we supplied timed-mated pregnant mice with diets containing 6% protein (6%P), 9% protein (9%P) or 18% protein (18%P; control) from day 0 of pregnancy. At embryonic days 11 (E11), 12 (E12) and 13 (E13), females were killed and fetuses removed. Gross morphological analysis revealed that fetal limb growth was impaired between E11 and E12 in 6%P animals, but this recovered by E13. Likewise, fetal liver growth and lung branching morphogenesis were seen to exhibit an initial growth impairment at E12 followed by a rapid recovery by E13. Coincident with the observed changes in fetal growth, we noted an elevation in maternal hepatic triglyceride content, expression of the ketogenic 3-hydroxy-3-methylglutaryl-CoA synthase 2 (Hmgcs2) and circulating plasma ß-hydroxybutyrate (BOHB). In addition, fetal liver Hmgcs2 expression was switched on by E13 in both 6%P- and 9%P-exposed animals. Exogenous BOHB did not influence branching morphogenesis in fetal lung explant cultures; however, we cannot rule out the possibility that this may occur in vivo. In conclusion, we find that disturbance of fetal growth by maternal dietary protein restriction is associated and therefore potentially indicated by changes in maternal and fetal ketone body metabolism.

Corticosteroid-induced kidney dysmorphogenesis is associated with deregulated expression of known cystogenic molecules, as well as Indian hedgehog.

An intact genome is essential for kidney growth and differentiation, but less is known about whether, and how, an altered fetal milieu modifies these processes. Maternal low-protein diets perturb growth of the metanephros, the precursor of the mature kidney. Fetal corticosteroid overexposure may, in part, mediate this, because such diets downregulate placental 11beta-hydroxysteroid dehydrogenase-2, which degrades maternal corticosteroids. We report that glucocorticoid and mineralocorticoid receptors are expressed in mouse metanephric epithelia. Metanephroi maintained in organ culture with hydrocortisone (1.4 or 14 microM) underwent a dose-dependant deceleration of overall growth accompanied by cyst formation. Dexamethasone, a glucocorticoid, reproduced these outcomes, but aldosterone, a mineralocorticoid, did not. Hydrocortisone upregulated transcripts levels of cadherin-11 and downregulated prospero-related homeobox-1, hence mimicking reported effects of maternal low-protein diet. Hydrocortisone also upregulated transcripts encoding Na(+)-K(+)-ATPase subunits and ligands for the epidermal growth factor receptor, all previously implicated in renal cyst growth. The most upregulated transcript, however, was indian hedgehog, and the encoded protein was immunodetected in metanephric cysts. Furthermore, in the presence of hydrocortisone, cystogenesis, but not whole organ growth, was significantly reduced by cyclopamine, a drug downregulating hedgehog signaling. Finally, both glucocorticoid receptor and indian hedgehog proteins were detected by immunohistochemistry in cystic tubules within human dysplastic kidneys, consistent with the hypothesis that these molecules modify the severity of this congenital malformation. Collectively, our observations raise the possibility that enhanced hedgehog signaling is an important stimulus for renal cyst formation. Furthermore, pharmacological inhibition of this pathway should be explored as a potential therapy for renal cystic diseases, starting with relevant animal models.

Trace Mineral Intake and Deficiencies in Older Adults Living in the Community and Institutions: A Systematic Review.

The global population is ageing with many older adults suffering from age-related malnutrition, including micronutrient deficiencies. Adequate nutrient intake is vital to enable older adults to continue living independently and delay their institutionalisation, as well as to prevent deterioration of health status in those living in institutions. This systematic review investigated the insufficiency of trace minerals in older adults living independently and in institutions. We examined 28 studies following a cross-sectional or cohort design, including 7203 older adults (≥60) living independently in 13 Western countries and 2036 living in institutions in seven Western countries. The estimated average requirement (EAR) cut-off point method was used to calculate percentage insufficiency for eight trace minerals using extracted mean and standard deviation values. Zinc deficiency was observed in 31% of community-based women and 49% of men. This was higher for those in institutional care (50% and 66%, respectively). Selenium intakes were similarly compromised with deficiency in 49% women and 37% men in the community and 44% women and 27% men in institutions. We additionally found significant proportions of both populations showing insufficiency for iron, iodine and copper. This paper identifies consistent nutritional insufficiency for selenium, zinc, iodine and copper in older adults.

Vegans, Vegetarians, and Omnivores: How Does Dietary Choice Influence Iodine Intake? A Systematic Review.

Vegan and vegetarian diets are becoming increasingly popular. Dietary restrictions may increase the risk of iodine deficiency. This systematic review aims to assess iodine intake and status in adults following a vegan or vegetarian diet in industrialised countries. A systematic review and quality assessment were conducted in the period May 2019-April 2020 according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Studies were identified in Ovid MEDLINE, Embase, Web of Science, PubMed, Scopus, and secondary sources. Fifteen articles met inclusion criteria. Participants included 127,094 adults (aged ≥ 18 years). Vegan groups presented the lowest median urinary iodine concentrations, followed by vegetarians, and did not achieve optimal status. The highest iodine intakes were recorded in female vegans (1448.0 ± 3879.0 µg day-1) and the lowest in vegetarians (15.6 ± 21.0 µg day-1). Omnivores recorded the greatest intake in 83% of studies. Seaweed contributed largely to diets of vegans with excessive iodine intake. Vegans appear to have increased risk of low iodine status, deficiency and inadequate intake compared with adults following less restrictive diets. Adults following vegan and vegetarian diets living in countries with a high prevalence of deficiency may be more vulnerable. Therefore, further monitoring of iodine status in industrialised countries and research into improving the iodine intake and status of adults following vegan and vegetarian diets is required.

Geographical and seasonal variation in iodine content of cow's milk in the UK and consequences for the consumer´s supply.

BACKGROUND: Dairy products provide a crucial source of dietary iodine for the majority of the UK population, contributing approximately 30-40 % of daily intake. Fluctuations in the iodine content of purchased milk both seasonally and annually implies potential fragility of iodine supply likely through fluctuating supplementation practices in cow herds. We set out to establish the level of national variation in herds and identify factors which might impact milk iodine content. METHODS: Milk samples were obtained from 98 herds across the UK via the National Milk Laboratories in August and December 2016. Iodine concentration of samples was measured using ICP-MS. Milk samples and feed intake data were additionally taken from 22 cows from the University of Nottingham (UON) dairy herd. RESULTS: There was considerable variation in milk iodine content from < 0.012 (Limit of Detection) to 1558 µg L-1, with a summer median of 197 µg L-1 and winter median 297 µg L-1. Overall, winter values were higher than summer counterparts (P < 0.001) and this held true for samples taken from the North West (P = 0.002) and South West (P = 0.006) but not for other regions studied. Data from the UON herd showed a negative relationship between iodine content and milk yield (P = 0.03) and we found that milk iodine content varied considerably despite apparently similar iodine intakes. CONCLUSIONS: Regional differences in milk iodine concentration between summer and winter suggests that feeding practices are far from uniform across the country. The negative association observed between iodine concentration and milk yield in UON samples, suggests that reduced summer values may be influenced by dilution in addition to seasonal differences in concentrate feed provision.

A novel liver specific isoform of the rat LAR transcript is expressed as a truncated isoform encoded from a 5'UTR located within intron 11.

BACKGROUND: The leukocyte common antigen related receptor (LAR) protein has been shown to modulate the signal transduction of a number of different growth factors, including insulin and insulin-like growth factor 1. Splice variants exhibit differing roles and are expressed according to tissue type and developmental stage. RESULTS: Using 5'RACE, we identified a 5'UTR within intron 11 of the rat LAR gene. We demonstrated that this gives rise to a novel isoform of the LAR transcript encoded from the identified region within intron 11. By priming across the site from exon 11 to exon 15 we show that the novel 5'UTR is not represented in the full-length transcript and thus, it produces a truncated form of the LAR mRNA. We examined the tissue distribution of this novel isoform and found it to be exclusively expressed in liver. We additionally identified a liver specific 150 kDa band with western blotting which we propose may represent the protein product of the novel transcript. Luciferase assays showed the region immediately upstream of the 5'UTR to possesses considerable promoter activity and that this may be conferred by the presence of a number of putative binding sites for liver enriched transcription factors. CONCLUSION: In summary, we describe a novel, liver specific, truncated isoform of the LAR transcript transcribed under the control of an intronic promoter, potentially representing a previously unidentified modulator of hepatic insulin signalling.

LIM kinase function and renal growth: Potential role for LIM kinases in fetal programming of kidney development.

AIMS: Maternal dietary restriction during pregnancy impairs nephron development and results in offspring with fewer nephrons. Cell turnover in the early developing kidney is altered by exposure to maternal dietary restriction and may be regulated by the LIM-kinase family of enzymes. We set out to establish whether disturbance of LIM-kinase activity might play a role in the impairment of nephron formation. MAIN METHODS: E12.5 metanephric kidneys and HK2 cells were grown in culture with the pharmacological LIM-kinase inhibitor BMS5. Organs were injected with DiI, imaged and cell numbers measured over 48h to assess growth. Cells undergoing mitosis were visualised by pH3 labelling. KEY FINDINGS: Growth of cultured kidneys reduced to 83% of controls after exposure to BMS5 and final cell number to 25% of control levels after 48h. Whilst control and BMS5 treated organs showed cells undergoing mitosis (100±11 cells/field vs 113±18 cells/field respectively) the proportion in anaphase was considerably diminished with BMS5 treatment (7.8±0.8% vs 0.8±0.6% respectively; P<0.01). This was consistent with effects on HK2 cells highlighting a severe impact of BMS5 on formation of the mitotic spindle and centriole positioning. DiI labelled cells migrated in 100% of control cultures vs 0% BMS5 treated organs. The number of nephrogenic precursor cells appeared depleted in whole organs and formation of new nephrons was blocked by exposure to BMS5. SIGNIFICANCE: Pharmacological blockade of LIM-kinase function in the early developing kidney results in failure of renal development. This is likely due to prevention of dividing cells from completion of mitosis with their resultant loss.

Acetylsalicylic acid interferes with embryonic kidney growth and development by a prostaglandin-independent mechanism.

AIM: To evaluate the effects of the non-selective, non-steroidal anti-inflammatory drug (NSAID) acetylsalicylic acid (ASA), on ex vivo embryonic kidney growth and development. METHODS: Pairs of fetal mouse kidneys at embryonic day 12.5 were cultured ex vivo in increasing concentrations of ASA (0.04-0.4 mg/mL) for up to 7 d. One organ from each pair was grown in control media and was used as the internal control for the experimental contralateral organ. In some experiments, organs were treated with ASA for 48 h and then transferred either to control media alone or control media containing 10 µmol/L prostaglandin E2 (PGE2) for a further 5 d. Fetal kidneys were additionally obtained from prostaglandin synthase 2 homozygous null or heterozygous (PTGS2-/- and PTGS2-/+) embryos and grown in culture. Kidney cross-sectional area was used to determine treatment effects on kidney growth. Whole-mount labelling to fluorescently detect laminin enabled crude determination of epithelial branching using confocal microscopy. RESULTS: Increasing ASA concentration (0.1, 0.2 and 0.4 mg/mL) significantly inhibited metanephric growth (P < 0.05). After 7 d of culture, exposure to 0.2 mg/mL and 0.4 mg/mL reduced organ size to 53% and 23% of control organ size respectively (P < 0.01). Addition of 10 µmol/L PGE2 to culture media after exposure to 0.2 mg/mL ASA for 48 h resulted in a return of growth area to control levels. Application of control media alone after cessation of ASA exposure showed no benefit on kidney growth. Despite the apparent recovery of growth area with 10 µmol/L PGE2, no obvious renal tubular structures were formed. The number of epithelial tips generated after 48 h exposure to ASA was reduced by 40% (0.2 mg/mL; P < 0.05) and 47% (0.4 mg/mL; P < 0.01). Finally, growth of PTGS2-/- and PTGS2+/- kidneys in organ culture showed no differences, indicating that PTGS2 derived PGE2 may at best have a minor role. CONCLUSION: ASA reduces early renal growth and development but the role of prostaglandins in this may be minor.

Maternal diet programs embryonic kidney gene expression.

Human epidemiological data associating birth weight with adult disease suggest that organogenesis is "programmed" by maternal diet. In rats, protein restriction in pregnancy produces offspring with fewer renal glomeruli and higher systemic blood pressures than controls. We tested the hypothesis that maternal diet alters gene expression in the metanephros, the precursor of the definitive mammalian kidney. We demonstrated that maternal low-protein diet initiated when pregnancy starts and maintained to embryonic day 13, when the metanephros consists of mesenchyme surrounding a once-branched ureteric bud, is sufficient to significantly reduce glomerular numbers in offspring by about 20%. As assessed by representational difference analyses and real-time quantitative polymerase chain reactions, low-protein diet modulated gene expression in embryonic day 13 metanephroi. In particular, levels of prox-1, the ortholog of Drosophila transcription factor prospero, and cofilin-1, a regulator of the actin cytoskeleton, were reduced. During normal metanephrogenesis, prox-1 protein was first detected in mesenchymal cells around the ureteric tree and thereafter in nascent nephron epithelia, whereas cofilin-1 immunolocalized to bud derivatives and condensing mesenchyme. Previously, we reported that low-protein diets increased mesenchymal apoptosis cells when metanephrogenesis began and thereafter reduced numbers of precursor cells. Collectively, these studies prove that the maternal diet programs the embryonic kidney, altering cell turnover and gene expression at a time when nephrons and glomeruli have yet to form. The human implication is that the maternal diet ingested between conception and 5- 6-wk gestation contributes to the variation in glomerular numbers that are known to occur between healthy and hypertensive populations.

Peptic digestion of beef myofibrils is modified by prior marination.

BACKGROUND: Preparatory steps such as seasoning, marination, and cooking may induce changes in meat which affects the ability of the stomach to adequately digest it. This may result in peptide chains reaching the colon intact where resident bacteria ferment them resulting in the formation of putative carcinogenic phenolic by-products. OBJECTIVE: In this study, we set out to determine whether peptic digestion of beef myofibrils was influenced by prior marination. DESIGN: Cubes of sirloin stewing steak were marinated in balsamic vinegar or left untreated at 4°C overnight. Samples were oven cooked and myofibrils were extracted. Myofibrils were subject to proteolytic digestion with pepsin and digestion products analysed spectrophotometrically and with gel electrophoresis. RESULTS: Both marination in balsamic vinegar and cooking significantly reduced the yield of myofibrils from shop-purchased beef (P<0.05). Digestion progressed in all samples as a function of time (P<0.01), varying depending on prior treatment. Marination induced resistance to the digestive effect of pepsin during the early to mid-phase of digestion, and we identified a protein band of ∼150 kDa which was protected from peptic digestion in samples which had been marinated and cooked, but not in any other groups. CONCLUSIONS: Pre-treatment of meat prior to cooking may influence specific peptides such that they become more resistant to the digestive actions of pepsin.

Excess maternal salt intake produces sex-specific hypertension in offspring: putative roles for kidney and gastrointestinal sodium handling.

Hypertension is common and contributes, via cardiovascular disease, towards a large proportion of adult deaths in the Western World. High salt intake leads to high blood pressure, even when occurring prior to birth - a mechanism purported to reside in altered kidney development and later function. Using a combination of in vitro and in vivo approaches we tested whether increased maternal salt intake influences fetal kidney development to render the adult individual more susceptible to salt retention and hypertension. We found that salt-loaded pregnant rat dams were hypernatraemic at day 20 gestation (147±5 vs. 128±5 mmoles/L). Increased extracellular salt impeded murine kidney development in vitro, but had little effect in vivo. Kidneys of the adult offspring had few structural or functional abnormalities, but male and female offspring were hypernatraemic (166±4 vs. 149±2 mmoles/L), with a marked increase in plasma corticosterone (e.g. male offspring; 11.9 [9.3-14.8] vs. 2.8 [2.0-8.3] nmol/L median [IQR]). Furthermore, adult male, but not female, offspring had higher mean arterial blood pressure (effect size, +16 [9-21] mm Hg; mean [95% C.I.]. With no clear indication that the kidneys of salt-exposed offspring retained more sodium per se, we conducted a preliminary investigation of their gastrointestinal electrolyte handling and found increased expression of proximal colon solute carrier family 9 (sodium/hydrogen exchanger), member 3 (SLC9A3) together with altered faecal characteristics and electrolyte handling, relative to control offspring. On the basis of these data we suggest that excess salt exposure, via maternal diet, at a vulnerable period of brain and gut development in the rat neonate lays the foundation for sustained increases in blood pressure later in life. Hence, our evidence further supports the argument that excess dietary salt should be avoided per se, particularly in the range of foods consumed by physiologically immature young.

Cell turnover in normal and abnormal kidney development.

As metanephric mesenchyme converts into nephrons, the first step is aggregation into a 'condensate'. Precursors inside this structure are proliferative and have a low rate of apoptosis, accompanied by expression of PAX-2 and BCL-2 survival molecules; conversely, cells at the borders of the structure have a high rate of apoptosis, probably a normal mechanism to regulate the number of cells in each nephron. Ureteric bud/collecting duct survival and mitosis may be determined partly by renal mesenchymal secreted molecules such as hepatocyte growth factor (HGF) and glial cell line-derived neurotrophic factor. Human kidney malformations often occur with lower urinary tract obstruction, e.g. cystic dysplastic kidneys caused by urethral valves. Deregulation of cell turnover occurs in these organs, with enhanced proliferation in cystic epithelium, accompanied by PAX-2, BCL-2 and HGF receptor expression, and apoptosis in surrounding mesenchyme, which transdifferentiates under the influence of transforming growth factor-beta1 into smooth muscle instead of forming nephrons. Similar abnormalities of cell turnover and gene expression can be generated by experimental fetal urinary flow impairment. Finally, renal mesenchymal apoptosis, associated with renal hypoplasia, can be induced experimentally by maternal low protein diet.

Protein restriction in pregnancy is associated with increased apoptosis of mesenchymal cells at the start of rat metanephrogenesis.

BACKGROUND: In rats, offspring born to mothers supplied low protein diets during pregnancy have fewer glomeruli than normal. We hypothesized that such nephron deficits are associated with altered cell turnover in the metanephros, the embryonic precursor of the adult kidney. METHODS: Wistar rats were supplied with one of three isocaloric diets from day 0 of pregnancy: control (18% protein) or low protein (9% or 6%) diets. All had a normal chow after birth. Groups were compared by multilevel statistical modeling. RESULTS: At two weeks postnatally, when nephrogenesis has finished, controls had 16.8 x 103 +/- 0.7 x 10(3) (mean +/- SEM) glomeruli/kidney, whereas offspring exposed to 9% diet had 5.1 x 10(3) +/- 1.2 x 10(3) fewer and those exposed to 6% diet had 6.9 x 10(3) +/- 1.7 x 10(3) fewer glomeruli/kidney (P < 0.001, both diets). At embryonic day 13 (E13), when the metanephros has just formed, control metanephroi contained 2.35 x 10(4) +/- 0.15 x 10(4) cells, with no significant differences in low protein groups. At E15, when mesenchyme begins forming primitive nephrons but glomeruli are still absent, controls had 2.00 x 10(6) +/- 0.13 x 10(6) cells. E15 embryos exposed to 9% protein had 1.09 x 10(6) +/- 0.36 x 10(6) fewer cells/metanephros than controls, while those exposed to 6% diet had 1.45 x 10(6) +/- 0.37 x 10(6) fewer (P < 0.01, both diets). Apoptotic cells were detected by molecular (in-situ end-labeling) and morphological (propidium iodide staining) techniques. In all diets, apoptosis was noted in condensing mesenchyme (nephron precursors) and loose mesenchyme (interstitial precursors). Control E13 metanephroi had 63 +/- 7 apoptotic cells/mm2, whereas those exposed to 9% diet had an increase of 77 +/- 26 cells/mm2 (P < 0.01) and those exposed to 6% diet had an increase of 55 +/- 26 cells/mm2 (P < 0.05). By E15, apoptosis was similar in all groups but metanephric mitosis was significantly increased in the 6% protein diet group. No change was found in the level of apoptosis in E13 mesonephroi. CONCLUSIONS: Maternal low protein diets reduce final numbers of glomeruli in association with enhanced deletion of mesenchymal cells at the start of kidney development. Whether aberrant nephrogenesis is a direct effect from deletion of nephron precursors, or an indirect effect from loss of supportive interstitial precursors, requires further investigation.